NE1030: Characterization and Mechanisms of Plant Responses to Ozone in the U.S.

(Multistate Research Project)

Status: Inactive/Terminating

Project Menu

SAES-422 Reports

Annual/Termination Reports:

[09/30/2008] [05/30/2009] [09/23/2010] [10/03/2011] [10/04/2012]

Date of Annual Report: 09/30/2008

Report Information

Annual Meeting Dates: 05/15/2008 - 05/16/2008
Period the Report Covers: 10/01/2007 - 09/01/2008

Participants

Best, Teodora Best (txo115@psu.edu) - Pennsylvania State University; Booker, Fitzgerald (fitz.booker@ars.usda.gov) - USDA-ARS, Raleigh, NC; Chappelka, Art (chappah@auburn.edu) - Auburn University; Grantz, David (david@uckac.edu) - University of California - Riverside; Manning, William (wmanning@microbio.umass.edu) - University of Massachusetts; Matyssek, Rainer (matyssek@wzw.tum.de) - Technical University of Munich; McGrath, Margaret (mtm3@cornell.edu) - Cornell University; Muntifering, Russell (rmuntife@acesag.auburn.edu) - Auburn University; Neufeld, Howard (neufeldhs@appstate.) - Appalachian State University; Ren, Wei renwei1@auburn.edu) - Auburn University; Sandermann, Heinrich (heinrich.sandermann@ctp.uni-freiburg.de) - Ecotox; Wiese, Cosima cwiese@misericordia.edu)- College Misericordia; Zilinskas, Barbara (zilinskas@aesop.rutgers.edu) - Rutgers University;

Brief Summary of Minutes

The Annual Meeting of the Technical Committee was held at Auburn University, May 15-16, 2008.

The meeting was hosted by Art Chappelka and Russell Muntifering of Auburn University and coordinated by outgoing Chair, Fitzgerald Booker of USDA/ARS in Raleigh NC.

Dr. Margaret Smith, Associate Director, Cornell Univ. Agricultural Experiment Station (attending on Mike Hoffmann's behalf) planned on attending the meeting but conflicts with travel arrangements prevented it.

Several members of the NE-1030 Technical Committee, along with Ray Knighton were unable to attend due to participation in the USDA-ARS Global Change program and Air Quality Task Force meetings.

Station reports were presented by project objective. Guest presentations were made at the conclusion of the station reports:

Rainer Matyssek - Spatio-Temporal Scaling of Ozone Uptake and Effective Dose in Forest Trees: Current Status and Perspectives;

Heinrich Sandermann - Bioactivation of Extracellular Ascorbate by Ozone.

Business Meeting

1. At the conclusion of the technical meeting, a business meeting was held. The Committee expressed satisfaction with the term of outgoing Chair, Fitz Booker who oversaw two excellent meetings and the completion of the Final Report for the NE-1013 Project.

2. NE-1030 Annual Reports for the period October 1, 2007 September 30, 2008 should be sent to D. Grantz.

3. Snap bean project status was discussed. Committee members involved in this project have finalized the experimental protocol and now have a few years of data from several locations. It is anticipated that there will soon be enough data for the modeling work.

4. Art Chappelka was nominated and elected Chair-elect (2010-2012). During this term the current project will terminate and a new proposal will need to be written. Meg McGrath agreed to continue serving as Secretary.

5. It was decided that the 2009 meeting of NE-1030 will be Riverside, CA, hosted by Nancy Grulke (US Forest Service, Riverside, CA) and coordinated by Chair, David Grantz of the University of California at Riverside. Meetings will continue to be rotated between the western, southern and northeastern regions of the US. Dennis Decoteau (Penn State University, State College, PA) offered to host a meeting, and it was selected for the meeting in 2010.

6. Rainer Matyssek and Teodora Best expressed an interest in becoming members of the NE-1030 project. The necessary steps are in process through the Administrative Advisor, Dr. Mike Hoffmann.

7. A number of other projects in the gestational stage, or with preliminary data not ready for public presentation, were discussed informally. The NE-1030 technical committee represents a large fraction of the ongoing research on the effects of ozone on vegetation. A few key projects have had a low profile in this group, which should be rectified if possible.

8. The NE-1013 website has been updated and revised for the NE-1030 project although the URL remains unchanged (http://www.ncsu.edu/project/usda-ne-1013/). Searches for NE-1030 are re-directed to this URL.

9. Chair Elect David Grantz assumed the Chair of the NE-1030 committee for the next two years (2008-2010).

Meeting adjourned at 11:30 a.m. on May 16, 2008.

Respectively submitted,
Meg McGrath, Secretary
August 4, 2008

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. (MA) A MS thesis project was conducted by Jennifer Albertine on soil warming, seedling emergence, and ozone injury. Beans grown in the warm soil (25 C) germinated faster, developed leaves faster, and had visible ozone injury 2 days earlier than plants grown at 20 C. Exposure to elevated ozone resulted in reduced leaf area and reduced stem length. Height was not affected by O3. (Bill Manning, University of Massachusetts). (NY) Plant response to ambient ozone on Long Island, NY, was examined in 2007 by growing ozone-sensitive and ozone-tolerant snap bean lines (S156 and R331) as done since 2004. There were three successive field plantings of beans to be able to assess the impact of ambient ozone occurring throughout the growing season (14 May, 12 June, and 11 July 2007). As they developed, bean pods were harvested repeatedly from some plants when immature and at a size typical for fresh-market consumption. Pods were harvested from the other plants when mature and dry. S156 yielded less than the tolerant line R331 when grown under ambient ozone conditions on Long Island in 2007. Total weight and number of bean pods harvested for fresh-market consumption from planting 1 (14 May) plants was 23% and 11% lower, respectively, for S156 compared to R331 (pods were harvested from 9 July through 30 July). There was a 30% and 18% reduction in these yield variables, respectively, for planting 2 (12 June) plants (30 July through 5 Sept). Reduction was 29% and 10%, respectively, for planting 3 (11 July) plants (29 Aug through 3 Oct). These differences were not always significant. Mature yield was also reduced for S156 compared to R331. For plants in planting 1, number of pods produced by S156 was reduced 10% compared to R331, number of seeds was reduced 22% and average seed weight was reduced 20%. There was a 17%, 24%, and 32% reduction in these yield variables, respectively, for planting 2 plants; and a 30%, 38%, and 28% reduction in these yield variables, respectively, for planting 3 plants. From emergence until the last fresh-market pod harvest, plants in the three plantings were exposed to O3 that was at least 40 ppb for 627, 791, and 605 hours, respectively. During these growth periods of 63, 79, and 78 days, O3 exposure expressed as AOT40 was 7,643 ppb.h, 10,451 ppb.h, and 6,827 ppb.h, respectively. These values greatly exceed the long-term critical level of ozone exposure for crops of 3000 ppb.h accumulated over three months. (Meg McGrath, Cornell University). 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. (AL) O3 effects on forage quality were examined. We completed the fourth year of a 7-year (20042010) experiment to determine effects of co-exposure to a range of current and projected levels of O3 and atmospheric N on productivity and nutritive quality of an extensively managed, species-rich (primarily Festuca, Nardus and Carex, numerous forb and few legume spp.) pasture located at Alp Flix, Switzerland. One hundred eighty plots (40 × 30 cm) representative of vegetation at the site were exposed during the AprilOctober growing seasons in 20042007 to one of three levels of O3 (ambient, ambient + 20 ppb O3, or ambient + 40 ppb O3, corresponding to 1.0, 1.2 or 1.6 × ambient O3, respectively) in a free-air fumigation system that comprised 9 exposure rings (3 rings/O3 level). Within each ring, 20 plots received biweekly applications of NH4NO3 solution that simulated five areal concentrations of atmospheric N deposition equivalent to 0, 5, 10, 25 or 50 kg/ha (4 plots/N concentration). Plots were harvested once each year in August by cutting forage at 2 cm above ground surface. Differences (P < 0.0001) were observed among years in forage concentrations of N and cell-wall constituents, and in relative nutritive quality calculated from forage concentrations of the latter. Across all four growing seasons and levels of N input, there was no systematic effect of O3 exposure level on grassland nutritive quality. However, there was an O3 × N interaction (P = 0.09) such that positive responses in forage quality to N inputs of 25 and 50 kg/ha were ablated by increased deposition, and lignification of cell-wall constituents associated with accelerated foliar senescence in the elevated-O3 treatments. Results indicate that excessive rates of N deposition may increase plant sensitivity to elevated O3, and further compound the phytotoxic effects of O3 on forage quality. In a second project just started, diet selection and nutrient utilization are being examined for a model herbivore (rabbit) receiving ozone-exposed forage. Clover, the current preferred forage over grass, is more sensitive to ozone, resulting in lower nutrient quality, which could lead to changes in feeding preference. (Russell Muntifering, Auburn University). (AL) provided an overview of a project on ozone impacts on native trees and wildflowers in the Great Smoky Mountains National Park that he is conducting with other NE1030 participants. This, the most visited park in the US, is considered a Class 1 Wilderness area, thus project findings can have policy implications. Results also were presented on a study investigating the effects of concurrent elevated carbon dioxide and ozone on leaf gas exchange characteristics. When European beech grown under ambient ozone and carbon dioxide was exposed to elevated ozone for 1 hr (55 or 95 ppb), stomatal control was reduced resulting in increased water loss. Ozone reduced transpiration at low but not elevated carbon dioxide. (Art Chappelka, Auburn University) (CA) O3 effects on stomatal behavior in tree species were described. Using a newly modified steady state gas exchange system it was shown that ozone exposure reduced the rate of stomatal response, and attenuated the typical closing responses in woody species. This led to increased long term average stomatal conductance and thus ozone flux, as well as degraded plant water relations. (Nancy Grulke; U.S. Forest Service, Riverside CA) (CA) Interactions between herbicide resistance and O3, comets, Grantz (CA) presented results on attempts to alter plant response to ozone by applying methyl jasmonate (o or 160 micrograms per plant applied in small droplets to youngest fully expanded leaves). These exogenous applications led to foliar symptoms that were similar to ozone symptoms. Jasmonate reduced growth and altered root to shoot ratios similarly to ozone. However, there was no interaction between jasmonate and ozone, except in the case of root respiration. In this case ozone had little effect on respiration in the absence of jasmonate, but in its presence ozone had a substantial inhibitory effect. Effects were generally additive. 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. Building on results presented in previous years, it was shown that ozone allows glyphosate-sensitive genotypes to escape the impact of the herbicide, potentially accelerating the fixation of alleles for glyphosate resistance in ozone impacted airbasins. (David Grantz, University of California Riverside) Objective 3. Examine the joint effects of O3 with other growth regulating factors (e.g., CO2, temperature) that are expected to vary with ongoing climate change on crop growth and productivity. (NC) The relative sensitivity to ozone of some tree species in the Great Smoky Mountains was evaluated. Black cherry, winged sumac, sycamore, and tulip popular were among the most sensitive. A graduate student is investigating impacts on lichens of long-term exposure to elevated carbon dioxide and ozone at the Rhinelander FACE site. Identification of individual lichens remains challenging. (Howard Neufeld, Appalachian State University) Objective 4. Examine the physiological and molecular basis of O3 toxicity and tolerance in plants. (NC) Isoprene emissions and O3: no interactions detected so far. 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 and ozone. It was found that isoprene biosynthesis in transgenic Arabidopsis had no influence on visible injury, decreased rosette diameter and lower biomass accumulation caused by 100 ppb of ozone for 21 days. Velvet bean (Mucuna pruriens) 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. Isoprene is synthesized in the chloroplast, so it was not unexpected that emission rates from velvet bean leaves declined with net photosynthesis following treatment with 70 ppb ozone for eight days. Inhibition of isoprene synthesis by fosmidomycin in hydroponically-grown velvet bean had no effect on suppression of net photosynthesis by 125 ppb ozone for eight days. There was no significant effect of fosmidomycin on photosynthesis rates in control plants. These results raise significant questions about the proposed role of isoprene in modifying ozone injury in isoprene-emitting plants. (Fitz Booker, USDA-ARS, Raleigh, NC). (PA) Results were presented to identify molecular and physiological mechanisms that confer enhanced tolerance to ozone stress in trees using black cherry and hybrid poplar. It is hypothesized that a network of genes exists whose expression confers resistance to ozone stress. A gene (OZO) was found that is highly expressed under high ozone. (Teodora Best, Pennsylvania State University). (PA) Phenolic antioxidant characterization in snap bean lines. The goal of this project, conducted in collaboration with Dr. Kent Burkey in the Plant Science Unit at USDA-ARS in Raleigh, NC, is to determine the role of the apoplast in plant defense responses to oxidative stress. To examine this, apoplastic wash fluid was extracted from three snap bean (Phaseolus vulgaris) cultivars (Provider, R123, S156), which differ in their sensitivity to O3, and analyzed using High Performance Liquid Chromatography (HPLC). Initial experiments primarily showed quantitative differences in apoplastic constituents at different leaf ages within one cultivar, indicating that the quantity of the chemical compounds changes as the leaves develop. Additional experiments were conducted during the summer of 2007, in which the R123 and S156 snap bean cultivars were exposed to O3 for 6 days and the first fully expanded leaf third was harvested at the end of the experiment. Initial analysis by HPLC did not show any significant effects of treatment or cultivar on the HPLC profiles of the apoplastic wash fluid. Currently, the HPLC separation procedure is being modified to optimize the separation of the unknown compounds in the samples. Additional experiments are planned to investigate the antioxidant activity of the insoluble cell wall fraction, and to determine whether changes in the apoplastic fluid occur sooner than six days into plant exposure to O3. (Cosima Wiese, College Misericordia). (NJ) The snap bean experiment in New Jersey resulted in final yield (both dry bean weight and seed weight) was 48-50% lower in the sensitive line compared to the tolerant line. The role of glutathione perosidase in the response of Arabidopsis thaliana to ozone was also examined. The hypothesis that GPX3 (glutathione peroxidase 3) is involved in the signal transduction pathway in response to ozone is being tested systematically. (Barbara Zilinskas, Rutgers University). (NC) Open-top chamber yield studies were completed for selected soybean ancestors exposed to season long treatments of either CF air (~25 ppb 12-hour seasonal mean) or CF+O3 (~75 ppb seasonal 12-hour mean). Analysis of three years of data is underway. (Kent Burkey, USDA-ARS, Raleigh). (Germany) The Free-Air ozone experiment at the Kranzberg Forest Site was described. Data are being evalulated to contribute to the use of site specific fluxes of ozone as a predictor of phytotoxicity and as a regulatory measure. It is widely agreed in Europe that mechanistically-based O3 risk assessment of trees and forests needs to be related to two components: (1) stomatal whole-tree O3 uptake and (2) effective O3 dose (i.e. responsiveness per unit of O3 uptake). The presentation highlighted current methodologies and perspectives towards spatio-temporal scaling of both components, exemplifying the combination of the sapflow and eddy covariance approaches in relation to (1), which provides a phytomedically relevant whole-tree and stand-level O3 dose while allowing to empirically derive the non-stomatal O3 flux (with perspectives towards the landscape level). Stable isotope analysis was suggested, as one option, in view of (2), providing a mechanistically based, long-term integration of metabolic O3 responsiveness and its temporal variation. Strategies were introduced towards proxies, which may be suitable for developing new risk modelling tools. The combination of whole-tree sap flow, stand-level eddy covariance, and experimental free-air O3 release methodologies was acknowledged as a promising strategy, in view of (1) and (2), of promoting cause/effect-based O3 risk assessment. (R. Matyssek, Freiburg) (Germany)Potential pro-oxidant reactions of ascorbic acid in response to O3 were evaluated from a theoretical chemistry perspective. The results, involving inadequate kinetics and contents, as well as previously undescribed toxic byproducts, make it somewhat problematic for ascorbic acid to perform the primary ozone defensive role that it has been commonly assumed to perform. Extracellular ascorbate is considered to be the first line of defense against ozone. Experimental evidence for this includes the Arabidopsis mutant vtc1, with only 30 % of normal total ascorbate, is ozone-sensitive. Ascorbate decomposes ozone at an extremely high rate. However, only a single and toxic decomposition product has been identified (singlet oxygen). Ozone might act mainly through secondary toxicants such as singlet oxygen or peroxy-compounds, some of which may be produced by reactions between ascorbate and ozone. Protection against ozone requires not only quenching of ozone but also scavenging of secondary toxicants. Ozone might still act directly, e.g. by attack on a sensitive SH group of some receptor protein. In view of multiple candidate mechanisms for protection or sensitivity, it is too early to define a simple ozone responsiveness parameter to be combined with an ozone flux parameter. (H. Sandermann Ecotox, Freiburg, Germany). EDU may protect plants from ozone injury by acting as an antioxidant. EDU has some similarities with chemicals used in rubber products to protect them from oxidation by ambient ozone and UV radiation. A new field of study is proposed that examines applications of this technology to plants and may be called "tire biology." (H. Sandermann Ecotox, Freiburg, Germany).

Publications

Ainsworth EA. 2008. Rice production in a changing climate: A meta-analysis of responses to elevated carbon dioxide and elevated ozone concentration. Global Change Biology, 14: 1642-1650. Ainsworth EA, Rogers A, Leakey ADB. 2008. Targets for crop biotechnology in a future high-CO2 and high-O3 world. Plant Physiology, 147: 13-19. Bergweiler, C, WJ Manning and BI Chevone. 2008. Seasonal and diurnal gas exchange differences in ozone-sensitive common milkweed (Asclepias syriaca L.) in relation to ozone uptake. Environmental Pollution 152:403-415. Bergweiler, C, H Carreras, E Wannaz, J Rodriguez, B Toselli, L Olcese and ML Pignata. 2008. Field surveys for potential ozone bioindicator plant species in Argentina. Environmental Monitoring and Assessment 138:305-312. Booker, FL, R Muntifering, M McGrath, KO Burkey, D Decoteau, EL Fiscus, W Manning, S Krupa, A Chappelka, DA Grantz. 2008. The ozone component of global change: Potential effects on agricultural and horticultural plant yield, product quality and interactions with invasive species. Journal of Integrative Plant Biology: In press. Feng, Z, K Kobayashi, EA Ainsworth. 2008. Impact of elevated ozone concentration on growth, physiology, and yield of wheat (Triticum aestivum L.): a meta-analysis. Global Change Biology: In press. E.A.Grantz, DA, A Shrestha and H-B Vu. 2008. Early vigor and ozone response in horseweed (Conyza canadensis) biotypes differing in glyphosate resistance. Weed Science 56:224-230. Grantz, DA, A Shrestha and H-B Vu. 2008. Ozone enhances adaptive benefit of glyphosate resistance in horseweed (Conyza canadensis). Weed Science 56:549-554. Handley T, Grulke NE. 2008. Interactive effects of O3 exposure on California black oak (Quercus kelloggii Newb.) seedlings with and without nitrogen amendment. Environmental Pollution xx:1-8.doi:10.1016/j.envpol.2008.01.002. Kline LJ, Davis DD, Skelly JM, Savage JE, Ferdinand J. 2008. Ozone sensitivity of 28 plant selections exposed to ozone under controlled conditions. Northeastern Naturalist 15:5766 Krupa, S, FL Booker, V Bowersox, C Lehmann and D Grantz. 2008. Uncertainties in the current knowledge of some atmospheric trace gases associated with US agriculture. Journal of Air & Waste Management Association 58:986-993. Matyssek, RH Sandermann, G Wieser, FL Booker, S Cieslik, R Musselman and D Ernst. 2008. The challenge of making ozone risk assessment for forest trees more mechanistic. Environmental Pollution:In press. Novak, K, M Schaub, J Fuhrer, JM Skelly, B Frey and N Kräuchi. 2008. Ozone effects on visible foliar injury and growth of Fagus sylvatica and Viburnum lantana seedlings grown in monoculture or in mixture. Environmental and Experimental Botany, Volume 62, 212-220 Orendovici-Best, T, JM Skelly, DD Davis, JA Ferdinand, JE Savage and RE Stevenson. 2008. Ozone uptake (flux) as it relates to ozone-induced foliar symptoms of Prunus serotina and Populus maximowizii × trichocarpa. Environmental Pollution 151:79-92 Paoletti, E., N. Contran, W.J. Manning, A. Castagna, A. Ranieri, and F. Tagliaferro. 2008. Protection of ash (Fraxinus excelsior) trees from ozone injury by ethylenediurea (EDU): Roles of biochemical changes and decreased stomatal conductance in enhancement of growth. Environmental Pollution 155:464-472. Percy, K and R Rittmaster. 2008. Clearing the Air on Forest Productivity. Impact Note No. 47, Natural Resources Canada, Canadian Forest Service-Atlantic Forestry Centre, Fredericton. (http://cfs.nrcan.gc.ca) Pregitzer, KS, AJ Burton, JS King and DR Zak. 2008. Soil respiration, root biomass, and root turnover following long-term exposure of northern forests to elevated atmospheric CO2 and tropospheric O3. New Phytologist doi: 10.1111/j.1469-8137.2008.02564.x Qiu, Q-S, JL Huber, FL Booker, V Jain, ADB Leakey, EL Fiscus, PM Yau, DR Ort and SC Huber. 2008. Increased protein carbonylation in leaves of Arabidopsis and soybean in response to elevated [CO2]. Photosynthesis Research 97:155-166. Reid, CD and EL Fiscus. 2008. Ozone and density affect the response of biomass and seed yield to elevated CO2 in rice. Global Change Biology. 14:60-76. Wang, X, Q Zheng, Z Feng, J Xie, Z Feng, Z Ouyang, and WJ Manning. 2008. Comparison of a diurnal vs. steady-state ozone exposure profile on growth and yield of oilseed rape (Brassica napus L.) in open-top chambers in the Yangtze Delta, China. Environmental Pollution In Press. 2007 Booker, FL, KO Burkey, WA Pursley and AS Heagle. 2007. Elevated carbon dioxide and ozone effects on peanut. I. Gas-exchange, biomass, and leaf chemistry. Crop Science 47:1475-1487. Burkey, KO, FL Booker, WA Pursley and AS Heagle. 2007. Elevated carbon dioxide and ozone effects on peanut. II. Seed yield and quality. Crop Science 47:1488-1497. Calfapietra, C, AE Wiberley, TG Falbel, AR Linskey, G Scarascia-Mugnozza, DF Karnosky, F Loreto, and TD Sharkey. 2007. Isoprene synthase expression and protein levels are reduced under elevated O3 but not under elevated CO2 (FACE) in field-grown aspen trees. Plant Cell Environment 30:654-661. Chen, X, C Tu, M Burton, D Watson, KO Burkey and S Hu. 2007. Plant nitrogen acquisition and interactions under elevated CO2: impact of endophytes and mycorrhizae. Global Change Biology. 13: 1238-1249. Cheng, FY, KO Burkey, JM Robinson and FL Booker. 2007. Leaf extracellular ascorbate in relation to O3 tolerance of two soybean cultivars. Environmental Pollution 150:355-362. Dubois, J.-J.B., EL Fiscus, FL Booker, MD Flowers and CD Reid. 2007. Optimizing the statistical estimation of the parameters of the Farquhar-von Caemmerer-Berry model of photosynthesis. New Phytologist 176:402-414. Farber, R.J. et. al. (Grantz is 15th out of 19 randomly ordered authors). 2007. Obliterating the dust in the Antelope Valley. Paper Number 384, Proceedings, Annual Meeting and Proceedings, Air and Waste Management Association. Fiscus, EL, FL Booker, J-JB Dubois, TR Rufty, JW Burton and WA Pursley. 2007. CO2 enhancement effects in container- versus ground-grown soybeans at equal planting densities. Crop Science 47:2486-2494. Flowers, MD, EL Fiscus, KO Burkey, FL Booker and J-J Dubois. 2007. Photosynthesis, chlorophyll fluorescence, and yield of snap bean (Phaseolus vulgaris L.) genotypes differing in sensitivity to ozone. Environmental and Experimental Botany 61:190-198. Grulke, NE, HS Neufeld, AW Davison, M Roberts, and AH Chappelka. 2007. Stomatal behavior of ozone-sensitive and insensitive coneflowers (Rudbeckia laciniata var. digitata) in Great Smoky Mountains National Park. New Phytologist 173: 100-109. Grulke NE, Paoletti E, Heath RL. 2007. Chronic vs. short term acute O3 exposure effects on nocturnal transpiration in two Californian oaks. The Scientific World 7(S1):134-140. DOI 10.1100/tsw.20007.33 Grulke, NE, Paoletti, E, Heath, RA. 2007. Comparison of calculated and direct measurements of foliar O3 uptake in crop and native tree species. Environmental Pollution 146:640-647. Holmes, WE, DR Zak, KS Pregitzer, and JS King. 2006. Elevated CO2 and O3 alter soil nitrogen transformations beneath trembling aspen, paper birch, and sugar maple. Ecosystems 9:1354-1363. Karnosky, DF, JM Skelly, KE Percy, and AH Chappelka. 2007. Perspectives regarding 50 years of research on effects of tropospheric ozone air pollution on U.S. Forests. Environmental Pollution 147:489-506. Karnosky, DF, H Werner, T Holopainen, K Percy, T Oksanen, E Oksanen, C Heerdt, P Fabian, J Nagy, W Heilman, R Cox, N Nelson, and R Matyssek. 2007. Free-air exposure systems to scale up ozone research to mature trees. Plant Biology 9:181-190. King, JS, CP Giardina, KS Pregtizer and AL Friend. 2007. Biomass partitioning in red pine (Pinus resinosa Ait.) along a chronosequence in the Upper Peninsula of Michigan. Canadian Journal of Forest Research 37:93-102. Kubiske, ME, VS Quinn, PE Marquardt, and DF Karnosky. 2007. Effects of elevated CO2 and/or O3 on intra- and interspecific competitive ability of aspen. Plant Biology 9:342-355. Lin, JC, M. Nosal, RB Muntifering, and SV Krupa. 2007. Alfalfa nutritive quality for ruminant livestock as influenced by ambient air quality in west-central Alberta. Environmental Pollution 149:99-103. Lin, JC, K Nadarajah, M Volk, RB Muntifering and J Fuhrer. 2007. Nutritive quality of a species-rich, extensively managed pasture exposed to elevated ozone in a free-air fumigation system. Journal of Animal Science 90 (Suppl. 1): 36. Long, SP, EA Ainsworth, ADB Leakey, DR Ort, J Nosberger and D Schimel. 2007. Crop models, CO2, and climate change Response. Science 315:460-460. McGrath, M.T. 2007. Assessing ambient ozone impact on plant productivity in NY with snap bean genotypes differing in sensitivity. Phytopathology 97: (presented 11/8/06). (http://www.apsnet.org/meetings/div/ne06abs.asp). Neufeld, HS and AH Chappelka. 2007. Air pollution and vegetation effects research in national parks and natural areas: Implications for science, policy and management. Environmental Pollution 149:253-255. Oncley, SP, T Foken, R Vogt, W Kohsiek, HAR DeBruin, C Bernhofer, A Christen, E van Gorsel, D Grantz, C Feigenwinter, I Lehner, D Liebethal, H Liu, M Mauder, A Pitacco, L Ribeiro and T Weidinger. 2007. The Energy Balance Experiment EBEX-2000. Part I: overview and energy balance. Boundary Layer Meteorology 123:1-28. Paoletti, E, A Bytnerowicz, C Andersen, A Augustaitis, M Ferretti, N Grulke, MS Günthardt-Goerg, J Innes J, DW Johnson, DF Karnosky, J Luangjame, R Matyssek, S McNulty, G Müller-Starck, R Musselman and KE Percy. 2007. Impacts of air pollution and climate change on forest ecosystems- emerging research needs. Scientific World 7:1-8. Paoletti, E and WJ Manning. 2007. Toward a biologically significant and usable standard for ozone that will also protect plants. Environmental Pollution 150:85-95. Paoletti, E, WJ Manning, J Spaziani and F Tagliaferro. 2007. Gravitational infusion of ethylenediurea (EDU) into trunks protected adult European ash trees (Fraxinus excelsior L.) from foliar ozone injury. Environmental Pollution 145:869-873. Percy, KE and DF Karnosky. 2007. Air quality in natural areas: Interface between the public, science and regulation. Environmental Pollution 149:256-267. Percy, KE, M Nosal, W Heilman, T Dann, AH Legge, J Sober, and DF Karnosky. 2007. New exposure-based metric approach for evaluating O3 risk to North American aspen forests. Environmental Pollution 147:554-566. Pregitzer, KS, DR Zak, WM Loya, JS King, and AJ Burton. 2007. The contribution of root systems to biogeochemical cycles in a changing world. In Z. Cardon and J. Whitbeck (eds) The rhizosphere-an ecological perspective. Elsevier, Boston, pp. 155-178. Ren, W, H Tian, G Chen, M Liu, C Zhang, AH Chappelka and S Pan. 2007. Influence of ozone pollution and climate variability on net primary productivity and carbon storage in China's grassland ecosystems from 1961 to 2000. Environmental Pollution 149:327-335. Sinclair, T, EL Fiscus, B Wherley, M Durham and T Rufty. 2007. Atmospheric vapor pressure deficit is critical in predicting growth response of cool season grass Festuca arundinacea to temperature change. Planta 227:273-276. Staszak J, Grulke NE, Prus-Glowacki W. 2007. Air pollution-driven genetic change in yellow pine in Sequoia National Park. Environmental Pollution 149:366-375. Szantoi, Z., A.H. Chappelka, R.B. Muntifering and G.L. Somers. 2007. Use of ethylenediurea (EDU) to ameliorate ozone effects on purple coneflower (Echinacea purpurea). Environmental Pollution 150: 200208. Tausz M, Grulke N, Weiser G. 2007. Plant defense and avoidance from ozone under global change. Environmental Pollution 147:525-531. Wang, X, W Manning, Z Feng, and Y Zhu. 2007. Ground-level ozone in China: Distribution and effects on crop yields. Environmental Pollution 147:394-400. Wang, X, Q Zheng, F Yao, Z Chen, Z Feng and WJ Manning. 2007. Assessing the impact of ambient ozone on growth and yield of a rice (Oryza sativa L.) and a wheat (Triticum aestivum L.) cultivar grown in the Yangtze Delta, China, using three rates of application of ethylenediurea (EDU). Environmental Pollution 148:390-395. Wittig VE, Ainsworth EA and Long SP. 2007. To what extent do current and projected increases in surface ozone affect photosynthesis and stomatal conductance of trees? A meta-analytic review of the last 3 decades of experiments. Plant, Cell & Environment, 30:1150-1162. Wullschleger, SD, ADB Leakey & SB St Clair. 2007. Functional genomics and ecology: A tale of two scales. New Phytologist 176:735-739. Zak DR, WE Holmes, KS Pregitzer, JS King, DS Ellsworth and ME Kubiske. 2007. Belowground competition and the response of developing forest communities to atmospheric CO2 and O3. Global Change Biology 13:2230-2238. Zhang, C, H Tian, AH Chappelka, W Ren, H Chen, S Pan, M Liu, DM Styers, G Chen and Y Wang. 2007. Impacts of climatic and atmospheric changes on carbon dynamics in the Great Smoky Mountains National Park. Environmental Pollution 149:336-347.

Impact Statements

Regulatory focus and attention of Agricultural Air Quality Task Force is directed to ongoing impacts of ambient ozone on vegetation. (27% reduction of fresh bean yield in the field in rural Long Island, NY, 49% reduction of dry bean yield in rural New Jersey, visible damage to native species in Class I Wilderness Areas in Great Smoky Mountains National Park, demonstrate ongoing welfare effects of ozone air pollution).
Tropospheric ozone is recognized as an important element of global change--ozone is a constituent of the changing atmosphere, has greenhouse gas potential, and interacts with other elements of global change. (warmer soil accelerated ozone damage to beans, nitrogen deposition increased ozone sensitivity of forage nutritive quality, ozone increased tree water loss which may alter stream flows and water supplies).
Growers in California are adopting new control measures for the native weed, horseweed which is acting like an invasive species. (ozone appears to accelerate the spread of a more competitive and herbicide resistant horseweed biotype).
Regulatory standards for ozone are being developed that are more vegetation-protective without simply requiring lower overall concentrations. (a combination of single plant water use and canopy flux measurements predicted effective ozone dose in trees, a key regulatory parameter that may be adopted in the future).
Public educational facilities are in operation in California and Pennsylvania to provide locally relevant information on ozone air pollution, its causes and effects, and feasible mitigation strategies.

Date of Annual Report: 05/30/2009

Report Information

Annual Meeting Dates: 05/05/2009 - 05/07/2009
Period the Report Covers: 10/01/2008 - 09/01/2009

Participants

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

Brief Summary of Minutes

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

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)

Publications

Albertine, JM, WJ Manning. 2009. Elevated night soil temperatures result in earlier incidence and increased extent of foliar ozone injury to common bean (Phaseolus vulgaris L.). Environmental Pollution 157:711-713. Booker, FL, R Muntifering, M McGrath, KO Burkey, D Decoteau, EL Fiscus, W Manning, S Krupa, A Chappelka, DA Grantz. 2009. The ozone component of global change: Potential effects on agricultural and horticultural plant yield, product quality and interactions with invasive species. Journal of Integrative Plant Biology 51:337-351. Burkey, KO and TE Carter. 2009. Foliar resistance to ozone injury in the genetic base of U.S. and Canadian soybean and prediction of resistance in descendent cultivars using coefficient of parentage. Field Crop Research 111:207-217. Davis, D.D., J.M. Skelly, D.R. Decoteau, L.J. Kline, J.A. Ferdinand, J.E. Savage, and T. Orendovici-Best. 2008. Susceptibility and Foliar Response of Broadleaved Species Exposed to Ozone. USDA Forest Service Forest Health Monitoring Program, 50 pp. Ditchkoff, S.S., J.S. Lewis, J.C. Lin, R.B. Muntifering, and A.H. Chappelka. 2009. Nutritive quality of highbush blackberry (Rubus argutus) exposed to tropospheric ozone. Rang. Ecol. & Mang. (In press, available online, DOI: 10.2111/08-222.1). Gonzalez-Fernadez, I, D. Bass, R. Muntifering, G. Mills and J. Barnes. 2008. Impacts of ozone pollution on productivity and forage quality of grass/clover swards. Atmos. Environ. 42: 8755-8769. Grantz, DA and H-B Vu. 2009. O3 sensitivity in a potential C4 bioenergy crop: Sugarcane in California. Crop Science 49:643-650. Kline, L.J., D.D. Davis. J.M. Skelly, and D.R. Decoteau 2009. Variation in Ozone Sensitivity Within Indian Hemp andCommon Milkweed Selections from the Midwest. Northeastern Naturalist 16:307-313. Leakey, ADB, F Xu, KM Gillespie, JM McGrath, EA Ainsworth and DR Ort. 2009. Genomic basis for stimulated respiration by plants growing under elevated carbon dioxide. Proceedings of the National Academy of Sciences 106:3597-3602. Leakey, ADB, EA Ainsworth, CJ Bernacchi, A Rogers, SP Long and DR Ort. 2009. Elevated CO2 effects on plant carbon, nitrogen, and water relations: six important lessons from FACE. Journal of Experimental Botany: In press. Liu, L, JS King, CP Giardina, FL Booker. 2009. The influence of chemistry, production and community composition on leaf litter decomposition under elevated atmospheric CO2 and tropospheric O3 in a northern hardwood ecosystem. Ecosystems 12:401-416. Liu, L, JS King, FL Booker, CP Giardina, HL Allen, S Hu. 2009. Enhanced litter input rather than changes in litter chemistry drive soil carbon and nitrogen cycles under elevated CO2: a microcosm study. Global Change Biology 15:441-453. Lucier, A., Ayres, M., Karnosky, D., Thompson, I., Loehle, C., Percy, K., Sohngen, B. 2009. Future environmental impacts and vulnerabilities. pp. 29-52 In Seppälä, R., Buck A., Katila, P. (Eds.) Adaptation of Forests and People to Climate Change A Global Assessment Report. International Union of Forest Research Organizations (IUFRO) World Series Vol. 22, Vienna, Austria. 224pp Paoletti, E, AM Ferrara, V Calatayud, J Cervero, F Giannetti, MJ Sanz, WJ Manning. 2009. Deciduous shrubs for ozone bioindication: Hibiscus syriacus as an example. Environmental Pollution 157:865-870. Paoletti, E, N Contran, WJ Manning, AM Ferrara. 2009. Use of the antiozonant ethylenediurea (EDU) in Italy: Verification of the effects of ambient ozone on crop plants and trees and investigation of EDU's mode of action. Environmental Pollution 157:1453-1460. Papinchak, H.L., E.J. Holcomb, T.O. Best and D.R. Decoteau. 2009. Effectiveness of houseplants in reducing the indoor air pollutant ozone. HortTechnology 19:286-290. Percy, KE, S Manninen, K-H Haberle, C Heerdt, H Werner, GW Henderson, R Matyssek. 2009. Effect of 3 years' free-air exposure to elevated ozone on mature Norway spruce (Picea abies (L.) Karst.) needle epicuticular wax physicochemical characteristics. Environmental Pollution 157:1657-1665. Percy, K.E., Nosal, M., Heilman, W., Dann, T., Karnosky, D.F. 2009. Standards-based ozone exposure-response functions that predict forest growth. pp. 269-293 In A. H. Legge (Ed.) Relating Atmospheric Source Apportionment to Vegetation Effects: Establishing Cause and Effect Relationships. Elsevier Environmental Science Series Vol. 9, Oxford, UK. Percy, Kevin E. , Sirkku Manninen, Karl-Heinz Haberle, C. Heerdt, H. Werner, Henderson, G.W., Rainer Matyssek, R. 2009. Effect of 3 years free-air exposure to elevated ozone on mature Norway spruce (Picea abies (L.) Karst.) needle epicuticular wax physicochemical characteristics. Environmental Pollution 157: 1657-1665 Rodolfo SE, BA Humberto, MA Violeta, SA Pablo, BL Emma, S Krupa. 2009. Levels and source apportionment of volatile organic compounds in southwestern area of Mexico City. Environmental Pollution 157:1038-1044. Suvi Nikula, Sirkku Manninen, Kevin Percy, Maarit Falck, Elina Oksanen and Toni Holopainen 2009. Elevated O3 induced minor changes in growth and foliar traits of European and hybrid aspen. Boreal Environment Research 14(A): 29-47. Szantoi Z, AH Chappelka, RB Muntifering, GL Somers. 2009. Cutleaf coneflower (Rudbeckia laciniata L.) response to ozone and ethylenediurea (EDU). Environmental Pollution 157:840-846.

Impact Statements

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

Date of Annual Report: 09/23/2010

Report Information

Annual Meeting Dates: 07/15/2010 - 07/17/2010
Period the Report Covers: 10/01/2009 - 09/01/2010

Participants

Best, Teodora (txo115@psu.edu) Penn State University, State College, PA;
Booker, Fitzgerald (fitz.booker@ars.usda.gov) USDA-ARS Plant Sciences, Raleigh, NC;
Vince Brisini, RRI Energy Corporation;
Burkey, Kent (kent.burkey@ars.usda.gov) - USDA-ARS Plant Science Unit, Raleigh, NC;
Carlson, John (jec16@psu.edu) Penn State University, State College, PA;
Maria Cazorla, Ph.D. student, The Penn State University;
Chappelka, Art (chappah@auburn.edu) - Auburn University, Auburn, Al;
Decoteau, Dennis (drd10@psu.edu) Penn State University, State College, PA;
Fiscus, Edwin (ed.fiscus@ars.usda.gov) USDA-ARS Plant Science Unit, Raleigh, NC;
Nicholas Gilliland (gillinj@auburn.edu) Auburn University, Auburn, AL;
Grantz, David (david@uckac.edu) - University of California - Riverside, Riverside, CA;
Herr, Josh (jrh408@psu.edu) Penn State University, State College, PA;
Knighton, Raymond (Rknighton@nifa.usda.gov) USDA-NIFA, Washington, DC;
Neufeld, Howard (neufeldhs@appstate.edu) Appalachian State University, Boone, NC;
Holly Salazer, Air Resource Manager, National Park Service, The Penn State University;
Savage, Jim (jes10@psu.edu) - Penn State University, State College, PA;
Skelly, John (jms34@psu.edu) Penn State University, State College, PA (retired);
Smith, Margaret (mes25@cornell.edu) - Cornell University, Ithaca, NY;
Mark Steinberg, Accuweather Corp. (tour leader);
Wiese, Cosima (cwiese@misericordia.edu) Misericordia University, Dallas, PA;
Zilinskas, Barbara (zilinskas@aesop.rutgers.edu) Rutgers Univ., New Brunswick, NJ

Brief Summary of Minutes

Chair David Grantz welcomed everyone to the meeting and thanked host, Dennis Decoteau, for organizing the meeting this year. We were reminded that the current project expires in 2012 and that we should be thinking about approaches for the new proposal. Grantz encouraged the group to think about possible collaboration activities, funding opportunities and the need to increase student participation. Dennis Decoteau welcomed the group and thanked Jim Savage for his help setting up for the meeting. Jim is the primary caretaker of the Demonstration Center and has done a great job maintaining and improving the facility.
Margaret Smith, Project Administrator, encouraged the group to continue meeting its multi-state project objectives. Collaborative research is a component of Hatch Fund projects, and 25% of each projects fund must be spent on it. Our annual report should highlight multi-state research activities. She went on to say that our project renewal process should begin now. The current project terminates on September 30, 2012. A request to write a proposal renewal is due to NIMSS by March 2011. The request should emphasize whats next in the projects objectives and how it will take advantage of the multi-state approach.
Ray Knighton, our USDA representative, described the four institutes in the newly formed NIFA office, formerly known as CSREES. The institute of Energy, Environment and Climate Change is where he is now assigned. The focus of the funding program has been narrowed compared to previous years but the scale of the projects is much larger. Resources allocated to each project were increased to promote multidisciplinary teams whose research will have impact, in this case, on climate change mitigation and agricultural production problems associated with climate change. Emphasis will be on projects that can develop approaches that will be viable for use in the field in short order.
David Grantz conducted the business meeting. A committee was formed, consisting of Art Chappelka, Fitz Booker and Dave Grantz to write the request to renew the project proposal, due for submission by March 2012. Location of the next meeting will either be on Long Island, hosted by Meg McGrath, or at Rutgers University, hosted by Barbara Zilinskas. Art Chappelka succeeded Dave as Chair of the project. The meeting was adjourned at 3:00 pm, July 16, 2010.

Accomplishments

Activities of the participating experiments are guided by the Objectives of the Current approved Project Proposal. These activities and achievements follow, organized by NE-1030 Project Objectives, and by approaches. 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. 1a. spatial analysis of ozone impacts on crops. A collaboration between Booker, Burkey, Fiscus and Ainsworth of USDA/ARS, NC State University and the University of Illinois, considered the elevated concentrations of ground-level O3 that are frequently measured over farmland regions in many parts of the world. While numerous experimental studies show that O3 can significantly decrease crop productivity, independent verifications of yield losses at current ambient O3 concentrations in rural locations are sparse. In this study, soybean crop yield data during a 5-year period over the Midwest of the United States were combined with ground and satellite O3 measurements to provide evidence that yield losses on the order of 10% could be estimated through the use of a multiple linear regression model. 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 chamber experiments conducted by ARS researchers in Raleigh, NC and an open-air experimental facility (SoyFACE) in central Illinois, conducted by ARS researchers there. Extrapolation of these findings supports previous studies that estimate the global economic loss to the farming community of more than $10 billion annually. (NC, IL,). 1b. diurnal trends in ozone sensitivity of vegetation. A collaboration of the University of California at Riverside (R. Heath) University of California Kearney Agricultural Center (D. Grantz), and USDA/ARS (K. Burkey) has addressed the factors that determine the sensitivity of extensive vegetation to ozone. In general these are only crudely characterized. In order to support efforts to model extensive regional impacts, it is necessary to parameterize the steps of ozone injury, and to characterize the variability among populations. There are three major steps for plant injury by ozone (O3). These are entrance of O3into the leaf (Flux, F), overcoming by O3 metabolic defenses (antioxidant capacity, A), and the actual attack of the effective dose of O3 (Deffective) on bioreceptors (Injury, I).This can be expressed mathematically. Current approaches model gs (stomatal conductance) and [O3] from meteorological data, the species composition of the ground cover, and air quality monitoring data. However, A is assumed to be constant. Recent use of flux (F) is an improvement over previous use of exposure ([O3]) to estimate injury. We have tested the hypothesis that defense capacity (A) may vary diurnally, and may therefore control the amount of injury caused by a given atmospheric ozone concentration. If so a specific O3 flux (F) will yield a different Deffective and thus a different I, at different times of the day, so that this will need to be considered in modeling of regional O3 impacts. From knowledge of how much O3 enters the leaf (F) and how much injury occurs (I), we can calculate the initial defense capacity of the leaf (A). Exposure must be rapid enough that tissues have insufficient time for induction of additional defense capacity. We have developed the first demonstration that defense capacity varies diurnally, and have explored the mechanism, using Pima cotton, cv. S-6, grown in a greenhouse. Injury was determined from digital photo analysis of necrosis, chlorophyll content (SPAD, Minolta) and summed abaxial and adaxial stomatal conductance (LiCor 1600) 6-7 days after exposure. Total antioxidant capacity, ascorbic acid and dehydroascorbic acid content were determined on non-exposed leaves at different times of day. Injury induced by an (interpolated) O3 dose of 19.8 mol m-2, exhibited a clear diurnal trend, shown by foliar necrosis, chlorophyll content (SPAD) and stomatal conductance, all obtained at 6 days after exposure for a 15 minute pulse. 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 ozone sensitivity is confirmed, the mechanism remains to be elucidated. These data indicate that parameterization of models of O3 injury to vegetation will require measures of inherent defense capability, for which time of day may be a key determinant. (CA, NC) 1c. Snapbean model system demonstrates ozone impacts on crops. The continuation of this study will strengthen our understanding of the impact of ambient ozone on plants and crop productivity. In NY, McGrath has been assessing impact on plant productivity of ambient ozone occurring on Long Island, where she is stationed, by growing the ozone-sensitive and ozone-tolerant snap bean lines that were developed for use in quantifying ozone impact. Each year there have been three successive field plantings to cover the entire growing period for beans in the area. As they developed, bean pods were harvested repeatedly from some plants when immature and at a size typical for fresh-market consumption. Pods were harvested from the other plants when mature and dry. Plants were examined routinely for ozone injury. Injury and defoliation due mainly to ozone injury were rated. Ozone concentration data were obtained from a monitor maintained at the research site (LIHREC) by the NYC DEC Air Quality Division. Results from 2009 research were analyzed and another experiment was conducted over the past year for this reporting period. In 2009, the ozone-sensitive snap bean line S156 yielded less than the tolerant line R331 when grown under ambient ozone conditions. Total weight and number of bean pods harvested for fresh-market consumption from planting 1 (22 May) plants was 28% and 19% lower, respectively, for S156 compared to R331 (pods were harvested from 17 July through 21 Aug). There was a 55% and 46% reduction in these yield variables, respectively, for planting 2 (22 June) plants (harvested 10 Aug through 9 Sept). Reduction was 33% and 16%, respectively, for planting 3 (16 July) plants (harvested 4 Sept through 6 Oct). These differences were similar to greater than in previous years. Mature yield data has not yet been collected. Exposure to ozone caused acute foliar injury in all three plantings. The visible symptom was bronzing. The sensitive line became more severely affected than the tolerant one. Severely affected leaves eventually died and dropped. For example, injury was first observed on Planting 1 plants on 19 June. Average percentage of leaf tissue with bronzing (determined by estimating the incidence or proportion of leaflets with symptoms and the average severity on affected leaves) was 0.04% and 5.6% for R331 and S156, respectively, on 11 July, which was 6 days before the first pods were ready for harvest. Average percentage of leaf tissue with bronzing had increased to 0.5% and 64% by 27 July and 2.4% and 70% by 1 Aug. (NY) In New Jersey, Zilinskas monitored the effects of ambient ozone on the productivity of two snapbean cultivars R331 (ozone-tolerant) and S156 (ozone-sensitive) over the 2009 growing season. Following a very wet month of June, we planted the two snapbean cultivars in East Brunswick, NJ, on July 1, 2009, two weeks after our usual planting date. We closely adhered 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, we made multiple harvests of marketable pods at 49, 56, 63, 69 and 76 days after planting. In the 2009 season, peak pod number and fresh weight (of both cultivars) occurred at the second harvesting date. A statistically significant decrease in total pod fresh weight of marketable snapbeans was observed in S156 relative to R331 in three of the five harvest dates, where the fresh weight of marketable pods of the ozone-sensitive cultivar was between 37% and 73% less than that of the ozone-tolerant cultivar. A final harvest of snapbeans was conducted at a uniform 84 days after planting. At this harvest date in the 2009 growing season, a significant proportion of the pods were immature (without seeds) or green. The number of seeds and the dry weight of seeds and pods from the two cultivars were significantly different, with yield reductions in the sensitive relative to the tolerant cultivar of 26%, 47% and 50%, respectively. There was no significant difference in the pod number of the sensitive cultivar relative to the tolerant cultivar. (NJ) 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. (MN) 1d. ozone impacts on plants with C4 photosynthetic systems. A number of food and potential biofuel crops in California utilize the C4 photosynthetic pathway. This is considerable desirable due to the inherent water use efficiency of this mode of carbon acquisition. In the studies of the National Crop Loss Assessment and more recently (maize reduced by 4-8%, relative to soybean of 22%, for example), these types of plants have been considered to be tolerant of ozone. We decided to investigate genotypes of the Saccharum complex which are 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, which are relatively low in sugar. Existing genotypes were not developed in areas subject to high ozone. 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 ozone 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 ozone sensitivity. It is not warranted to assume that C4 crops will exhibit the high levels of ozone tolerance observed in early studies. (CA) 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. Previous work underway on competition between horseweed, purple nutsedge and the crops cotton and tomato has been concluded. Extension efforts are taking this information to growers in Californias Central Valley (CA) 2a. impacts of ozone on growth, competition and nutritional value of forages. A study was initiated in 2009 to determine the effects of tropospheric ozone 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). Twelve, large (4.8 m ht. × 4.5 m dia.) open-top chambers (modified with rain-exclusion caps) located at the Auburn University Atmospheric Deposition Site were used in this study. 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 regrowth 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. Data are currently being statistically analyzed. 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 were fabricated into 50-g cubes that were fed to New Zealand White rabbits in a nutrient-utilization/diet-selection experiment beginning in December 2009. Sixteen, 8 wk-old rabbits were initially obtained, from which 10 were used in the experiment and divided into two groups: 1) fed NF forage and 2) fed 2X forage for 10 days. Orts for each rabbit were collected each day before new forage was introduced and pooled for future chemical analysis for all 10 days. The final six days consisted of fecal and urinary output collection for each rabbit. Orts, feces, and urine were collected from steel trays located under each rabbits cage. Samples were analyzed for various nutrient constituents. Preliminary results indicate that digestible dry matter intake was lower for 2X rabbits compared with NF-fed rabbits, primarily as a result of decreased digestibility of cell-wall constituents. The decrease in fiber digestibility cannot be explained on the basis of lignifications solely as originally hypothesized. Further analysis is being conducted investigating the possible role of soluble phenolics.(AL) 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. 3a. drought x ozone interactions. A five-year (2004 2008) field study by Decoteau on the effects of ambient ozone levels on the growth and production of two wine grape varieties was terminated in 2009. We have determined through ambient ozone exposures over the past four years at our research site located at the Penn State Fruit Research and Extension Center (Biglerville, Adams Co.) that the grape cultivar Charbourcin is sensitive to ozone levels typically experienced during the summer months in Pennsylvania. Ozone injury to Charbourcin grape included adaxial stipple and yellowing and defoliation of the older leaves. The amount of injury observed on the grape foliage varied from year to year and appears to be influenced by other weather conditions. Injury was more significant during the 2004 and 2006 growing seasons. The droughts of 2005 and 2008 resulted in less injury than in other years. The Vidal variety of grape, which is considered tolerant to ozone injury, typically exhibited little to no foliar injury to ambient ozone levels. Each year, berry harvests were made in early October and fruit quality evaluations determined. Berry weight, pH, brix levels and fruit acidity varied from year to year and among treatments and varieties. Grape plants in the open plots tended to have less berry weight and more acid than the other treatments. Further evaluations need to look at the influence of recorded ozone levels and its variability during the season on fruit results. (PA) A model was developed to predict stomatal conductance under well watered and water stressed conditions in soybean using soil moisture and common atmospheric inputs. By including field site measurements of atmospheric O3, the midday leaf influx of O3 may be calculated and related to yield and visible damage effects. (NC) 3b. effect of houseplants on indoor ozone exposures. Three common indoor houseplants, the snake plant, spider plant, and golden pothos, continued to be evaluated for their species effectiveness in reducing ozone concentrations in a simulated indoor environment. Ozone was injected into the continuously stirred tank reactor chambers housed within a greenhouse equipped with a charcoal filtration air supply system chambers and when concentrations reached 200 ppb (+/- 5 ppb), the ozone generating system was turned off and ozone concentrations over time (ozone was monitored every 5 to 6 min in each chamber) were recorded until approximately < 5 ppb was measured in the treatment chamber. On average, ozone depletion time (time from when the ozone generating system was turned off at approximately 200 ppb to < 5 ppb in the chamber) ranged from 38 to 120 min per evaluation. Ozone depletion rates were higher within chambers that contained plants than within control chambers without plants, but there were no plant species differences. New studies are focusing on the impact of total surface area within the chambers on the influence of plant reduction in ozone levels. (PA) 3c. ozone by carbon dioxide interactions. Agricultural soils are thought to be a C sink in a changing global climate because rising CO2 often enhances plant-derived C inputs belowground. However, elevated tropospheric O3 concentrations may offset the CO2-fertilization effect. Unfortunately, limited information is available on long-term belowground responses to elevated CO2 and O3 in agricultural systems. The objective of our five-year experiment was to determine the separate and combined effects of elevated CO2 and O3 on soil respiration, microbial biomass, nutrient availability and soil C levels in a no-till soybean-wheat cropping system using open-top field chambers. Soil respiration during four growing seasons was stimulated by 21% with elevated CO2, but the O3 effect was not statistically significant. Microbial soil respiration and biomass were higher with elevated CO2 at later stages of the study but similar to the control in the O3 treatment. Soil delta 13C values decreased with elevated CO2, showing that elevated CO2 influenced soil C concentrations, but soil C levels were not significantly affected by either elevated CO2 or O3. It is hypothesized that increased residue and root C and N inputs with elevated CO2 accelerated decay rates, as evidenced by increased soil and microbial respiration rates. High N availability due to increased input from an N-fixing legume, along with increased C and adequate supplies of other mineral nutrients and water, may accelerate organic C turnover at elevated CO2, thus constraining potential C sequestration in highly-managed agroecosystems. (NC). 3d. ozone by vapor pressure interactions. Differential atmospheric vapor pressure deficit experiments showed that high vpd substantially reduced seed yields in the S156/R123 bioindicator snapbean pair. At low vpd the addition of O3 reduced yield by 55% (R123) and 72% (S156). High vpd in clean air reduced yields by 30% in both genotypes while the addition of O3 at high vpd had no further significant yield effect in either genotype. However, in O3 air, high vpd had a salutary affect, increasing yields by 43% (R123) and 83% (S156) when compared to the high O3-low vpd treatment. However, these interactions, did not substantially affect the ratio (S156/R123) of responses to O3, thus preserving their usefulness as an O3 bioindicator pair. (NC) 3e. ozone x herbivorous insect interactions. We are also comparing genome-level responses to ozone in two hybrid poplar clones that are known to vary in sensitivity to ozone. In the summer of 2009 the O3-sensitive (NE 388) and O3-tolerant (NE 245) clones were exposed to damaging levels of ozone, with half of the plants also being subjected to gypsy moth caterpillars, Lymantria dispar, a common defoliator of Populus spp. We conducted a full factorial, time course microarray experiment to investigate global gene expression patterns in a spatially and temporally robust manner. Microarray hybridizations were conducted with the recently developed whole genome microarray for Populus from Nimblegen. A first study 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 ozone, while only 15% of the herbivore-regulated genes were activated independent of ozone. We are now in the process of analyzing the data from the second study. Our results should 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. This study should reveal genome-level interactions and will be useful in developing strategies that might provide tolerance to both abiotic and biotic stresses in plants.This project has led to the production of more expressed gene sequences for Black Cherry than are currently available for any other single Rosaceae species. (PA) OBJECTIVE 4. Examine the physiological and molecular basis of O3 toxicity and tolerance in plants. 4a. role of antioxidant defense mechanisms. A collaboration between USDA/ARS in NC and Misericordia University in PA further examined the antioxidant relationship with ozone injury to determine the role of the apoplast in plant defense responses to oxidative stress.. Upon entry into leaves, O3 and related ROS must pass through the leaf extracellular space before reacting with plasma membrane components to initiate plant injury responses. Therefore, O3 and ROS detoxification reactions localized in the leaf cell wall represent a first line of defense against O3 injury. Cellular events caused by O3 exposure include the production of reactive oxygen species (ROS), which can lead to foliar injury and suppressed biomass production. However, plants possess biochemical mechanisms that regulate ROS concentrations, such as peroxidases and superoxide dismutases. In Arabidopsis, stimulation of two cationic peroxidases was observed following exposure to moderate levels of O3. Through the use of protein sequencing, gene expression arrays, quantitative real-time PCR and insertion mutants, identity of the peroxidases was determined, along with confirmation that one gene was transcriptionally regulated in response to O3. Knock-out mutants for the peroxidase genes are being used to investigate possible functional significance of peroxidase activity changes in responses to O3. In a related experiment, Arabidopsis mutants transformed with a superoxide reductase gene, which have shown increased tolerance to heat stress and a ROS-generating herbicide, did not exhibit increased tolerance to O3, suggesting that protection from superoxide in the cytoplasm was relatively unimportant in counteracting O3 toxicity in this experimental system. (NC). Insoluble cell wall material was isolated from leaves of O3-sensitive and tolerant genotypes of soybean and snap bean to assess differences in cell wall chemistry that may relate to observed differences in O3 sensitivity. Snap bean and soybean plants were exposed to charcoal-filtered (CF) air or elevated O3. Leaf intercellular wash fluid (IWF) was recovered and analyzed by reversed-phase HPLC using a column selected for separation of highly polar aromatic compounds.HPLC analysis following alkaline hydrolysis of the cell wall material revealed relatively simple chromatograms consisting of 4 to 6 unknown compounds that varied in quantity depending upon species, genotype, and O3 treatment. Snap bean and soybean plants contained unique profiles of soluble compounds in the leaf apoplast demonstrating significant species differences. In snap bean, no major genotype or ozone treatment effects were observed. In soybean, significant differences in quantities of apoplast constituents were observed when the two genotypes were compared, often with greater amounts present in O3-tolerant Fiskeby than in the O3-sensitive Mandarin Ottawa, regardless of treatment. Several peaks present in both soybean genotypes were reduced by O3 treatment in Mandarin Ottawa but not in Fiskeby. Future work will be directed toward identification of the major apoplast constituents and their role, if any, in O3 tolerance. (NC, PA). Molecular genomic analyses of previously identified ozone tolerant and ozone sensitive families of black cherry and Populus are allowing us to characterize and understand the physiological and molecular basis of ozone toxicity and tolerance in trees. We are currently studying both Populus and Prunus species, taking advantage of previous research that has identified ozone tolerant and sensitive genotypes in these species. For black cherry, a wide range of O3-sensitivity is known, with some genotypes being so sensitive as to serve as ozone indicator plants. We conducted four years of O3 treatments at normal ambient stress levels (80 ppb O3 for 8 hr, 7 day/wk) in three large half-sib (open-pollinated) black cherry families which were known to have low, medium, and high family means for ozone damage. The treatments permitted us to identify the most ozone-sensitive and ozone-tolerant individuals within each family. We prepared two cDNA libraries from RNA isolated from O3-treated leaves of ozone-sensitive and ozone-tolerant seedlings, from which we generated app. 82 million bases of DNA sequence in 2008, and 250 million bases of sequence in 2010. Functionally, 13% of the DNA sequences of the sensitive family are from genes known to be involved in response to biotic and / or abiotic stresses (Figure below). 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 ozone tolerance. About 1% of genes in black cherry are involved in signal transduction. The full-sib black cherry families that we have selected for mapping will be maintained as reference populations for future research. We have also shown recently that the new DNA markers can be used to identify pollination patterns within the black cherry seed orchard at Penn Nursery. 4b. role of anatomical defense mechanisms. A collaboration between Neufeld of Appalachian State University and many others has characterized impacts of ozone on understory vegetation of Great Smokies National Park. The factors responsible for determining the ozone sensitivity of a plant may range from the physical to the biological. 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 ozone sensitivity in plants. In this study, leaf anatomical properties were investigated for ozone-sensitive and ozone-insensitive individuals of the ozone bioindicator plant cutleaf coneflower (Rudbeckia laciniata var. digitata) at Purchase Knob in Great Smoky Mountains National Park. Plants growing at Purchase Knob in either a field (full sun) or beneath a forest (shade) were studied. Both young and old leaves on the same plant were studied, but because results were similar for the two age classes in most cases, leaf age is not addressed further in this report. Leaves were sampled in mid-August of 2003, and then preserved in FAA before undergoing dehydration in EtOH. They were fixed in plastic, sectioned on a microtome, stained and viewed using TEM and light microscopy. Image J was used to measure various parameters (described below). There were no statistically significant differences in either adaxial or abaxial stomatal densities between the two groups of plants, or between those growing in sun or shade. Leaves in the sun were thicker than those in the shade, but there were no sensitivity differences. There were also no sensitivity differences for palisade or spongy mesophyll layer thicknesses, but these layers were thinner for leaves grown in shade. There was a significant trend for narrower epidermal wall/cuticle thicknesses in insensitive plants compared to sensitive ones, in both the sun and shade, but not so for either palisade or spongy mesophyll wall thicknesses. Total cell areas appeared to be slightly less in insensitive plants than sensitive ones, mainly due to reduced spongy mesophyll areas, but not palisade mesophyll areas. The amount of airspace did not differ among the sensitivity types. The tortuosity of the diffusional pathway appeared greater in sun plants than shade, and there was a light x sensitivity interaction, mainly due to the fact that tortuosity was higher in ozone-sensitive plants when growing in the sun, but not in any other condition. In the two cases where 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. Thicker cell walls would have a greater capacity to store anti-oxidant compounds, and would inhibit the diffusion of ozone the cell membrane, so if this parameter is involved with determining sensitivity, it should be thinner in the sensitive individuals, not the insensitive ones as found. Therefore, we conclude that in cutleaf coneflower, leaf anatomical differences do not contribute to the observed sensitivity differences in this species. Because earlier studies on cutleaf coneflower have shown no differences in gas exchange or anti-oxidant activity in uninjured leaves, we suggest that the basis for sensitivity variation might reside elsewhere, possibly at the molecular level, where ozone may differentially affect gene regulation and/or transcriptional regulation between sensitive and insensitive individuals. (NC) 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. 5a. community involvement in annual meetings. The NE-1030 Technical Committee conducts outreach in the individual states and communities, and through the annual meeting attempts to forge linkages with local agencies, institutions and personnel. At this years Annual Meeting in State College PA a number of such linkages were developed. A field trip to Accuweather, the headquarters of a weather-forecasting company, was incorporated in the afternoons activities. We wish to express our appreciation to Mark Steinberg, our tour group leader, and Accuweather for a most interesting and informative tour. John Skelly, now retired but a long-time member of the Technical Committee, opened the technical portion of the meeting with a presentation on the history of the Air Quality Learning/Demonstration Center. He emphasized that success of the center was due in part to cooperation between Penn State University and private industry, notably RRI Energy. John said it was great to see how far the center had progressed from his initial efforts in getting it started. John brought Vince Brisini to the meeting, where he was able to meet with the currently active Ozone researchers gathered. Vince Brisini, from RRI Energy Corporation, talked about their participation in establishing the Air Quality Demonstration Center and reviewed the current status of emission control activities at power generation facilities. He stated that emission controls were quite effective now and that attaining further reductions will be difficult. Economic incentives must be available for businesses to comply with stricter regulations. (PA) Holly Salazer, Air Resource Manager, National Park Service, located at Penn State, presented an overview of the park services goals for improving air quality in National Parks. By law, they are mandated to prevent significant deterioration in air quality in National Parks. Specifically, by 2064, the goal of the Regional Haze Regulation is to reduce haze in the parks to pre-impact levels. Hollys office helps coordinate analysis of air quality monitoring data and comments on permit applications for development that may affect air quality in national parks. (PA) Maria Cazorla, a Ph.D. student at Penn State, presented results of her research on a new instrument that measures ozone production rates for use in air quality monitoring in the field. 5b. Public education centers on air quality issues. Decoteau at 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. During 2009 approximately 250 individuals attended course lectures or public presentations on air pollution effects on terrestrial plants at the Learning Center and in 2010 we hosted the NE 1030 project participants. (PA) Grantz in California maintains field and greenhouse exposure chambers that are shown to visiting school and industry groups and international visitors from commodity outreach programs. (CA) 5c. Expert involvement in local educational and cooperative extension programs. The USDA/ARS group in Raleigh NC (Burkey, Booker, Fiscus) has developed some direct participation programs for NCSU students in the Environmental Technology courses, Plants Soils and Natural Systems (ET202) and Crop Physiology (CS714). Demonstrations were presented using the ozone-sensitive and ozone-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. Photosynthesis measurements at a range of CO2 concentrations showed that C fixation by the enzyme Rubisco was inhibited by O3 in the sensitive line, but not in the resistant line. This finding demonstrated one biochemical mechanism of O3 effects on plants that likely accounts in part for suppressed biomass accumulation and yield of plants in the field. (NC). Middle school students at Rosman High School, Rosman, NC were provided seeds of ozone-sensitive and ozone-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 ozone. (NC). McGrath (NY) shared information on ambient ozone and its impacts during a class on vegetable diseases that is presented annually during the Master Gardener Training Program conducted during spring each year in Suffolk County.(NY) Grantz addressed school groups and industry organizations, and worked with individual commodity groups to 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. (CA) 5d. Maintenance of a web educational presence. Booker of USDA in NC, the Web Master for the NE-1030 Project, developed and maintained a project web page at (http://www.ncsu.edu/project/usda-ne-1013/index.htm). This web page is frequently updated with current news items, project annual report and news of the project such as minutes of the 2009 annual meeting. (NC).

Publications

Booker, F., R. Muntifering, M. McGrath, K. Burkey, D. Decoteau, E. Fiscus, W. Manning, S. Krupa, A. Chappelka, and David Grantz. 2009. The Ozone Component of Global Change: Effects on Agricultural and Horticultural Plant Yield, Product Quality and Interactions With Invasive Species, Journal of Integrative Plant Biology 51: 337-351. Ditchkoff, S.S., J.S. Lewis, J.C. Lin, R.B. Muntifering, and A.H. Chappelka. 2009. Nutritive quality of highbush blackberry (Rubus argutus) exposed to tropospheric ozone. Rang. Ecol. & Mang. (In press, available online, DOI: 10.2111/08-222.1). Fishman, J, JK Creilson, PA Parker, EA Ainsworth, GG Vining, J Szarka, FL Booker, X Xu. 2010. An investigation of widespread ozone damage to the soybean crop in the upper Midwest determined from ground-based and satellite measurements. Atmospheric Environment 44:2248-2256. Gould, Kevin S., Dana A. Dudle and Howard S. Neufeld. 2010. Why some stems are red. Photoprotective roles for anthocyanins in internodes. Journal of Experimental Botany 61:2707-2717. Grantz, D.A., Shrestha, A., Vu, H. Ozone Impacts on Assimilation and Allocation to Reproductive Sinks in the Vegetatively Propagated C4 Weed, Yellow Nutsedge. Crop Science 50:246-252. Grantz, DA, Vu, H-B. 2009. O3 Sensitivity in a Potential C4 Bioenergy Crop: Sugarcane in California. Crop Science. 49:18. Grantz, D.A., Vu, H., Aguilar, C., Rea, M.A. No Interaction Between Methyl Jasmonate and Ozone in Pima Cotton: Growth and allocation respond independently to both. Plant Cell and Environmen. 33, 717728. Grantz, Da Vu. H-B, Heath, RL, Burkey K. 2010. Temporal Sensitivity Key to Modeling Ozone Impacts on Vegetation. Extended Abstract 2010-EE-208-AWMA. Proceedings Annual Meeting, Air and Waste Management Association, Calgary. June 2010. Kline, L.J., D.D. Davis. J.M. Skelly, and D.R. Decoteau 2009. Variation in ozone sensitivity within Indian Hemp and common milkweed selections from the Midwest. Northeastern Naturalist 16:307-313. Papinchak, H.L., E.J. Holcomb, T.O. Best and D.R. Decoteau. 2009. Effectiveness of houseplants in reducing the indoor air pollutant ozone. HortTechnology 19:286-290. Szantoi, Z., A.H. Chappelka, R.B. Muntifering, G.L. Somers. 2009. Cutleaf coneflower (Rudbeckia laciniata L.) response to ozone and ethylenediurea (EDU). Environ. Pollut. 157: 840-846. Temple, P.J., Grantz, D.A. 2010. Air Pollution Stress. Physiology of Cotton. Editors: J. McD. Stewart, D. Oosterhuis, J.J. Heitholt, J. Mauney. Springer. Chapter 15 Tu, C., FL Booker, KO Burkey and S. Hu. 2009. Elevated atmospheric CO2 and O3 differentially alter nitrogen acquisition in peanut. Crop Science 49:1827-1836.

Impact Statements

Agricultural producers, modelers and regulators were provided with multiple, redundant demonstrations of agricultural yield loss due specifically to ozone, over broad geographic areas. Additionally, modeling of ozone impacts were improved by incorporation of diurnal changes in sensitivity of ozone to vegetation. This information is useful in cost-benefit analyses of ozone control measures and in yield prediction domestically and internationally. These efforts have informed USEPA in formulating the evolving ozone standards.
Ozone reduces crop yield, and is here shown to reduce nutritional value. This allows a more accurate estimation of the economic impact of ozone damage to vegetation, by using the product of percent yield loss by percent reduction in nutritive value. These evaluations are useful in cost benefit analyses of regulatory activities, have been used by USEPA in formulating regulations, and reports from this group have been incorporated into agricultural industry publications, such as guidelines published for growers by the Almond Board of California.
Ozone levels are declining only slowly, and are increasing in some locations. Protection of agriculture will require improving ozone tolerance of key cultivars. This group has demonstrated interactions of ozone with many environmental factors, each of which provides a potential means of improving ozone tolerance. This is facilitated by the analysis of biochemical mechanisms of response involving antioxidant metabolism, and by genomic analysis of these interactions. These activities directly facilitate plant improvement in support of sustainable agriculture in the U.S.
The NE1030 group has educated numerous high school, undergraduate, and graduate students through outreach efforts, in-class teaching, student mentoring, public education, and informal Cooperative Extension activities. These led to public participation in annual meetings at all locations where held. Members of the group have been sought for formal and informal teaching and or information on numerous occasions. These activities lead to an informed regulated community, and informed electorate, as well as to improved activities with respect to air quality impacts.

Date of Annual Report: 10/03/2011

Report Information

Annual Meeting Dates: 07/27/2011 - 07/29/2011
Period the Report Covers: 10/01/2010 - 09/01/2011

Participants

" Best, Teodora Best (txo115@psu.edu) - Pennsylvania State University
" Booker, Fitzgerald (fitz.booker@ars.usda.gov) - USDA-ARS, Raleigh, NC (Connected Remotely)
" Burkey, Kent (kent.burkey@ars.usda.gov) - USDA-ARS Plant Science Unit, Raleigh, NC
" Chappelka, Art (chappah@auburn.edu) - Auburn University
" Grantz, David (david@uckac.edu) - University of California - Riverside
" Grulke, Nancy (ngrulke@fs.fed.us) USFS-Bend, OR (Connected Remotely)
" Knighton, Raymond (Rknighton@nifa.usda.gov) USDA-NIFA, Washington, DC (Connected Remotely)
" Kohut, Robert (rjk9@cornell.edu)- Cornell University (retired)
" McGrath, Margaret (mtm3@cornell.edu) - Cornell University
" Neufeld, Howard (neufeldhs@appstate.) - Appalachian State University (Connected Remotely)

Brief Summary of Minutes

The 2011 meeting of the NE-1030 Multi-State Project Technical Committee was held at Schoodic Environmental Center in Acadia National Park, Winter Harbor, ME July 27-29. The meeting was called to order at 8:10 am 27 July 2011 by chairman Art Chappelka. The meeting agenda was discussed followed by introductions by those attending the meeting in person and remotely via conference telephone and GoToMeeting web connection for presentations.

Station reports were then presented. Kent Burkey (NC) reported on soybean introductions previously shown to exhibit differential ozone tolerance were subjected to season long ozone exposures in open-top-chambers. Using these data certain soybean varieties were selected for crossing. A genetic cross has been made between Fiskeby III and Mandarin Ottawa and a set of 240 random inbred lines developed for future studies to map ozone tolerance genes in soybean. Teodora Best (PA) reported on data used to develop genomic resources for the analysis of traits related to ozone response in black cherry. There were two objectives 1) Develop a sequence database for expressed genes in black cherry using RNA isolated from the ozone-treated tissues of ozone-tolerant/-sensitive trees. Two cDNA libraries derived from Prunus serotina tissues were sequenced using Roche 454 technology, 2) Construct a genetic linkage map for two full-sib black cherry populations segregating for ozone sensitivity, which will serve as the reference map for the species. David Grantz (CA) described recent research focusing on diurnal trends in ozone sensitivity of vegetation. While the diurnal nature of ozone sensitivity is confirmed, the mechanism remains to be elucidated. These data indicate that parameterization of models of ozone injury to vegetation will require measures of inherent defense capability, for which time of day may be a key determinant. Ozone impacts on plants with C4 photosynthetic systems is also being investigated. Application of methyl jasmonate was shown to have an additive, but no protective effect, on ozone tolerance of Pima cotton. Continued studies of genotoxicity of ozone in Pima cotton, using the Comet assay (alkaline single cell electrophoresis) demonstrated that fine roots do not exhibit chromosomal damage. Art Chappelka (AL) reported that clover biomass and nutritive quality were affected by ozone, however, grasses growing the same community were not. Rabbits fed elevated ozone forages were found to have lower DM, ADF, and NDF digestibility. Forages treated with elevated ozone increased bound phenolic composition which were correlated with lower DM, ADF, and NDF ingestibility. Digestibility depression in forages contributed to lower ingestion and utilization of energy. Dennis Decoteau (PA) presented information regarding a teaching module for demonstrating ozone effects on plants that has been developed at The Air Quality Learning and Demonstration Center at Penn State. The ozone module has been tested and is already being used in high school classrooms in Pennsylvania. The module is available for download by educators and the general public at various public websites. Research was continued in 2010 on Long Island, NY, (McGrath, NY) at Cornells research center (LIHREC) in Riverhead to further an investigation of the impact of ambient ozone on plant productivity using the snap bean system developed by project cooperators. Ozone injury occurred in all plantings. Quaking aspen, cutleaf coneflower and spreading dogbane were set up in plots in national parks and monitored during 2006-10 (Kohut, guest). Plots with elevated soil moisture were selected. Different symptoms of ozone injury were observed on cutleaf coneflower in the Rocky Mt and Smoky Mt national parks.

After the presentations a general discussion occurred regarding the station reports. Reduced funding for ozone research and challenges experienced obtaining funding were also discussed. Concern was voiced and discussed about the future of the study of plant response to ozone as a whole generation of scientists is being lost now due to lack of funding with no new scientists being trained, while this air pollutant continues to be an increasing problem. Ray Knighton gave a presentation via conference call. General budgetary constraints and funding challenges at the federal level were described. Lack of funding for travel was the reason Ray was only able to attend the meeting remotely, as was also the case for most committee members who did not attend in person. The Agriculture and Food Research Initiative (AFRI) was described, including opportunities in this program for research pertaining to air pollution. Mechanisms of plant response to stresses is a priority under Biology of Agriculture Plants. A challenge with the APRI program is the need for large, multi-institutional grants that integrate research with extension and/or education. Climate Change Program is a Challenge Area that should be an appropriate fit for a proposal from our group.

A field trip to Acadia National Park was taken by the members on July 28. Sites used in previous ozone bioinidicator studies were examined and a presentation was made by park personnel regarding their research in the park.

Business Meeting (27 July and 29 July)

The future of this Multistate Ozone Effects Project was discussed. Our proposal was approved to renew the project as a Coordinating Committee. The project expires on 30 September 2012. The final report probably is due 6 months after this date; this needs to be confirmed. Preparing the report was discussed. Fitz Booker, David Grantz and Art Chappelka agreed to take leadership on preparing the final report. Grant proposal topics and potential funding sources were discussed. There was a lengthy discussion of collaborations that could work with the range of expertise among the committee members. Some topics included: 1. examining the effect modern variety development has had on ozone sensitivity by comparing heirloom versus modern lines of vegetable, 2. building on interesting results from initial work undertaken by committee members on ozone and herbivore interactions, 3. relationship to ozone sensitivity and defense mechanisms of the timing and seasonality of high ozone episodes, and 4. the relationship between ozone episodes and drought stress comparing natural and agricultural ecosystems.

Bend, OR in May June 2012 was selected as the next meeting site and time period, which Nancy Grulke agreed to host.

Meeting adjourned at 10:30 am on 29 July 2011.

Minutes prepared by Margaret McGrath, Cornell University.

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

Publications

Aspinwall, MJ, JS King, FL Booker and SE McKeand. 2011. Genetic effects on total phenolics, condensed tannins and non-structural carbohydrates in loblolly pine (Pinus taeda L.) needles. Tree Physiology 31:831-842. Betzelberger AM, Gillespie KM, McGrath JM, Koester RP, Nelson RL, Ainsworth EA (2010) Biochemical, physiological and yield variation in soybean cultivar responses to chronic elevated ozone concentration. Plant, Cell Environment 33: 1569-1581. Cheng, L., Booker, F.L., Burkey, K.O., Tu, C., Shew, H.D., Rufty, T., Fiscus, 0000251160E.L., Hu, S. 2011. Soil microbial responses to elevated CO2 and O3 in a wheat-soybean agroecosystem. PLoS One. 6:e21377. Chrzanowski, S., D. D. Davis, and D. R. Decoteau, 2011. The Air Quality Learning and Demonstration Center at Penn States Teaching Module for Demonstrating Ozone Effects on Plants. US EPA AirNow Conference (http://airnow.gov/index.cfm?action=naq_conf_2011.aq3) Decoteau, D.R. 2011. Air Pollution Symptoms, 2011 Mid-Atlantic Fruit & Vegetable Convention Proceedings pages 10 11. Grantz, D.A., Vu, H. B., Heath, R. L., Burkey, K. (2011). Diel trends in plant sensitivity to ozone: Toward parameterization of the defense component of effective flux. Abstract American Geophysical Union, Annual Meeting. San Francisco, December 2011. Neufeld, HS, SJ Peoples, AW Davison, AH Chappelka, GL Somers, JE Thomley and FL Booker. 2011. Ambient ozone effects on gas exchange and total non-structural carbohydrate levels in cutleaf coneflower (Rudbeckia laciniata L.) growing in Great Smoky Mountains National Park. Environmental Pollution:In press. Niyogi, D., Mera, R., Xue, Y., Wilkerson, G., Booker, F.L. 2011. The use of 0000254469 Alpert-Stein Factor Separation Methodology for climate variable interaction studies in hydrological land surface models and crop yield models. In: Factor Separation in the Atmosphere. Cambridge University Press. Book Chapter. 171--183. Ren, W., H. Tian, B. Tao, A. Chappelka, G. Sun, C. Lu, M. Liu, G. Chen and X. Xu. 2011. Impacts of tropospheric ozone and climate change on net primary productivity and net carbon exchange of Chinas forest ecosystems. Global Ecol. Biogeogr. 20: 391-406. Tian, H. G. Chen, M. Liu, C. Zhang, G. Sun, C. Lu, X. Xu, W. Ren, S. Pan, A. Chappelka. 2010. Model estimates of net primary productivity, evapotranspiration, and water use efficiency in the terrestrial ecosystems of the southern United States during 18952007. For. Ecolog. & Mang. 259: 1311-1327.

Impact Statements

1. Personnel from this Multi-State Project have provided data to state and federal regulatory bodies and Agricultural Air Quality Task Force as air quality standards and policies are revised. Wilderness and National Park managers have utilized Project data to document long term impacts of ozone on unmanaged vegetation. Across a broad spectrum of stakeholders, tropospheric ozone is recognized as an element of global change that interacts with other elements, such as temperature, moisture and nitrogen.
2. Arrangements are being made with Carolina Biological Supply Co. in Burlington, NC to market the ozone-sensitive and ozone-tolerant snap bean lines commercially. Seeds and an instructional manual for conducting experiments with the snap beans in ozone projects will be made available.
3. Public educational facilities are in operation in California and Pennsylvania, and a comprehensive web presence is maintained in North Carolina to provide information that is relevant locally, nationally and internationally, with respect to ozone air pollution.
4. Ozone-sensitive and ozone-tolerant snap bean lines were used in laboratory exercises in two classes at NC State University (Environmental Technology 202 and Crop Physiology 714) to teach students about the impacts of ozone on plants.

Date of Annual Report: 10/04/2012

Report Information

Annual Meeting Dates: 07/10/2012 - 07/12/2012
Period the Report Covers: 10/01/2007 - 09/01/2012

Participants

Betzelberger, Amy - University of Illinois;
Burkey, Kent - USDA, Agricultural Research Service;
Chappelka, Arthur - Auburn University;
Decoteau, Dennis - The Pennsylvania State University;
Grantz, David - University of California Riverside;
Grulke, Nancy - USDA, Forest Service;
Minocha, Subhash - University of New Hampshire (by telephone 10 July and 12 July);
Smith, Margaret - Cornell University (by telephone, 12 July);
Knighton, Raymond - USDA, NIFA (by telephone, 12 July)

Brief Summary of Minutes

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

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.

Publications

Ainsworth, EA. 2008. Rice production in a changing climate: A meta-analysis of responses to elevated carbon dioxide and elevated ozone concentration. Global Change Biology, 14: 1642-1650. Ainsworth EA, A Rogers, ADB Leakey. 2008. Targets for crop biotechnology in a future high-CO2 and high-O3 world. Plant Physiology, 147: 13-19. Ainsworth EA, CR Yendrek, S Sitch, WJ Collins, LD Emberson LD. 2012. The effects of tropospheric ozone on net primary production and implications for climate change. Annual Review of Plant Biology 63: 637-661. Albertine, JM, WJ Manning. 2009. Elevated night soil temperatures result in earlier incidence and increased extent of foliar ozone injury to common bean (Phaseolus vulgaris L.). Environmental Pollution 157: 711-713. Aspinwall, MJ, JS King, FL Booker, SE McKeand. 2011. Genetic effects on total phenolics, condensed tannins and non-structural carbohydrates in loblolly pine (Pinus taeda L.) needles. Tree Physiology 31: 831-842. Bergweiler, C, WJ Manning, BI Chevone. 2008. Seasonal and diurnal gas exchange differences in ozone-sensitive common milkweed (Asclepias syriaca L.) in relation to ozone uptake. Environmental Pollution 152: 403-415. Bergweiler, C, H Carreras, E Wannaz, J Rodriguez, B Toselli, L Olcese, ML Pignata. 2008. Field surveys for potential ozone bioindicator plant species in Argentina. Environmental Monitoring and Assessment 138: 305-312. Betzelberger AM, KM Gillespie, JM McGrath, RP Koester, RL Nelson, EA Ainsworth. 2010. Biochemical, physiological and yield variation in soybean cultivar responses to chronic elevated ozone concentration. Plant Cell Environment 33: 1569-1581. Booker, FL, KO Burkey, WA Pursley, AS Heagle. 2007. Elevated carbon dioxide and ozone effects on peanut. I. Gas-exchange, biomass, and leaf chemistry. Crop Science 47:1475-1487. Booker, FL, R Muntifering, M McGrath, KO Burkey, D Decoteau, EL Fiscus, W Manning, S Krupa, A Chappelka, DA Grantz. 2009. The ozone component of global change: Potential effects on agricultural and horticultural plant yield, product quality and interactions with invasive species. Journal of Integrative Plant Biology 51:337-351. Burkey, KO, FL Booker, WA Pursley, AS Heagle. 2007. Elevated carbon dioxide and ozone effects on peanut. II. Seed yield and quality. Crop Science 47:1488-1497. Burkey, KO, TE Carter. 2009. Foliar resistance to ozone injury in the genetic base of U.S. and Canadian soybean and prediction of resistance in descendent cultivars using coefficient of parentage. Field Crop Research 111:207-217. Burkey KO, FL Booker, EA Ainsworth, RL Nelson RL. 2012. Field assessment of a snap bean ozone bioindicator system under elevated ozone and carbon dioxide in a free air system. Environmental Pollution 166: 167-171. Calfapietra, C, AE Wiberley, TG Falbel, AR Linskey, G Scarascia-Mugnozza, DF Karnosky, F Loreto, TD Sharkey. 2007. Isoprene synthase expression and protein levels are reduced under elevated O3 but not under elevated CO2 (FACE) in field-grown aspen trees. Plant Cell Environment 30: 654-661. Chen, X, C Tu, M Burton, D Watson, KO Burkey, S Hu. 2007. Plant nitrogen acquisition and interactions under elevated CO2: impact of endophytes and mycorrhizae. Global Change Biology 13: 1238-1249. Cheng, FY, KO Burkey, JM Robinson, FL Booker. 2007. Leaf extracellular ascorbate in relation to O3 tolerance of two soybean cultivars. Environmental Pollution 150: 355-362. Cheng, L., FL Booker, KO Burkey, C Tu, HD Shew, T Rufty, EL Fiscus, S Hu. 2011. Soil microbial responses to elevated CO2 and O3 in a wheat-soybean agroecosystem. PLoS One 6:e21377. Cheng, L., FL Booker, C Tu, KO Burkey, L Zhou, HD Shew, TW Rufty, S Hu. 2012. Arbuscular mycorrhizal fungi increase organic carbon decomposition under elevated CO2. Science 337: 1084-1087. Chrzanowski, S, DD Davis, DR Decoteau. 2011. The Air Quality Learning and Demonstration Center at Penn States Teaching Module for Demonstrating Ozone Effects on Plants. US EPA AirNow Conference (abstract accepted and paper presented on March 10, 2011, San Diego, CA). The presentation powerpoint is published at http://airnow.gov/index.cfm?action=naq_conf_2011.aq3. Davis, DD, JM Skelly, DR Decoteau, LJ Kline, JA Ferdinand, JE Savage, T Orendovici-Best. 2008. Susceptibility and Foliar Response of Broadleaved Species Exposed to Ozone. USDA Forest Service Forest Health Monitoring Program, 50 pp. Decoteau, DR. 2011. Air Pollution Symptoms, 2011 Mid-Atlantic Fruit & Vegetable Convention Proceedings pages 10-11. Ditchkoff, SS, JS Lewis, JC Lin, RB Muntifering, AH Chappelka. 2009. Nutritive quality of highbush blackberry (Rubus argutus) exposed to tropospheric ozone. Rangeland Ecology and Management 62: 364-370. Dubois, J-JB, EL Fiscus, FL Booker, MD Flowers, CD Reid. 2007. Optimizing the statistical estimation of the parameters of the Farquhar-von Caemmerer-Berry model of photosynthesis. New Phytologist 176: 402-414. Farber, RJ et. al. (Grantz is 15th out of 19 randomly ordered authors). 2007. Obliterating the dust in the Antelope Valley. Paper Number 384, Proceedings, Annual Meeting and Proceedings, Air and Waste Management Association. Feng, Z, K Kobayashi, EA Ainsworth. 2008. Impact of elevated ozone concentration on growth, physiology, and yield of wheat (Triticum aestivum L.): a meta-analysis. Global Change Biology 14: 2696-2708. Fiscus, EL, FL Booker, J-JB Dubois, TR Rufty, JW Burton, WA Pursley. 2007. CO2 enhancement effects in container- versus ground-grown soybeans at equal planting densities. Crop Science 47: 2486-2494. Fishman, J, JK Creilson, PA Parker, EA Ainsworth, GG Vining, J Szarka, FL Booker, X Xu. 2010. An investigation of widespread ozone damage to the soybean crop in the upper Midwest determined from ground-based and satellite measurements. Atmospheric Environment 44:2248-2256. Flowers, MD, EL Fiscus, KO Burkey, FL Booker, J-J Dubois. 2007. Photosynthesis, chlorophyll fluorescence, and yield of snap bean (Phaseolus vulgaris L.) genotypes differing in sensitivity to ozone. Environmental and Experimental Botany 61: 190-198. Galant, A, RP Koester, EA Ainsworth, LM Hicks, JM Jez. 2012. From climate change to molecular response: redox proteomics of ozone-induced responses in soybean. New Phytologist 194: 220-229. Gillespie, KM, F Xu F, KT Richter, JM McGrath, RJ Markelz, DR Ort, ADB Leakey, EA Ainsworth. 2012. Greater antioxidant and respiratory metabolism in field-grown soybean exposed to elevated O3 under both ambient and elevated CO2 concentrations. Plant Cell & Environment 35: 169-184. Gilliland, NJ, AH Chappelka, RB Muntifering, FL Booker, SS Ditchkoff. 2012. Digestive utilization of ozone-exposed forage by rabbits (Oryctolagus cuniculus). Environmental Pollution 163: 281-286. Gonzalez-Fernadez, I, D Bass, R Muntifering, G Mills, J Barnes. 2008. Impacts of ozone pollution on productivity and forage quality of grass/clover swards. Atmospheric Environment 42: 8755-8769. Gould, KS, DA Dudle, HS Neufeld. 2010. Why some stems are red. Photoprotective roles for anthocyanins in internodes. Journal of Experimental Botany 61: 2707-2717. Grantz, DA, A Shrestha, H-B Vu. 2008. Early vigor and ozone response in horseweed (Conyza canadensis) biotypes differing in glyphosate resistance. Weed Science 56: 224-230. Grantz, DA, A Shrestha, H-B Vu. 2008. Ozone enhances adaptive benefit of glyphosate resistance in horseweed (Conyza canadensis). Weed Science 56: 549-554. Grantz, DA, H-B Vu. 2009. O3 sensitivity in a potential C4 bioenergy crop: Sugarcane in California. Crop Science 49: 643-650. Grantz, DA, A Shrestha, H. Vu. 2010. Ozone impacts on assimilation and allocation to reproductive sinks in the vegetatively propagated C4 weed, yellow nutsedge. Crop Science 50: 246-252. Grantz, DA, H Vu, C Aguilar, MA Rea. 2010. No interaction between methyl jasmonate and ozone in pima cotton: growth and allocation respond independently to both. Plant Cell and Environment 33: 717-728. Grantz, DA, H-B Vu, RL Heath, K Burkey. 2010. Temporal Sensitivity Key to Modeling Ozone Impacts on Vegetation. Extended Abstract 2010-EE-208-AWMA. Proceedings Annual Meeting, Air and Waste Management Association, Calgary. June 2010. Grantz, DA, H-B Vu, RL Heath, K Burkey. 2011. Diel trends in plant sensitivity to ozone: Toward parameterization of the defense component of effective flux. Abstract American Geophysical Union, Annual Meeting. San Francisco, December 2011. Grantz, D A, H-B Vu, TL Tew, JC Veremis. 2012. Sensitivity of gas exchange parameters to ozone in diverse C4 sugarcane hybrids. Crop Science 52: 1-11. Grantz, DA, H-B Vu. 2012. Root and shoot gas exchange respond additively to moderate ozone and methyl jasmonate without induction of ethylene: ethylene is induced at higher O3. Journal of Experimental Botany 63: 43034313. Grulke, NE, HS Neufeld, AW Davison, M Roberts, AH Chappelka. 2007. Stomatal behavior of ozone-sensitive and insensitive coneflowers (Rudbeckia laciniata var. digitata) in Great Smoky Mountains National Park. New Phytologist 173: 100-109. Grulke NE, E Paoletti, RL Heath. 2007. Chronic vs. short term acute O3 exposure effects on nocturnal transpiration in two Californian oaks. The Scientific World 7(S1):134-140. DOI 10.1100/tsw.20007.33 Grulke, NE, E Paoletti, RL Heath. 2007. Comparison of calculated and direct measurements of foliar O3 uptake in crop and native tree species. Environmental Pollution 146: 640-647. Handley T, NE Grulke. 2008. Interactive effects of O3 exposure on California black oak (Quercus kelloggii Newb.) seedlings with and without nitrogen amendment. Environmental Pollution 156: 53-60. Haydt, SC, DD Davis, T Hoover, DR Decoteau. 2011. A teaching module on ozone as an air pollutant and its effect on plants. NACTA J. Teaching Tips. December: 107 109. Holmes, WE, DR Zak, KS Pregitzer, JS King. 2006. Elevated CO2 and O3 alter soil nitrogen transformations beneath trembling aspen, paper birch, and sugar maple. Ecosystems 9: 1354-1363. Karnosky, DF, JM Skelly, KE Percy, AH Chappelka. 2007. Perspectives regarding 50 years of research on effects of tropospheric ozone air pollution on U.S. Forests. Environmental Pollution 147: 489-506. Karnosky, DF, H Werner, T Holopainen, K Percy, T Oksanen, E Oksanen, C Heerdt, P Fabian, J Nagy, W Heilman, R Cox, N Nelson, R Matyssek. 2007. Free-air exposure systems to scale up ozone research to mature trees. Plant Biology 9: 181-190. King, JS, CP Giardina, KS Pregtizer, AL Friend. 2007. Biomass partitioning in red pine (Pinus resinosa Ait.) along a chronosequence in the Upper Peninsula of Michigan. Canadian Journal of Forest Research 37: 93-102. Kline LJ, DD Davis, JM Skelly, JE Savage, J Ferdinand. 2008. Ozone sensitivity of 28 plant selections exposed to ozone under controlled conditions. Northeastern Naturalist 15: 57-66. Kline, LJ, DD Davis, JM Skelly, DR Decoteau. 2009. Variation in ozone sensitivity within Indian hemp and common milkweed selections from the Midwest. Northeastern Naturalist 16: 307-313. Kubiske, ME, VS Quinn, PE Marquardt, DF Karnosky. 2007. Effects of elevated CO2 and/or O3 on intra- and interspecific competitive ability of aspen. Plant Biology 9: 342-355. Krupa, S, FL Booker, V Bowersox, C Lehmann, D Grantz. 2008. Uncertainties in the current knowledge of some atmospheric trace gases associated with US agriculture. Journal of Air & Waste Management Association 58: 986-993. Leakey, ADB, F Xu, KM Gillespie, JM McGrath, EA Ainsworth, DR Ort. 2009. Genomic basis for stimulated respiration by plants growing under elevated carbon dioxide. Proceedings of the National Academy of Sciences 106: 3597-3602. Leakey, ADB, EA Ainsworth, CJ Bernacchi, A Rogers, SP Long, DR Ort. 2009. Elevated CO2 effects on plant carbon, nitrogen, and water relations: six important lessons from FACE. Journal of Experimental Botany 60: 2859-2876. Leisner CP, EA Ainsworth. 2012. Quantifying the effects of ozone on plant reproductive growth and development. Global Change Biology 18: 606-616. Liu, L, JS King, CP Giardina, FL Booker. 2009. The influence of chemistry, production and community composition on leaf litter decomposition under elevated atmospheric CO2 and tropospheric O3 in a northern hardwood ecosystem. Ecosystems 12: 401-416. Liu, L, JS King, FL Booker, CP Giardina, HL Allen, S Hu. 2009. Enhanced litter input rather than changes in litter chemistry drive soil carbon and nitrogen cycles under elevated CO2: a microcosm study. Global Change Biology 15: 441-453. Lin, JC, M Nosal, RB Muntifering, SV Krupa. 2007. Alfalfa nutritive quality for ruminant livestock as influenced by ambient air quality in west-central Alberta. Environmental Pollution 149: 99-103. Lin, JC, K Nadarajah, M Volk, RB Muntifering, J Fuhrer. 2007. Nutritive quality of a species-rich, extensively managed pasture exposed to elevated ozone in a free-air fumigation system. Journal of Animal Science 90 (Suppl. 1): 36. Long, SP, EA Ainsworth, ADB Leakey, DR Ort, J Nosberger, D Schimel. 2007. Crop models, CO2, and climate change response. Science 315: 460-460. Lucier, A, M Ayres, D Karnosky, I Thompson, C Loehle, K Percy, B Sohngen. 2009. Future environmental impacts and vulnerabilities. pp. 29-52 In Seppälä, R., Buck A., Katila, P. (Eds.) Adaptation of Forests and People to Climate Change A Global Assessment Report. International Union of Forest Research Organizations (IUFRO) World Series Vol. 22, Vienna, Austria. 224pp Matyssek, R, H Sandermann, G Wieser, FL Booker, S Cieslik, R Musselman, D Ernst. 2008. The challenge of making ozone risk assessment for forest trees more mechanistic. Environmental Pollution 156: 567-582. McGrath, M.T. 2007. Assessing ambient ozone impact on plant productivity in NY with snap bean genotypes differing in sensitivity. Phytopathology 97: (presented 11/8/06). (http://www.apsnet.org/meetings/div/ne06abs.asp). Neufeld, HS, AH Chappelka. 2007. Air pollution and vegetation effects research in national parks and natural areas: Implications for science, policy and management. Environmental Pollution 149: 253-255. Neufeld, HS, SJ Peoples, AW Davison, AH Chappelka, GL Somers, JE Thomley, FL Booker. 2012. Ambient ozone effects on gas exchange and total non-structural carbohydrate levels in cutleaf coneflower (Rudbeckia laciniata L.) growing in Great Smoky Mountains National Park. Environmental Pollution 160: 74-81. Nikula, S, S Manninen, K Percy, M Falck, E Oksanen, T Holopainen 2009. Elevated O3 induced minor changes in growth and foliar traits of European and hybrid aspen. Boreal Environment Research 14(A): 29-47. Niyogi, D, R Mera, Y Xue, G Wilkerson, FL Booker. 2011. The use of Alpert-Stein Factor Separation Methodology for climate variable interaction studies in hydrological land surface models and crop yield models. In: Factor Separation in the Atmosphere. Cambridge University Press. Book Chapter. 171-183. Novak, K, M Schaub, J Fuhrer, JM Skelly, B Frey, N Kräuchi. 2008. Ozone effects on visible foliar injury and growth of Fagus sylvatica and Viburnum lantana seedlings grown in monoculture or in mixture. Environmental and Experimental Botany 62: 212-220. Oncley, SP, T Foken, R Vogt, W Kohsiek, HAR DeBruin, C Bernhofer, A Christen, E van Gorsel, D Grantz, C Feigenwinter, I Lehner, D Liebethal, H Liu, M Mauder, A Pitacco, L Ribeiro, T Weidinger. 2007. The Energy Balance Experiment EBEX-2000. Part I: overview and energy balance. Boundary Layer Meteorology 123: 1-28. Orendovici-Best, T, JM Skelly, DD Davis, JA Ferdinand, JE Savage, RE Stevenson. 2008. Ozone uptake (flux) as it relates to ozone-induced foliar symptoms of Prunus serotina and Populus maximowizii × trichocarpa. Environmental Pollution 151: 79-92. Paoletti, E, N Contran, WJ Manning, A Castagna, A Ranieri, F Tagliaferro. 2008. Protection of ash (Fraxinus excelsior) trees from ozone injury by ethylenediurea (EDU): Roles of biochemical changes and decreased stomatal conductance in enhancement of growth. Environmental Pollution 155: 464-472. 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Impact Statements

This Multi-State Project provided data to state and federal regulatory bodies and Agricultural Air Quality Task Force as air quality standards and policies are revised. Wilderness and National Park managers have utilized Project data to document long term impacts of ozone on unmanaged vegetation. Across a broad spectrum of stakeholders, tropospheric ozone is recognized as an element of global change that interacts with other elements, such as temperature, moisture and nitrogen.
Modeling studies that combined soybean crop yield data with ground and satellite ozone measurements from the Midwest of the United States provided evidence that yield losses from ozone on the order of 10%. Extrapolation of these findings supports previous studies that estimate the global economic loss to the farming community of more than $10 billion annually.
Modern U.S. soybeans are susceptible to yield losses from ozone. Cultivars that are major players in North American soybean breeding efforts were subject to significant yield losses when exposed to elevated ozone concentrations at the SoyFACE site. This finding was supported by a study that screened soybean ancestors representing 92% of the genetic base of North American soybean and found that ancestors with the greatest ozone resistance were not major contributors to current US cultivars.
Ozone-tolerant (Fiskeby III) and ozone-sensitive (Mandarin Ottawa) soybean ancestors were used parents to develop a population consisting of 240 random inbred lines for mapping ozone tolerance genes. 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 rare opportunity to simultaneously map and compare genes for a wide range of abiotic stress factors.
Ozone-sensitive and ozone-tolerant snap bean random inbred lines were shown to be an effective ozone bioindicator in both irrigated and non-irrigated environments at multiple locations across the U.S. The results suggest that this snap bean system could be used to quantify ozone effects in specific locations with potential applications in diverse environments including agricultural fields.
A mixture of common Southern Piedmont grassland species were grown under elevated ozone and the forage fed to rabbits in a digestibility experiment. Elevated ozone resulted in decreased digestible dry matter intake. These findings suggest that ozone air pollution can have a negative impact on forage quality, resulting in decreased nutrient utilization by mammalian herbivores.
Native plant species continue to be indentified and evaluated for use as bioindicators to document ambient ozone effects on natural ecosystems. Species studied by NE-1030 include cutleaf coneflower, Tree of Heaven (Ailanthus altisima), staghorn sumac, black cherry, common milkweed, and dogbane.
Ozone sensitivity of cotton was found to vary diurnally. If confirmed in other species, these findings suggest that the next generation of flux based models for setting ozone standards must incorporate the concept that plant sensitivity to ozone is variable.
Ambient ozone concentrations adversely affected the foliage of the most important red grape variety grown for wine in Pennsylvania. This research continues to provide needed information concerning the relationships between ambient ozone exposures and induced foliar injury more commonly observed on ozone sensitive plants.
A USDA NRI Plant Genome Program funded project lead to identification of more expressed gene sequence resources for Black Cherry than any other single Rosaceae species. Results of network analysis of the genes differentially expressed in black walnut and green ash will soon be available as well. The comparisons will to a more general understanding of the similarities and differences between hardwood tree species in their response to ozone stress.
Sequestering additional carbon in agricultural soils to offset rising carbon dioxide levels may be more difficult than originally thought. Elevated carbon dioxide enhances plant-derived carbon inputs into the soil, but it also appears to stimulate microbial decomposition in the presence of nitrogen inputs required in cropping systems. The interactive effects of carbon and nitrogen on microbial activities must be considered when projecting soil carbon balance under future climate scenarios.
Public educational facilities are in operation in California and Pennsylvania, and a comprehensive web presence is maintained in North Carolina to provide information that is relevant locally, nationally and internationally, with respect to ozone air pollution.
Ozone-sensitive and ozone-tolerant snap bean lines were used in laboratory exercises in two classes at NC State University (Environmental Technology 202 and Crop Physiology 714) to teach students about the impacts of ozone on plants.
Growers and extension educators in California are recognizing ozone impacts as part of climate change on the dynamics of important agricultural weeds, including horseweed and yellow nutsedge. This influences regulatory acceptance, and may lead to altered vegetation management protocols.