Annual Report 2003

CSREES Multistate Research Project

Project No. NE-1013

Mechanisms of Plant Responses to Ozone in the Northeastern US

2003 Annual Report

Executive Summary

Demonstrable progress was made in 2003 toward achieving the objectives of the NE-1013 project. The proposal’s milestones for 2003 were fully met.

First, a common experimental protocol was developed for a field project using an ozone-sensitive and ozone-tolerant cultivar of snap bean, and initial field trials were begun at five locations nationwide. Results from this study will aid in understanding how environmental factors affect plant sensitivity to ambient ozone.

Second, a variety of investigative approaches continued to show that ambient ozone concentrations in many regions of the United States suppress crop yields, damage native vegetation and impair forest health.

Third, advances in molecular biology research, in conjunction with physiological measurements, further revealed mechanisms of ozone action on plants. These results will aid in our understanding of ozone injury and the development of ozone-tolerant plants.

Several of the Technical Committee Members fulfilled an important outreach function by serving as peer reviewers of the US. EPA’s Draft Criteria Document for Ozone (the first step in the process addressing any revisions in the National Ambient Air Quality Standards for Ozone to protect human health and welfare of the environment).

U.S. Department of Agriculture AD-421 Progress Report (Investigator Copy) U.S. Dept. of Agriculture, State Agricultural Experiment Stations and Other Institutions			Date (Month, Day, Year) 12/22/2003
1. Accession 0194827	Agency Identification No. 2. CSREES 3. ALA	5. Work Unit/Project No. ALA011-030	6. Status Progress
7. Title Mechanisms of Plant Responses to Ozone in the Northeastern US
12. Investigator Name(s) (Last Name and Intitials) Chappelka, A. H.; Muntifering, R. B.
20. Termination Date 09/30/2007		40. Period Covered (mo/da/year): 01/01/2003 to 12/31/2003
41. Progress Report: Studies investigating the effects of ambient ozone on native forest and wildflower communities growing in the Great Smoky Mountains National Park were initiated during 2003. Leaves were collected during July for a population genetics study with cutleaf coneflower at 23 different locations in the Park. Leaves are currently being sampled for genetic similarities/differences using various DNA fingerprinting techniques. In addition, preliminary analyses of the nutritive quality of cutleaf coneflower at one site indicated that as visible injury increases, fiber and lignin concentration increases causing a reduction in relative food value. Results were inconclusive at another location regarding nutritive quality. A more thorough investigation is planned in the future. A study to determine the effectiveness of ethylenediurea (EDU) to native plant species was initiated in 2003 using open-top chambers (OTCs) in Auburn, AL. Initial toxicological studies were conducted with two species: purple coneflower and lance-leaf coreopsis. Both were found to be sensitive to elevated ozone concentrations. EDU did not appear to alleviate visible injury symptoms significantly for either species. However, nutritive quality appeared to be increased with the addition of EDU in the presence of elevated ozone. Another study was initiated to investigate effects of early-season ozone exposure and N status on nutritive quality of annual grass and legume species representative of an extensively managed dehesa community in the Iberian Peninsula. Seedlings of three legume and three grass species were exposed to various ozone treatments in OTCs. Ozone decreased aboveground and root biomass yield in all Trifolium species. Early-season ozone exposure decreased nutritive quality of T. subterraneum and T. cherleri, but not of T. striatum. Early-season ozone exposure also decreased nutritive quality of Briza maxima and Cynosurus echinatus, but not Bromus hordaceus, as predicted from concentration of cell-wall constituents.
42. Impact Our results will provide a fundamental understanding of ozone effects to individual plants as well as community and ecosystem responses. In addition, these results will provide information to decision makers so adequate protective standards can be developed.
43. Publications Chappelka, A.H., H.S. Neufeld, A.W. Davison, G.L. Somers and J.R. Renfro. 2003. Ozone injury on cutleaf coneflower (Rudbeckia laciniata) and crown-beard (Verbesina occidentalis) in Great Smoky Mountains National Park. Environ. Pollut. 125: 53-59. Davison, A. W., H. S. Neufeld, A. H. Chappelka, K. Wolff, and P. L. Finkelstein, 2003. Interpreting spatial variation in ozone symptoms shown by cutleaf coneflower, Rudbeckia laciniata L. Environ. Pollut.: s61-70. Powell, M.C., R.B. Muntifering, J.C. Lin and A.H. Chappelka. 2003. Yield and nutritive quality of sericea lespedeza (Lespedeza cuneata) and little bluestem (Schizachyrium scoparium) exposed to ground-level ozone. Environ. Pollut. 122: 313-322.
Approved (Signature)		Title	Date

U.S. Department of Agriculture AD-421 Progress Report (Investigator Copy) U.S. Dept. of Agriculture, State Agricultural Experiment Stations and Other Institutions			Date (Month, Day, Year) 12/31/2003
1. Accession 0194827	Agency Identification No. 2. CSREES 3. CA	5. Work Unit/Project No.	6. Status Progress
7. Title Mechanisms of Plant Responses to Ozone in the Northeastern US
12. Investigator Name(s) (Last Name and Intitials) Grantz D.A.
20. Termination Date 09/30/2007		40. Period Covered (mo/da/year): 01/01/2003 TO 12/31/2003
41. Progress Report: (Example shown) We have developed an experimental system of potted plants grown in sintered clay and exposed to ozone in open top field exposure chambers. Using this system we have demonstrated that in both Pima cotton and in muskmelon, ozone-induced inhibition of carbon assimilation in the shoot does not cause the expected reduction of root respiratory activity. As source strength and transport of carbohydrate to sink tissues declined, root respiration per unit fresh weight increased. The mechanism of this linkage remains unknown. However, the hypothesis that substrate control of root respiration, as it is modulated by ozone impacts above ground, may be rejected. Various hypotheses associated with signaling, translocation of damage, and alteration of substrate species available to root respiratory metabolism, must now be tested. We have built on our previous work demonstrating reduced root extension and impaired hydraulic conductance in response to ozone, to investigate the impact of ozone on the competitive interactions of a C4 weed, yellow nutsedge, with cotton and tomato. We found that ozone induced a large shift in the allocation strategy of nutsedge. Increasing ozone concentration to a moderate level caused nutsedge to produce fewer reproductive tubers, but further increase in ozone concentration caused a substantial increase in tuber production, almost to control levels. Ozone caused an increase in the root to shoot biomass ratio in nutsedge, but a decline in tomato. The relative decline in total root biomass with increasing ozone was less in nutsedge than in tomato, suggesting a deleterious impact of ozone on the competitiveness of tomato with respect to nutsedge below ground. We did not observe a significant interaction between ozone and weed pressure on tomato growth in our experimental system of adequate soil volume and irrigation. Future experiments will add nutritional status as a third factor, as we develop an increasingly complex system analysis. Tomato and nutsedge are both moderately tolerant to ozone. Pima cotton, in contrast, is quite sensitive to ozone. Preliminary results indicate that an important interaction between nutsedge and ozone may lead to increasing competitiveness of nutsedge in future environments of increasing ground level ozone. Further experiments are in progress. We have obtained samples of high repetition experiments to determine impacts on the carbohydrate speciation of source, sink and transport tissue of cotton and melon. These samples have been analyzed chromatographically, but not yet evaluated. This is underway at this time. In summary, our research program is developing increasing complex systems in which to evaluate the interactions of which ozone is an important component. Results in the current reporting period suggest that the significance of these interactions will depend substantially on the specific components of the system under investigation.
42. Impact Tropospheric ozone is an increasing problem in the San Joaquin Valley of California, recently surpassing the notorious South Coast (Los Angeles) airbasin as the worst in the country. As the San Joaquin Valley is dominantly agricultural, a major impact is on crop production. The mechanism of ozone damage to vegetation remains mostly unknown. Our results contribute to a fundamental understanding of ozone effects on individual plants as well as on community and ecosystem responses. To this end we have identified below ground processes as critical determinants of between-plant competition, of shoot gas exchange behavior, and of crop growth and yield. As these relationships become more fully developed, a realistic protocol for crop loss prediction, based on systems analytical approaches, will become feasible. In addition, these results will provide to policy makers information on the modes and severity of plant damage due to ozone, so that adequate protective standards can be developed. By maintaining experimental evidence of such damage in an accessible location at Kearney Agricultural Center, the program provides outreach to public, regulatory, and policy maker stakeholders, so that appropriate public policy may be promulgated.
43. Publications Grantz, D.A., Silva, V., Toyota, M., Ott, N. 2003. Ozone increases root respiration but decreases leaf CO2 assimilation in cotton and melon. Journal of Experimental Botany 54: 2375-2384. Grantz, D.A. 2003. Ozone impacts on cotton: towards an integrated mechanism. Environmental Pollution 126: 331-344. Grantz, D.A., Garner, J.H.B., Johnson, D.W. 2003. Ecological effects of particulate matter. Environment International 29: 213-239. Farber, R.J., Kim, B.M., Grantz, D.A. et al. 2003. Crushing the dust in the Antelope Valley. Proceedings of the Air and Waste Management Association. Paper No. 71037.
Approved (Signature)		Title	Date

U.S. Department of Agriculture AD-421 Progress Report (Investigator Copy) U.S. Dept. of Agriculture, State Agricultural Experiment Stations and Other Institutions			Date (Month, Day, Year) 12/31/2003
1. Accession 0194827	Agency Identification No. 2. CSREES 3. MD	5. Work Unit/Project No.	6. Status Progress
7. Title Mechanisms of Plant Responses to Ozone in the Northeastern US
12. Investigator Name(s) (Last Name and Intitials) Momen, Bahram
20. Termination Date 09/30/2007		40. Period Covered (mo/da/year): 01/01/2003 TO 12/31/2003
41. Progress Report: (Example shown) We studied the effects of ozone on two snap bean cultivars (S156 and R331) that have differential ozone sensitivity. Response variables studied were stem and leaf metabolic heat rates (q), stem and leaf total biomass, and total q per total stem and leaf mass. We used micro-calorimetry to measure stem and leaf q (W per unit of dry mass) at 25 ^oC. In micro-calorimetry, tissue metabolic heat rate is measured to assess respiration rate. Metabolic heat rate indicates total metabolism of the tissue with an emphasis on the oxidation reaction. Measure of q detects heat loss due to thermodynamic inefficiency in metabolic reaction. Heat rates are proportional to metabolic rates and energy use efficiency. This makes q a more general and informative measure of respiration rate compared to a measure of CO₂ production or O₂ consumption rate. Our O₃ exposure chambers consisted of eight CSTRs. Two O₃ levels (25 and 100 nmol mol^-1) were applied randomly to these chambers 8 hrs. daily. The two bean cultivars were sub-plots within each O₃ main plot. After one and two weeks of treatment, no cultivar-by-O₃ interaction for stem and leaf q or leaf dry mass was observed. This indicates that the assumed differential sensitivity of the two cultivars to O₃ was not manifested based on these responses. Averaged over both O₃ levels, leaf and stem q of S156 was higher than those of R331, but the two cultivars did not differ in their total leaf dry mass. Elevated O₃ increased leaf q of both cultivars, had no effect on stem q, and decreased leaf dry mass. Variety-by-O₃ interactive effect was significant for stem dry mass and for a measure of total q of total stem or total leaf dry mass. This implies that the O₃ effect on such responses was not the same for both varieties. Compared with R331, S156 had smaller stem dry mass under reduced O₃, but this difference disappeared under elevated O₃. Also, the trend of cultivar differences observed under reduced O₃ was reversed by elevated O₃ when q was expressed based on total stem or total leaf mass.
42. Impact Our results will provide a fundamental understanding of ozone effects to individual plants as well as community and ecosystem responses. In addition, these results will provide information to decision makers so adequate protective standards can be developed.
43. Publications None
Approved (Signature)		Title	Date

U.S. Department of Agriculture AD-421 Progress Report (Investigator Copy) U.S. Dept. of Agriculture, State Agricultural Experiment Stations and Other Institutions			Date (Month, Day, Year) 11/17/2003
1. Accession 0194493	Agency Identification No. 2. CSREES 3. MA	5. Work Unit/Project No. MAS00880	6. Status Progress
7. Title Mechanisms of Plant Responses to Ozone in the Northeastern US
12. Investigator Name(s) (Last Name and Intitials) Manning, W.J.
20. Termination Date 09/30/2007		40. Period Covered (mo/da/year): 01/01/2003 TO 12/31/2003
41. Progress Report: (Example shown) The behavior of bean (Phaseolus vulgaris L.) selections R331 (tolerant to ozone) and S156 (susceptible to ozone) were compared in a field plot. This is a cooperative effort with several other members of NE-1013. One harvest was made when all pods had matured and yellowed. R331 had significantly more pods and greater fresh and dry weights of pods and dry weights of shoots than S156. These two selections are better suited for comparisons than are R123 and S156. Comparative stomatal conductance was determined for abaxial leaf surfaces in open-top chambers (OTCs) supplied with either charcoal-filtered air (CF) or non-filtered air (NF) and for plants in non-chambered ambient air plots (AA). Stomatal conductance was measured at weekly intervals between 17 July and 15 August, 2002 (at 43, 52, 58, 65 and 72 days after sowing) during morning hours. Genotype did not significantly affect stomatal conductance. Conductance rates between R123 and S156 plants in CF-air were not significantly different. In a CF-filtered greenhouse in November, 2002, S156 had significantly higher values for stomatal conductance for the abaxial leaf surface, but not for the adaxial surface. Stomatal density and aperture measurements were done on abaxial and adaxial leaf surfaces of R123 and S156 plants in CSTRs containing CF-air, CF+30 ppb ozone or CF+60 ppb ozone. For R123, no differences were seen for aperture size on both leaf surfaces. For S156, aperture size on the adaxial leaf surface was significantly larger for plants exposed to 30 ppb ozone. For R123, there were no treatment-related differences in stomatal density for either leaf surface. Stomatal density was increased on abaxial leaf surfaces for S156 at 60 ppb ozone. When compared with each other, R123 had higher stomatal densities on adaxial leaf surfaces than S156.
42. Impact Our results are fundamental to an understanding of ozone effects on plants. They have a direct bearing on the development of air quality standards for ambient ozone for plants and for humans.
43. Publications Manning, W.J., Flagler, R.B. and Frenkel, M.A. 2003. Assessing plant response to ambient ozone: growth of ozone-sensitive loblolly pine seedlings treated with ethylenediurea or sodium erythorbate. Environmental Pollution 126:73-81. Manning, W.J. 2003. Detecting plant effects is necessary to give biological significance to ambient ozone monitoring data and predictive ozone standards. Environmental Pollution 126:375-379.
Approved (Signature)		Title	Date

U.S. Department of Agriculture AD-421 Progress Report (Investigator Copy) U.S. Dept. of Agriculture, State Agricultural Experiment Stations and Other Institutions			Date (Month, Day, Year) 12/31/2003
1. Accession 0194827	Agency Identification No. 2. CSREES 3. MN	5. Work Unit/Project No.	6. Status Progress
7. Title Mechanisms of Plant Responses to Ozone in the Northeastern US
12. Investigator Name(s) (Last Name and Intitials) Krupa, S.
20. Termination Date 09/30/2007		40. Period Covered (mo/da/year): 01/01/2003 to 12/31/2003
41. Progress Report: (Example shown) A multivariate, non-linear statistical model was developed to simulate passive ozone (O₃) sampler data to mimic the hourly frequency distributions of continuous measurements using climatologic O₃ indicators and passive sampler measurements. The main meteorological parameters identified by the model were air temperature, relative humidity, solar radiation and wind speed, although other parameters were also considered. Together, air temperature, relative humidity and passive sampler data by themselves could explain 62.5–67.5% (R²) of the corresponding variability of the continuously measured O₃ data. The final correlation coefficients (r) between the predicted hourly O₃ concentrations from the passive sampler data and the true, continuous measurements were 0.819–0.854, with an accuracy of 92–94% for the predictive capability. With the addition of soil moisture data, the model can lead to the first order approximation of atmospheric O₃ flux and plant stomatal uptake. Additionally, if such data are coupled to multi-point plant response measurements, meaningful cause-effect relationships can be derived in the future.
42. Impact Inexpensive passive ambient ozone samplers are being increasingly used, in lieu of continuous electronic monitors. The work described herein is highly relevant in relating passive sampler data (mean values) to the dynamics of biological responses.
43. Publications Grünhage, L., Krupa, S. V., Legge, A. H., & Jäger, H.-J. (2003). Ambient flux-based critical values of ozone for protecting vegetation: differing spatial scales and uncertainties in risk assessment. In Proc. UN-ECE Conf. Ozone: Critical Levels II, Gothenburg, Sweden, 19-23 November 2002. IVL, Gothenburg, Sweden. pp 19-34. Krupa, S. V., Nosal, M., Ferdinand, J.A., Stevenson, R.E. & Skelly, J.M. (2003). A multi-variate statistical model integrating passive sampler and meteorology data to predict the frequency distributions of hourly ambient ozone (O₃) concentrations. Environ. Pollut. 124, 173-178.
Approved (Signature)		Title	Date

U.S. Department of Agriculture AD-421 Progress Report (Investigator Copy) U.S. Dept. of Agriculture, State Agricultural Experiment Stations and Other Institutions			Date (Month, Day, Year) 12/31/2003
1. Accession 0194827	Agency Identification No. 2. CSREES 3. NJ	5. Work Unit/Project No.	6. Status Progress
7. Title Mechanisms of Plant Responses to Ozone in the Northeastern US
12. Investigator Name(s) (Last Name and Intitials) Zilinskas, B. A.
20. Termination Date 09/30/2007		40. Period Covered (mo/da/year): 01/01/2003 TO 12/31/2003
41. Progress Report: (Example shown) Ascorbic acid (known also as vitamin C) is present in millimolar concentrations in plant cells. Ascorbic acid can directly quench singlet oxygen and superoxide and hydroxyl radicals, and thus it is critical in protecting plants against oxidative stress. It is also important in maintaining vitamin E in its reduced state by regenerating it from its alpha-tocopherol radical. Ascorbate also serves as substrate for ascorbate peroxidase and violaxanthin de-epoxidase. Its presence in the apoplast has been shown to be important in minimizing damage by the air pollutant, ozone. Our laboratory is attempting to evaluate the contribution of ascorbic acid to tolerance against ozone by genetically manipulating the content of ascorbic acid by overexpression of key enzymes in the ascorbate biosynthetic pathway in tobacco. These are GDP-mannose pyrophosphorylase and L-galactone-1,4-lactone dehydrogenase. Results to date have shown only small changes in ascorbate content in some of the transgenic lines. The tolerance of these plants to ozone exposure is currently being analyzed.
42. Impact This research should provide insight into the contribution of ascorbic acid to the tolerance of plants to ozone and help to determine whether ascorbic acid content can be genetically manipulated in plants.
43. Publications Mittler, R. and B.A. Zilinskas 2003 Activated oxygen species in multiple stress situations and protective systems. In: Molecular Ecotoxicolgy of Plants, Ecological Studies, Vol. 170, H. Sandermann (Ed.), Springer-Verlag, Berlin Heidelberg, pp.51-73.
Approved (Signature)		Title	Date

U.S. Department of Agriculture AD-421 Progress Report (Investigator Copy) U.S. Dept. of Agriculture, State Agricultural Experiment Stations and Other Institutions			Date (Month, Day, Year) 12/22/2003
1. Accession 0194827	Agency Identification No. 2. CSREES 3. NY	5. Work Unit/Project No.	6. Status Progress
7. Title Mechanisms of Plant Responses to Ozone in the Northeastern US
12. Investigator Name(s) (Last Name and Intitials) Kohut, R.
20. Termination Date 09/30/2007		40. Period Covered (mo/da/year): 01/01/2003 to 12/31/2003
41. Progress Report: Research for the National Park Service is being conducted on two distinct, but related, tasks: assessment of potential ozone risk at specific park service units, and development of protocols to assess ozone impacts to vegetation in the field. TASK I: Ozone Risk Assessment This task will provide the National Park Service with an assessment of the risk of ozone injury on vegetation at approximately 270 units within the Vital Signs Monitoring Networks. Each unit will use the findings of the risk assessment as it decides which indicators it will include in its monitoring program. The approach employed in conducting the risk assessments is based on the triad concept of ozone injury that holds that foliar ozone injury on plants is the result of the interaction of the plant, ambient ozone, and the environment. The production of foliar injury is maximized when certain properties of these three interacting entities are optimized. Deviation of any of the properties from the optimum reduces the risk of injury, and extreme deviation may preclude injury. Optimization of the three entities involves having species of plants that are genetically predisposed to ozone, concentrations of ambient ozone that exceed thresholds required for injury, and environmental conditions that foster gas exchange and the uptake of ozone. The risk assessment is based on evaluation of the states of these entities on a site-specific basis to produce a qualitative risk rating of high, medium, or low. The plant species, ozone exposure, and soil moisture data will be integrated to assess the level of risk of foliar injury for each site, and recommendations will be made on bioindicator species that can be used in a foliar injury assessment program. Task II: Ozone Injury Field Assessment Handbook A handbook is being developed that will serve as the National Park Service’s standard reference document for the assessment of ozone injury on plants in the field. Appendices will provide background on ozone and its impacts to plants, and added information and materials to support the field assessment. The field assessment handbook is composed of two major sections: field assessment protocols, and appendices. The protocol section contains a discussion of the response triad concept, descriptions of foliar ozone injury, and a discussion of bioindicator species. This information allows the reader to understand how ozone interacts with plants, and creates a foundation for making informed decisions about establishing an ozone injury assessment program. Subsequent sections address personnel and training for field assessment, data compilation, analysis, and maintenance, and quality assurance. Consideration of these elements is essential if a field assessment plan is to be successfully executed. The appendices provide illustrations of ozone injury on bioindicator species and technical information for use in sampling the plots and recording response data.
42. Impact This research for the National Park Service will assess potential ozone risk at specific park service units, and aid in the development of protocols to assess ozone impacts to vegetation in the field. The results will provide information to decision makers so adequate protective standards can be developed.
43. Publications
Approved (Signature)		Title	Date

U.S. Department of Agriculture AD-421 Progress Report (Investigator Copy) U.S. Dept. of Agriculture, State Agricultural Experiment Stations and Other Institutions			Date (Month, Day, Year) 12/31/2003
1. Accession 0194827	Agency Identification No. 2. CSREES 3. NY	5. Work Unit/Project No.	6. Status Progress
7. Title Mechanisms of Plant Responses to Ozone in the Northeastern US
12. Investigator Name(s) (Last Name and Intitials) McGrath, M.T.
20. Termination Date 09/30/2007		40. Period Covered (mo/da/year): 01/01/2003 TO 12/31/2003
41. Progress Report: (Example shown) Research conducted in 2003 was undertaken to test experimental protocols developed for the NE-1013 collaborative field project on ozone sensitive and tolerant cultivars of bean and to obtain additional information on the impact of ozone on plants growing on Long Island, NY. From 1 May to 30 Oct, ozone concentration was >80 ppb for 40 hrs on 8 days. The highest concentration (118 ppb) was reached on 27 June. This was the lowest level in 8 years. Ozone was >80 ppb for 60, 124, 121, 184, 77, at least 67, and 94 hrs in 1996, 1997, 1998, 1999, 2000, 2001, and 2002, respectively. Ozone caused acute, visible injury to leaves of the sensitive crops studied. Ozone-sensitive and ozone-resistant snap bean lines, S156 and R331, were grown in pots (seeded on 19 Jun) and in the field (27 Jun). In the field, weeds were managed by applying herbicides, Eptam and Treflan, on 17 Jun, mechanically cultivating and hand-weeding. Leafhoppers were managed by applying Di-Syston in furrow with the seed and Orthene to foliage on 15 Jul. Bean pods were harvested when immature for fresh-market consumption from some plants and when mature and dry from the rest of the plants. Ozone injury was first observed on 4 Aug when plants were flowering. Incidence of affected leaflets and severity of injury were similar for pot- and field-grown plants. Average incidence and severity on 18 Aug were 0.5% and 1% for pot-grown R331, 9% and 10% for field-grown R331, 34% and 30% for pot-grown S156, and 41% and 44% for field-grown S156. Fresh-market yield and dry bean yield were lower for S156 than R331 indicating that ambient ozone affected plant productivity. Weight of fresh-market pods was 39% lower for pot-grown S156 than R331 (P-value=0.035) and 35% lower for field-grown S156 (P-value=0.18). Number of fresh-market pods was 20% lower for both pot- and field-grown S156 compared to R331, but these differences were not statistically significant. Weight of mature seed was 31% lower (P-value=0.0007) for pot-grown S156 than R331 and 29% lower for field-grown S156 (P-value=0.067). Number of seed was reduced only for field-grown S156 (48%, P-value=0.039). Ozone-sensitive and ozone-resistant clones of the commercial white clover line ‘Regal’ were rooted from cuttings on 17 April, then moved outdoors and transplanted to 15-liter pots on 21 May. In contrast with previous years, the sensitive clone grew better after planting than the resistant clone, thus these plants were larger when transplanted yielded 44% more forage dry weight than the resistant clone on 23 Jun. Ozone injury was first observed on clover on 6 Jun. The sensitive clone was more severely injured, with 9.8% of leaves having symptoms and average severity on these leaves of 30.5% on 23 Jun, compared to 0.1% and 0.6%, respectively, for the resistant clone. Animal feeding subsequently prevented harvesting every 28 days. The forage dry weight ratio (sensitive/resistant) was 0.81 for tissue harvested on 4 Sep. This value is similar to that obtained in previous years when ozone concentrations have been high. In conclusion, although ambient ozone concentrations were lower than during the previous 8 years, ozone reached sufficiently high concentrations on Long Island in 2003 to affect productivity of sensitive snap bean and clover lines.
42. Impact Results will provide a fundamental understanding of ozone effects to individual plants as well as community and ecosystem responses. In addition, these results will provide information to decision makers so adequate protective standards can be developed.
43. Publications
Approved (Signature)		Title	Date

U.S. Department of Agriculture AD-421 Progress Report (Investigator Copy) U.S. Dept. of Agriculture, State Agricultural Experiment Stations and Other Institutions			Date (Month, Day, Year) 12/22/2003
1. Accession 0194827	Agency Identification No. 2. CSREES 3. NC	5. Work Unit/Project No.	6. Status Progress
7. Title Mechanisms of Plant Responses to Ozone in the Northeastern US
12. Investigator Name(s) (Last Name and Intitials) Neufeld, H.S.
20. Termination Date 09/30/2007		40. Period Covered (mo/da/year): 01/01/2003 TO 12/31/2003
41. Progress Report: During 2003, studies on the effects of ozone on native wildflowers in Great Smoky Mountains National Park continued. This past year, we continued our studies of the effects of stand structure on the deposition of ozone to leaves of cutleaf coneflower, Rudbeckia laciniata var. laciniata. This research was done at the Highlands Science Learning Center at Purchase Knob in conjunction with Dr. Pete Finkelstein, EPA. It appears that a simple model can explain much of the variation in ozone with depth into the canopy of these plants, and this may have great utility for modeling fluxes of ozone to individual leaves of plants in the field. In collaboration with Dr. Alan Davison, we have been continuing our work on the relationship between photosynthesis and chlorophyll fluorescence. There appears to be a strong correlation between maximum rates of photosynthesis and the amount of chlorophyll fluorescence, as expressed by the ratio of F_v/F_m. This may turn out to be a useful tool for identifying ozone impacts on plants in the field. Dr. Davison has also shown a very strong linear relationship between the leaf area index and the penetration of ozone into coneflower stands. For each unit increase in LAI, ozone drops by ~15%. This relationship holds for a variety of weather conditions, and is very similar to that developed for other species by Finkelstein. In addition to these studies, we have also completed a two year project on the effects of ozone on the growth of coneflowers at the greenhouse facilities at Appalachian State University. There, coneflowers were grown for two seasons in outdoor chambers receiving either filtered or non-filtered air. Each season, plants were rated for foliar injury and at the end of the second growing season, plants were harvested for biomass determinations. Results indicate little or no effect of ambient ozone on biomass accumulation, but there were small differences in foliar injury amounts, with those in filtered air having higher injury ratings. We could not detect any differences in any responses between sensitive and non-sensitive individuals in the chambers. Additional gas exchange studies were done at Purchase Knob on sensitive and insensitive individuals of coneflower. Preliminary investigations of the sensitivity of stomatal conductance to vapor pressure deficit showed large decreases in conductance as the vapor pressure deficit increased. These data will provide the necessary parameters to model the responses of leaf-level gas exchange to changing microclimatic conditions, furthering our ability to predict ozone uptake by these plants. Lastly, we have measured the impacts of foliar injury on the absorbance and transmission of light in leaves of these plants. When injury ratings are 5 or 6, absorbance is greatly reduced compared to leaves with no injury, particularly from 500 to 700 nm, while transmission is concomitantly increased. Over the entire photosynthetically active spectrum (400-700 nm) the amount of energy absorbed is reduced by 50%. This shows that not only does ozone cause foliar injury to leaves, it also greatly alters the energy balance of leaves, which may have direct effects on leaf temperature regulation, transpiration and photosynthesis.
42. Impact Our results will provide a fundamental understanding of ozone effects to individual plants as well as community and ecosystem responses. In conjunction with studies at the leaf and stand levels, others in our research group are investigating biochemical responses and trophic interactions, such that our studies should greatly add to the knowledge base about the impacts of ozone on native wildflowers and their herbivores. In addition, these results will provide information to decision makers so adequate protective standards can be developed.
43. Publications Chappelka, A.H., H.S. Neufeld, A.W. Davison, G.L. Somers and J.R. Renfro. 2003. Ozone injury on cutleaf coneflower (Rudbeckia laciniata) and Crown-beard (Verbesina occidentalis) in Great Smoky Mountains National Park. Environ. Pollut. 125: 53-59. Davison, A. W., H. S. Neufeld, A. H. Chappelka, K. Wolff, and P. L. Finkelstein, 2003. Interpreting spatial variation in ozone symptoms shown by cutleaf coneflower, Rudbeckia laciniata L. Environ. Pollut. 61-70.
Approved (Signature)		Title	Date

U.S. Department of Agriculture AD-421 Progress Report (Investigator Copy) U.S. Dept. of Agriculture, State Agricultural Experiment Stations and Other Institutions			Date (Month, Day, Year) 12/31/2003
1. Accession 0194827	Agency Identification No. 2. CSREES 3. VA	5. Work Unit/Project No.	6. Status Progress
7. Title Mechanisms of Plant Responses to Ozone in the Northeastern US
12. Investigator Name(s) (Last Name and Intitials) Chevone, Boris
20. Termination Date 09/30/2007		40. Period Covered (mo/da/year): 01/01/2003 TO 12/31/2003
41. Progress Report: (Example shown) Photosynthetic function (Pn) in ozone-sensitive (BCS) and ozone-tolerant (BCT) black cherry trees were measured in July and August in Giles County, VA during 2003. Irradiance-assimilation and CO₂-assimilation curves were generated to assess the effect of ambient ozone levels on the photosynthetic-carbon fixation apparatus in sensitive trees. Ozone levels were low throughout the summer with daily peak hour concentrations in the 50 to 60 ppb range. A late June episode produced peak levels of 100 ppb for a few hours. Foliar injury was first apparent on sensitive trees in mid July and increased moderately throughout the summer. Maximum Pn at ambient CO2 was 35% less in BCS compared to BCT in July. From July to August, quantum efficiency for CO₂ fixation declined by 16 % in BCS and by 12% in BCT. Carboxylation efficiency in BCT and BCS did not differ between July and August but was 25% lower in the sensitive genotypes. Maximum Pn at saturating CO₂ levels was 65% higher in BCT than BCS in August. The effect of ambient ozone on BCS photosynthetic function was less than that observed in 2002 when ozone concentrations were considerably higher. However, these data demonstrate that even low ozone levels can damage both CO₂ assimilation processes in sensitive black cherry in the forests of western Virginia.
42. Impact Ambient ozone, at relatively low concentrations, can significantly reduce photosynthetic function in leaves of sensitive black cherry trees in western Virginia.
43. Publications None
Approved (Signature)		Title	Date

U.S. Department of Agriculture AD-421 Progress Report (Investigator Copy) U.S. Dept. of Agriculture, State Agricultural Experiment Stations and Other Institutions			Date (Month, Day, Year) 12-31-03
1. Accession 0194827	Agency Identification No. 2. USDA 3. Federal	5. Work Unit/Project No.	6. Status Progress
7. Title Mechanisms of Plant Responses to Ozone in the Northeastern US
12. Investigator Name(s) (Last Name and Intitials) Booker, F.L.
20. Termination Date 09/30/2007		40. Period Covered (mo/da/year): 01/01/2003 TO 12/31/2003
41. Progress Report: (Example shown) Recent studies suggest that ozone reacts in the extracellular space of plant leaves to form activated oxygen species that can be propagated by the plant. If not effectively scavenged by plant metabolites, toxic metabolites are formed and plant responses are induced that compound plant injury. Since GTPases (G-proteins) are involved in induced plant defense responses, Arabidopsis thaliana G-protein mutants were tested for ozone sensitivity. After exposure to ozone concentrations ranging from 0 to 250 ppb for five days, the leaf epinasty usually occurring in Columbia wild type plants was not present in null mutants for the alpha subunit of the heterotrimeric G-protein. Leaf mass per unit leaf area of the G-protein mutants was suppressed less by ozone exposure compared with wild type plants and was not due to lower ozone flux. It is possible that plant responses are mediated by phytohormone interactions involving G-proteins. However, these results indicate, for the first time, that G-proteins are critically involved in plant responses to ozone.
42. Impact Our results will provide a fundamental understanding of ozone effects to individual plants as well as aid in the development of ozone-tolerant plants. In addition, these results will provide information to decision makers so adequate protective standards can be developed.
43. Publications Fuhrer, J. and F.L. Booker. 2003. Ecological issues related to ozone: agricultural issues. Environment International 29: 141-154.
Approved (Signature)		Title	Date

U.S. Department of Agriculture AD-421 Progress Report (Investigator Copy) U.S. Dept. of Agriculture, State Agricultural Experiment Stations and Other Institutions			Date (Month, Day, Year) 12/31/2003
1. Accession 0194827	Agency Identification No. 2. USDA 3. Federal	5. Work Unit/Project No.	6. Status Progress
7. Title Mechanisms of Plant Responses to Ozone in the Northeastern US
12. Investigator Name(s) (Last Name and Intitials) Burkey, K.O.
20. Termination Date 09/30/2007		40. Period Covered (mo/da/year): 01/01/2003 TO 12/31/2003
41. Progress Report: (Example shown) Significant variation in ozone sensitivity was observed for three snap bean genotypes based on mid-season foliar injury and suppression of final pod dry mass. Plants were grown in pots in open-top chambers under charcoal-filtered (CF), non-filtered (NF), and ambient air (AA) treatments. The ozone impact was greatest for S156, moderate for Oregon-91, and was not significant for R123. For the most sensitive genotype S156, an increase in ozone from 30 ppb (CF seasonal mean) to 50 ppb (AA seasonal mean) was sufficient to reduce pod yield by 50%, suggesting that the snap bean system has the potential to detect ambient ozone effects at present-day ozone concentrations. Ozone tolerant tobacco (Bel B, Bel C), ozone-sensitive tobacco (Bel W3), and a supersensitive tobacco clone (SS) isolated in the laboratory of John Sun were selected from tissue culture for comparison of leaf antioxidant status. Plants were rooted in Metro-Mix 200 and grown in a greenhouse. Exposure to 100 ppb ozone for 5 hours in greenhouse chambers induced flecking injury in the Bel W3 and SS clones, but not Bel B and Bel C. The differential ozone response was not related to leaf ascorbic acid and glutathione contents because all clones contained similar levels. Reduced ascorbate in the leaf apoplast was very low in all clones relative to total antioxidant capacity, suggesting a role for unidentified antioxidant compounds in the cell wall.
42. Impact Our results will provide a fundamental understanding of ozone effects to individual plants. In addition, these results will provide information to decision makers so adequate protective standards can be developed.
43. Publications Burkey, K.O., G. Eason, E.L. Fiscus (2003) Factors that affect leaf extracellular ascorbic acid content and redox status. Physiologia Plantarum 117: 51-57.
Approved (Signature)		Title	Date

U.S. Department of Agriculture AD-421 Progress Report (Investigator Copy) U.S. Dept. of Agriculture, State Agricultural Experiment Stations and Other Institutions			Date (Month, Day, Year) 12/31/2003
1. Accession 0194827	Agency Identification No. 2. USDA FS 3. Federal	5. Work Unit/Project No. RWU 4451	6. Status Progress
7. Title Mechanisms of Plant Responses to Ozone in the Northeastern US
12. Investigator Name(s) (Last Name and Intitials) Bytnerowicz, A.
20. Termination Date 09/30/2007		40. Period Covered (mo/da/year): 01/01/2003 TO 12/31/2003
41. Progress Report: (Example shown) During 2003, we continued analyzing data from the 2002 monitoring of ambient ozone, nitric acid and ammonia with passive samplers in selected areas of the Sierra Nevada and the San Bernardino Mountains during photochemical smog season (May – October). In 2003, an air pollution monitoring network for ozone, nitric acid, ammonia and nitrogen oxides with passive and active monitors was developed for the ongoing King’s River Project conducted by the Forest Service PSW Research Station (west-central part of the Sierra Nevada). Pollution monitoring in that study was conducted during the June-October period and will be continued in future years. The San Bernardino monitoring network was extended from 12 to 18 locations in the 2003 season. Our preliminary results show: (1) elevated levels of ozone and nitric acid in the Lake Tahoe Basin are mostly caused by local generation, (2) ozone moves across Sierra Nevada along the San Joaquin River Valley and across southern Sierra Nevada near Lake Isabella, (3) elevated concentrations of ozone in the eastern Sierra Nevada are caused by long-range transport of polluted air masses, (4) elevated levels of ozone, nitric acid and ammonia in large areas of the Sierra Nevada are caused by forest fires (McNalley and Walker fires), agricultural activities and traffic in the California central Valley, and (5) complex patterns of ozone and nitrogenous pollutants distribution in the San Bernardino Mountains are quite different from the patterns observed in the 1970s and 1980s.
42. Impact Our results provide new information on landscape-level distribution of ambient ozone and nitrogenous pollutants in the California mountain forests and other ecosystems. Our data has high importance for understanding patterns of air pollution distribution in complex mountain terrain and in areas where traditional electronic air pollution monitoring equipment cannot be used. Such information is needed for scientists, land managers and air quality specialists responsible for managing federal lands. Our results help to evaluate potential risks caused by air pollutants to health of forests and other ecosystems as well as people residing and visiting California mountains. Our data will also be used for recommendations regarding revisions of air pollution regulations in California.
43. Publications: A. Bytnerowicz, M. Arbaugh, R. Alonso (eds.) 2003. Ozone Air Pollution in the Sierra Nevada: Distribution and Effects on Forests. Elsevier, Developments in Environmental Science 2, Amsterdam, 402 pp, ISBN 0 08 044193 9.
Approved (Signature)		Title	Date

U.S. Department of Agriculture AD-421 Progress Report (Investigator Copy) U.S. Dept. of Agriculture, State Agricultural Experiment Stations and Other Institutions			Date (Month, Day, Year) 12/31/2003
1. Accession 0194827	Agency Identification No. 2. USDA 3. Federal	5. Work Unit/Project No.	6. Status Progress
7. Title Mechanisms of Plant Responses to Ozone in the Northeastern US
12. Investigator Name(s) (Last Name and Intitials) Robinson, M.
20. Termination Date 09/30/2007		40. Period Covered (mo/da/year): 01/01/2003 TO 12/31/2003
41. Progress Report: (Example shown) The soybean cultivar Forrest was more ozone sensitive than Essex when compared on the basis of photosynthetic rates in mature leaves. This appeared to be because of the ability of Essex to maintain a higher foliar ascorbate to dehydroascorbate redox status than could Forrest. The research provided whole plant-whole leaf evidence implicating the role of the ascorbate-glutathione enzyme cycle in protecting the photosynthetic apparatus from ozone-induced active oxygen species. We also collaborated with Dr. John Lydon, Sustainable Agricultural Systems Laboratory, USDA-ARS Beltsville to examine the role of photosynthesis in maintaining levels of ascorbic acid in leaf cells of sunflower plants. The results were interpreted to imply that carbohydrate products of photosynthetic carbon dioxide assimilation such as glucose, and one or more functions of mitochondrial respiration, are concurrently required for the maintenance of normal levels of ascorbic acid in green leaves. In collaboration with Dr. Richard Sicher, USDA-ARS Beltsville, Alternate Crops Systems Laboratory, we demonstrated that exposure of 2 to 3 week old d barley seedlings to elevated CO₂ lowers the levels of the leaf antioxidants ascorbic acid and reduced glutathione in the primary leaves. This has proven to be because the primary leaves of barley senesce more rapidly during exposure to elevated CO₂ compared with plants which were concurrently exposed to ambient levels of CO₂. Ascorbate levels are lower because photosynthetic CO₂ assimilation and photosynthetically generated glucose production, apparently required to support ascorbate precursor levels, are also reduced over time. This work again contributes evidence supporting the hypothesis that photosynthetic carbon dioxide assimilation is required to support the maintenance of ascorbic acid levels. The study demonstrates that exposure of some plants to elevated CO₂ is harmful to plants; in this case, elevated CO₂ level must be viewed as an air pollutant. Further, it is now known that wheat and barley appear to be species of plants that are negatively influenced by elevated CO₂ during there early growth stages. An experiment was conducted to evaluate the role of cell wall phenolic compounds in O₃ tolerance in soybean cultivars and to investigate the long-term effects of elevated O₃ on leaf cell wall phenolics in two cultivars of soybean, cv Essex (O₃ tolerant) and cv Forrest (O₃ sensitive). Experiments were conducted to determine if a relationship exists between the differential sensitivity of these two cultivars to O₃, and postulated differential antioxidant enzyme activity and differential oxidative modification of phenolic cell wall components. Soybean cv Forrest (O₃-sensitive) consistently had higher quantities of p-coumaric and ferulic acids and lower quantities of the benzaldehyde isomer in the cell wall bound fraction as compared to cv Essex. Exposure to O₃ caused an increase in the quantities of the cell wall bound phenolic compounds.
42. Impact Our results will provide a fundamental understanding of ozone effects to individual plants. In addition, these results will provide information to decision makers so adequate protective standards can be developed.
43. Publications
Approved (Signature)		Title	Date