NE-176: Characterization and Mechanisms of Plant Responses to Ozone in the Northeastern U.S.

Technical Committee Report of Progress and Accomplishments in 1999

Annual Progress Report: January 1, 1999 – December 31, 1999

COOPERATING AGENCIES & PRINCIPAL LEADERS: C. Andersen (US EPA), F. Booker (USDA-ARS, NC), S. Britz (USDA-ARS, MD), K. Burkey (USDA-ARS, NC), A. Chappelka (AL), B. Chevone (VA), A. Heagle (USDA-ARS, NC), R. Kohut (Boyce Thompson Institute, NY), S. Krupa (MN), W. Manning (MA), M. McGrath (NY, LI), J. Miller (USDA-ARS, NC), C. Mulchi (MD), E. Pell (PA), J. Rebbeck (USDA Forest Service, OH), M. Robinson (USDA-ARS, MD), J. Sinn (PA), B. Zilinskas (NJ).

Advisors: P. Logan (RI), D. Jones (USDA-CSREES)

PROGRESS OF THE WORK & PRINCIPAL ACCOMPLISHMENTS, January - December, 1999

Objective 1. Characterize whole plant responses to O₃, including carbon assimilation and allocation, growth and productivity.

Carbon budgets in ponderosa pine forests. Carbon budgets for two ponderosa pine stands in Eastern Oregon that differ in age (>250 yrs vs 20 yr old) were constructed in collaboration with researchers at Oregon State University. Eddy flux measurements, estimates of root productivity, and the contribution of deep and shallow water sources were evaluated over the growing season. Stable isotopes were used to separate soil heterotrophic from autotrophic respiration. The results provide important inputs for tree and stand level models that are being used to evaluate ozone effects at the ecosystem level. (C. Andersen, US EPA)

Root growth in forests. A US Forest Service collaboration on several sites across a natural ozone gradient in Southern California showed that root growth is lowest at the most polluted site. (C. Andersen, US EPA)

Ecosystem response using native species. Exposure-response relationships between ambient ozone and visible injury on mature black cherry in two National Parks in the eastern US were developed. Percent injured trees increased with elevation, and was positively correlated with SUM06 and W126. The relationship was strengthened significantly by combining the data from both parks. A new study has been initiated investigating the response of tall milkweed to ozone along an elevational gradient in the Great Smoky Mountains National Park. A study is being developed to investigate the effects of ozone on semi-natural grassland communities. (A. Chappelka, AL)

Modeling ozone impacts on forest growth and composition. Research involves the use of mechanistically-based simulation models to address the long-term, complex impacts of air quality and climate change on forest growth and composition. Effects of air quality on plant population genetics is being investigated. An on-going study is assessing the effects of escalating levels of CO₂ on the phenotypic, physiological, and genetic properties of plant populations over 10 generations of exposure. (R. Kohut, BTI, NY)

Ozone exposure dynamics. A stochastic, Weibull frequency distribution, probability generator was tested and applied for replacing missing data on hourly atmospheric concentrations of ozone and other trace gases (oxides of nitrogen and sulfur dioxide). The method provided highly accurate and realistic results for the replacement of as many as 100 missing values on either side of a measured data sub-set, without altering the overall characteristics of the true frequency distribution of the entire data set. This method will be used independently to theoretically reconstruct the exposure dynamics underlying single, time-integrated values obtained by the use of passive samplers. (S. Krupa, MN)

Snap bean and white clover yield loss. Ambient ozone was shown to be high enough to greatly reduce growth and yield of sensitive plants on Long Island, one of the most important agricultural counties in New York. Sensitive snap bean lines exhibited yield reductions of 25% in weight of beans harvested for fresh-market consumption and 40% in number of seeds at maturity compared to ozone-tolerant snap beans. Growth of an ozone-sensitive white clover clone was reduced by an average of 27% compared to an ozone-tolerant clone from May through early Sep, then the two clones grew at a similar rate when ozone concentrations were lower. Average daily 12-hr ozone mean was 52.5, 48.4, 62.0, 48.9, and 35.9 ppb for the five growth periods. (M. McGrath, NY)

Screening soybean for ozone tolerance. Twenty-four cultivars of soybean (eight each from maturity groups III, IV and V) were grown full-season in 5-m diameter open-top chambers purged continuously with charcoal filtered (CF) air or non-filtered (NF) air + 35 ± 5 nL O₃ L^-1 for 7 h day^-1. In addition to grain yields per plant at maturity, leaf discs were examined for relative changes in chlorophyll content and ion leakage in response to chronic ozone exposure. The goals of the program are: 1) screen modern soybean cultivars for their relative O₃ tolerance in terms of productivity; and 2) develop a leaf assay procedure that may be useful to assess the relative tolerance of soybeans to O₃ stress. The studies will be repeated in 2000. (C. Mulchi, MD)

Objective 2. Identify & delineate factors, both biotic & environmental, that determine plant O₃ response.

Ponderosa pine competitiveness. In a three-year open top chamber study, ponderosa pine seedling growth was reduced by the combination of ozone and grass competition relative to grass competition alone. The reduced competitiveness was associated with altered nitrogen metabolism because ozone-exposed seedlings were unable to either take up or retain as much nitrogen when growing in the presence of grass as when growing alone. (C. Andersen, US EPA)

Tree species O₃/CO₂ interaction responses. Growth was significantly stimulated by twice ambient CO₂ in seedlings of black cherry, green ash, and yellow-poplar; although there were significant growth responses following exposure to twice ambient O₃ or the combination of twice ambient CO2 plus twice ambient O₃. Interactive effects of O₃ and CO₂ on photosynthesis and stomatal conductance on all three species were limited. Following a 5-year exposure of white pine to elevated ozone and carbon dioxide, analysis of many growth and biomass variables showed that ambient and twice ambient O₃ reduced seedling biomass relative to CF seedlings, and additions of twice ambient CO₂ ameliorated those effects. Findings support the hypothesis that future productivity of eastern US forests will not be negatively impacted by elevated O₃ in the presence of elevated ambient CO₂. (J. Rebbeck, USDA Forrest Service)

Ozone/CO₂ interaction mechanisms. A major factor in O₃/CO₂ interactions is reduced ozone flux into leaves in the presence of elevated CO₂. In soybean, amelioration of ozone injury by elevated CO₂ also appeared to involve additional physiological and biochemical responses. However, there was no measurable effect of elevated CO₂ on antioxidant metabolism. Only ozone injury correlated with increased ascorbate, glutathione, glutathione reductase and peroxidase activities. Superoxide dismutase activities were not affected by either ozone or elevated CO₂. (F. Booker, ARS, NC)

Environmental factors that influence ozone uptake. A series of field experiments has resulted in determination of some important environmental factors that control ozone uptake by plants and hence expression of foliar injury under real-time ambient conditions. The degree of solar radiation intensity (PAR at and above 500 mmol m^-2 s^-1), available soil moisture (greater than 25 %), ozone concentration and degree of stomatal conductance determine ozone uptake and result in foiliar ozone injury to Bel-W3 tobacco. (W. Manning, MA)

Objective 3. Determine the mechanisms of O3 action and plant defense systems, using cultivars and genotypes characterized in whole plant experiments.

Glutathione-S-transferase associated with ozone tolerance. Ozone exposure at 200 ppb for 4 hrs caused comparable reductions in maximum net photosynthesis (35-42%) and apparent quantum yield (15-27%) in a tolerant (Bel B) and sensitive (Bel W3) tobacco cultivar immediately after fumigation. By 24 hrs, photosynthetic efficiency had recovered in Bel B, but not in Bel W3, demonstrating that tolerant genotypes are significantly affected by ozone. The mRNAs of two glutathione-S-transferase genes increased more than tenfold in Bel B, but were unchanged in Bel W3 after ozone treatment, indicating a possible role for ozone tolerance for this enzyme. (B. Chevone, VPI)

Drought effects on leaf ascorbic acid. During the summer of 1999, we examined the influence of drought-induced water deficit on the steady-state levels of ascorbic acid (ASC), dehydroascorbate (DHA) and the ASC:DHA redox status in leaflets of the soybean cultivars Essex and Forrest. This approach was taken as a first step into the eventual examination of the influence of simultaneous drought stress and elevated ozone exposure on soybean mesophyll leaf cell ASC synthesis and redox turnover. In this present study, done with plants grown and drought stressed for 5 to 7 days either in the growth chamber or in the greenhouse, low leaflet water potential values (‰ -3.00 to -3.95 megaPascals) were accompanied by little or no loss of ASC and maintenance of most of the total ascorbate (ASC+DHA) as ASC. The results suggested that during drought-induced water stress, activities of enzymes associated with ASC synthesis and ASC-DHA redox turnover were sufficient to sustain ASC levels. (M. Robinson, ARS, MD)

Cellular localization of ascorbic acid associated with ozone tolerance. A comparison of antioxidant metabolites (ascorbic acid, glutathione and a-tocopherol) in ozone-sensitive and tolerant snap beans showed that high levels of leaf extracellular ascorbate are associated with ozone tolerance, presumably through protection of plasma membranes from damage via antioxidant reactions that detoxify ozone and activated oxygen intermediates. (K. Burkey, ARS, NC)

Transgenic plants for testing the role of ascorbic acid in ozone tolerance. We have initiated a new collaborative project with Dr. M. Robinson (ARS, MD), Dr. K. Burkey (ARS, NC) and Dr. S. Jones-Held (King's College). Ascorbate (vitamin C) serves as an important antioxidant, and recent evidence suggests that it is critical for providing protection against ozone damage. Our plan is to produce transgenic plants with altered ascorbate content by manipulating the expression of two enzymes in the ascorbate biosynthetic pathway, namely GDP-D-mannose pyrophosphorylase and L-galactono-1,4-lactone dehydrogenase. We have made sense and antisense constructs for each of these enzymes and will soon be introducing them into plants to test the hypothesis that higher ascorbate content will provide tolerance to ozone and conversely lower ascorbate content will deem plants more ozone-sensitive. (B. Zilinskas, NJ)

Ozone responses elicited by cell wall components. Cell wall oxidation products were recovered from tomato leaves after ozone exposure. Infiltration of these compounds into leaves can induce the expression of ozone-responsive genes without producing visible leaf injury. (E. Pell & J. Sinn, PA)

Role of ethylene in plant response to ozone. In Arabidopsis, the expression of ozone-responsive genes was not consistently associated with the evolution of ethylene by exposed plants. Potato plants which under-express selected genes in the ethylene biosynthetic pathway are being used to further examine ethylene’s role in plant ozone response. (E. Pell & J. Sinn, PA)

Ozone effects on stomatal function. Studies of membrane ion channels and whole-plant physiology in V. faba have demonstrated that ozone can have direct effects on stomatal function distinct from impacts on the photosynthetic apparatus. (E. Pell & J. Sinn, PA)

USEFULLNESS OF FINDINGS: Tree species and crop cultivars for use as biological monitors of ambient ozone have been identified. Knowledge of factors that affect plant response to ozone (e.g. soil moisture, elevated CO₂, altered plant competition) is required to develop response models. Predictive models are needed by regulatory agencies to establish air quality standards for the protection of crops and natural ecosystems. Information about the molecular basis of plant response to ozone is required to effectively use biotechnology as an approach in the development of ozone tolerant plants.

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