Brief Summary of Minutes of Annual Meeting

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

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).

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