Annual Report 1998

INSTITUTIONAL ACTIVITIES

Barbara Zilinskas - Rutgers University:

We have initiated two new collaborative projects dealing with the response of plants to ozone. Briefly, the first project, a collaboration with Dr. C. Mulchi, is a characterization of antioxidants in two wheat cultivars grown in open-top chambers in response to variations in atmospheric ozone, carbon dioxide and soil moisture. The field work and sampling was done in Beltsville during the spring-summer of 1996 and 1997. Statistical analyses of yield, stomatal conductivity for one sampling period, and monodehydroacorabate reductase and glutathione reductase activity have been completed for all 1997 sampling periods. Ascorbate peroxidase activity and AsA/DHA and GSH/GSSG ratios will be measured soon. The second project is a collaboration with Dr. T. Leustek. We found that exposure of plants to ozone induces a rapid and large increase in 5'-adenylsulfate (APS) reductase activity. The APS reductase activity correlated directly with the oxidized state of the glutathione pool and inversely with the level of cysteine and total glutathione. Our work suggests that redox regulation of APS reductase may provide a mechanism for rapid response to oxidative stress.

Boris Chevone - Virginia Tech (VPI):

Two clones of white clover, (cv. Regal), one that is ozone sensitive (NC-S) and one that is ozone tolerant (NC-R) have been utilized to investigate mechanisms involved in oxidative tolerance. During the past several years, clover has been grown in pots at the Horton Research Center, Giles County, VA and foliage/stem tissue was harvested at 28-day intervals. The ratio of NC-S/NC-R dry forage biomass was calculated for growing periods during the summer. The clover clones were grown under the same conditions at two other sites, Amherst, MA and Raleigh, NC. Forage dry mass was analyzed to determine more fully the performance of the two clones as indicators of ozone-induced plant growth reductions on a regional scale. There was no clear decreasing trend in dry mass ratios from harvest to harvest, indicating no residual biomass response to ambient ozone concentrations. Differential variation in forage ratios relative to cumulative ozone exposures indicated that environmental factors, specific to each site, modulated the dominant ozone effect. The nature of these growth regulating components is not known.

Two clones of clover also have been utilized examining biochemical mechanisms of oxidative tolerance. We have identified 2-4 low molecular mass proteins that are induced by ozone to a greater extent in NC-R than in NC-S. Amino acid analysis of the most abundant, gel-purified protein indicated that the N-terminal amino acid was blocked and a sequence could not be obtained. In-gel protease digestion of this protein produced four major fragments; however, the peptide yield was insufficient for amino acid sequencing. Because of the difficulty in regenerating and transforming clover, research of ozone tolerance mechanisms has shifted to tobacco cultivars Bel-W3 and NC88 (ozone sensitive), Bel-B (ozone tolerant) and Xanthi (moderately ozone tolerant). We have examined HMGR promoter:GUS fusions transformed into Xanthi and have observed promoter activation in response to 200 ppb ozone exposure for 3 hr. GUS activity was localized around necrotic lesions on the leaf tissue.

Mike Robinson and Steve Britz - USDA-ARS-BARC:

A study was continued to further determine whether there was a positive correlation between the ability of soybean plants to remain productive during prolonged exposure to moderate ozone levels and the level of ascorbate in the leaflets of those plants. In studies conducted in the summer of 1997 at Beltsville, an O₃-tolerant soybean cultivar (cv Essex)

and an O₃-sensitive cultivar (cv Forrest) were grown, concurrently, from the seedling stage through bean harvest. Plants were grown in soil plots within open‑top chambers aerated continuously either with carbon-filtered air (CF) (average daily O₃ was »30 ppb), or with non‑filtered air supplemented with O₃ (NF+ O₃) (average O₃ was »60 ppb). During the same period, both cultivars were grown in ambient air (AMB) in plots just adjacent to the chambers. Elevated O₃ exposure decreased both the biomass per plant and the bean yield per plant of cv Forrest by approximately 20%, while neither biomass yield per plant nor the bean yield per plant of cv Essex appeared to be affected by O₃. Upper canopy leaflets of plants of both cultivars were sampled at the vegetative, flowering and pod‑fill stages. On designated days of each growth stage, leaf disk samples were excised from mature leaflets at »0830, »1130, and »1430 hrs. Ascorbate (ASC) and dehydroascorbate (DHA) were measured enzymatically in leaflet disk extracts. At each each growth stage, CF, NF+O₃, and AMB cv Essex leaflets were able to maintain a high steady‑state ASC level and ASC mole fraction (»0.8‑0.9), especially during the mid-day to afternoon period. In comparison with cv Essex plants, leaflets of CF, NF+O₃ and AMB cv Forrest plants did not maintain as high a leaflet steady‑state ASC level and ASC mole fraction (»0.6‑0.7) during the mid-day to afternoon period. It is concluded that cv Essex was more vegetatively and reproductively successful than cv Forrest during prolonged O₃ exposure, in part, because cv Essex leaflet cells had a higher steady‑state ASC level. The higher steady-state ASC/DHA redox status in cv Essex leaflets, compared with that of cv Forrest leaflets, was interpreted to reflect that there was a more rapid turnover of DHA to ASC in cv Essex leaflet cells. Further, the results are interpreted to indicate that during O₃ decomposition to reactive oxygen species, cv Essex leaflet cells were better able to meet the demands for ASC exerted by the ascorbate‑glutathione cycle in the process of removing the H₂O₂.

Meg McGrath - Cornell University:

Plant response to ambient ozone on Long Island, NY was examined by growing ozone-sensitive and ozone-tolerant white clover (from Heagle, NC) and snap beans. Ozone was ³ 80 ppb for 121 hrs between 1200 and 2400 on 30 days in 1998: 16, 28, and 30 May; 2, 13, 16, 17, 20, 25, and 26 June; 4, 13, 20-23, and 29 July; 3, 4, 18, 21, 23-25, 29, and 30 Aug.; 6, 7, 12, and 27 September. The highest concentration (129 ppb) was reached on 26 June. Ozone reduced growth of the sensitive clover clone starting in June. Symptoms of ozone injury (white flecking) were observed on leaves of the sensitive clone on 18 June. Forage dry weight ratios (sensitive/resistant) were 1.03, 0.76, 0.72, 0.74, and 0.82 for tissue harvested from clover growing in pots on 5 June, 2 July, 30 July, 28 August, and 29 September, respectively. Thus, the two clones were growing at the same rate in May, then growth of the sensitive clone was reduced by an average of 24% for the rest of the season. Ozone had a similar impact on field-grown clover (growth reduction of 27%). The 12 h (1000-2200) means were 0.048, 0.049, 0.052, 0.046, and 0.043 for the five growth periods. Foliar injury and yield were compared for 14 snap beans. Pods were harvested for fresh market yield from some plants and for mature seed from others. Ozone-induced injury was significantly more severe on Oregon 91, ozone-sensitive variety, and four of five ozone-sensitive lines (from Reinert, NC) than on three ozone-tolerant varieties and three of five ozone-tolerant lines. Ozone had the greatest impact on mature seed weight, which averaged 34% less for the ozone-sensitive beans than for the ozone-tolerant beans (mean dry weights were 0.158 g and 0.238 g, respectively). Only one of the ozone-tolerant lines did not have significantly heavier seed than all of the ozone-sensitive beans. Ozone-sensitive lines did not produce significantly fewer pods than the ozone-tolerant lines, but the weight of pods harvested for fresh market was 21% lower. These studies document that ozone levels were high enough to greatly reduce growth and yield of sensitive plants on Long Island where the most important agricultural county in the state is located.

Charles Mulchi, Moon Kim and Tanya Chernikova - University of Maryland,

College Park:

Modeling Gaseous Exchange in Soybean

Using leaf photosynthesis, stomatal conductance, transpiration rates, ambient and elevated chamber O₃ and CO₂ concentrations obtained during field studies with soybeans conducted during the summers of 1994 through 1997, gas exchange responses were modeled under well-watered and moisture-deficient conditions. The results were combined over cultivars. Under high moisture, Pn rates were increased linearly in response to CO₂ and decreased linearly in response to elevated O₃ during pre- and post-flowering. Under low moisture, Pn responses behaved similarly to high moisture conditions during pre-flowering but were strongly curvilinear downward under post-flowering with increased O₃ concentration at all CO₂ concentrations. Stomatal conductance showed slight curvilinear downward patterns with increases in both CO₂ and O₃ during pre-flowering under high moisture, but showed linear increases in response to both gases during post-flowering. Under low moisture, r_s values were curvilinear downward in response to elevated O₃ at all CO₂ levels and curvilinear upward under elevated CO₂ during pre-flowering but were typically reversed under post-flowering. O₃ uptake or flux into the leaves increased linearly with O₃ concentrations during pre-flowering at all CO₂ concentrations and were only slightly decreased in response to elevated CO₂. However, during post-flowering, O₃ uptake rates were curvilinear with increased O₃ under low O₃ and high moisture but downward under high O₃ and low moisture, clearly illustrating the effects of reduced stomatal conductance during post-flowering under low moisture. Transpiration (E) values were largely decreased in response to elevated CO₂ under low O₃ during both pre- and post-flowering but were unaffected by CO₂ under elevated O₃. However, under low moisture, E values were curvilinear downward in response to increased CO₂ at all O₃ levels during pre-flowering but strongly curvilinear downward in response to O₃ at all CO₂ levels. WUE values were increased in response to CO₂ at all O₃ levels with the sharpest increases occurring during low moisture and moderate to low O₃ conditions. Under high moisture, WUE values were largely unaffected by O₃ concentration but were slightly increased by O₃ under low CO₂ and low moisture during post-flowering, suggesting O₃ has a role in water economy during drought.

Leaf Fluorescence

The primary objective was to characterize fluorescence responses as a noninvasive detection method for crop species subjected to elevated atmospheric CO₂ and O₃ under two soil moisture regimes. A laboratory-based fluorescence imaging system (FIS) was developed to capture and process images of plant leaves at the blue (F450), green (F550), red (F680), and far-red (F740) regions of the spectrum. Full surface imaging was superior to point source measurements in assessing fluorescence characteristics of plant leaves.

Field experiments were conducted at the USDA Beltsville Agricultural Research Center during 1997 and 1998 using soybean cultivars “Essex” and “Forrest” grown full-season in open-top chambers exposed to four combinations of tropospheric CO₂ and O₃ under well-watered (WW) vs. restricted (RM) conditions. The gaseous environments included: 1) Charcoal-filtered (CF) ambient air; 2) CF air plus 150±10 mL L^-1 CO₂; 3) Non-filtered (NF) ambient air plus 35 ± 5 nL L^-1 O₃; and 4) NF air plus 150 ± 10 mL L^-1 CO₂ and 35 ± 5 nL L^-1 O₃. Soil moisture levels were 0 to -0.05 Mpa (WW) vs. -1.0 ± 0.5 Mpa (RM).

The results from this investigation demonstrated that chronic exposures of soybean leaves to air quality treatments varying in O₃ and CO₂ concentrations produced significant alterations in steady-state fluorescence values at F450, F550, F680 and F740 even in the absence of visual symptoms or significant changes in gas exchange parameters. Likewise, soil moisture treatments caused significant changes in fluorescence responses throughout the spectrum. The FIS detected the effects of elevated O₃, partial compensation of elevated O₃ effects in response to elevated CO₂, and positive physiological effects of elevated CO₂ on plants under WW and RM regimes. Cultivar sensitivities in response to air quality and soil moisture treatments were also differentiated with the FIS. Among the more significant findings in this investigation was that blue-green fluorescence emission from leaves was highly responsive to oxidative O₃ stress at levels that were sub-lethal.

On the basis of findings from this research in conjunction with advances in detector and laser technologies, a laser-induced fluorescence imaging system (LIFIS) capable of in situ canopy level measurements was proposed and is currently being developed jointly by NASA/GSFC and USDA/BARC.

Future research considerations are addressed for the use of steady-state fluorescence as a viable early remote detection method for assessing plant stress factors.

Joe Miller, Al Heagle, Fitz Booker and Kent Burkey - USDA-ARS - Raleigh, NC:

Field experiments to determine combined effects of elevated CO₂ and O₃ were performed with two wheat cultivars — one O₃-sensitive (NK-9904) and one O₃-resistant (NK-9835). Plants were grown in open-top chambers and treated with all combinations of three CO₂ and three O₃ concentrations in a replicated design. Carbon dioxide treatments [374 (ambient), 541, and 707 ppm] were given for 24 hr/day to obtain designated concentrations. Ozone treatments were charcoal-filtered air (CF; 27 ppb), nonfiltered air (NF; 44 ppb), and NF with O₃ added in proportion to ambient O₃ (NF+; 87 ppb) for 12 hr/day (0800 to 2000 hours EST). Elevated CO₂ caused little or no effect on plant growth or yield at low O₃ (CF) or near ambient O₃ (NF) concentrations. At ambient CO₂, the highest O₃ concentration (NF+) suppressed yield of wheat compared to yield in CF air, especially in the more O₃ sensitive cultivar, NK-9904. With this cultivar, yield response to increased CO₂ concentrations was highly dependent upon the O₃ concentrations to which the plants were exposed, i.e., the yield enhancement caused by elevated CO₂ was greater when yield was suppressed by O₃. Essentially, the greater yield response to elevated CO₂ in the presence of the higher O₃ concentrations is because elevated CO₂ prevents or partially prevents yield suppression by O₃ (i.e., plants grown at elevated CO₂ appear less sensitive to O₃ than plants grown at ambient CO₂). With plants grown in high CO₂ air, the yield of NF+-treated plants approached the yield of CF-treated plants. Photosynthesis was usually greater in flag leaves of both wheat cultivars grown in elevated CO₂, especially when the leaves were young. Ozone suppressed photosynthesis only in the O₃-sensitive cultivar (NK-9904) at ambient CO₂ concentration, but when the plants were grown in elevated CO₂, this suppression was prevented. As a whole, these results show that the effects of rising CO₂ concentrations on crop productivity depend upon the level of O₃ stress to which the plants are exposed and upon the O₃ sensitivity of the plants.

A field experiment showed that ambient concentrations of tropospheric O₃ in North Carolina during the summer decreased forage yield of sensitive white clover (Trifolium repens L.) by 39%. Field experiments with the Colorado potato beetle showed that larval growth, survival to the adult stage, and weight of emergent adults were affected little by stressing host potato plants with elevated O₃. However, preliminary field and greenhouse results suggest that a small amount of O₃ stress can increase egg laying by newly emerged adults.

Ascorbic acid present in leaf apoplastic fluid may limit ozone injury by participating in reactions that detoxify ozone prior to plasma membrane damage. To assess the relationship between apoplast ascorbate and ozone stress, seven genotypes of snap bean (Phaseolus vulgaris L.) known to differ in ozone sensitivity were compared in an open top chamber study. Plants were then subjected to either CF conditions (29 nmol mol^-1ozone) or elevated ozone (CF+O₃, 67nmol mol^-1 ozone). Following an eight-day treatment period, vacuum infiltration methods were employed to separate leaf ascorbic acid into apoplast and cytoplasm fractions. Under CF conditions, afternoon apoplast ascorbic acid levels in the fourth trifoliate leaf were in the range of 100 to 150 nmol g^-1FW for all genotypes, representing 3 to 5% of total leaf ascorbate. In general, apoplast ascorbate increased in ozone-treated plants relative to CF controls with ozone-sensitive genotypes exhibiting larger increases than tolerant genotypes. The results suggest that ascorbic acid in the cell wall plays a role in plant response to ozone stress.

Keith Davis, Mulpuri V. Rao and Jennifer Riehl Koch - Ohio State University:

The long-term goal of this research is to identify molecular mechanisms controlling the effects of ozone on plants. These studies will utilize a combination of molecular and genetic approaches to examine the effects of ozone on the model plant systems, Arabidopsis and hybrid poplar. Our studies, along with others, have clearly shown that these model plants offer significant advantages for applying genetic and molecular genetic approaches to the study of ozone-induced responses to plants. The long-term goal of this project is to identify the regulatory mechanisms underlying ozone-induced responses in plants, particularly with respect to the signal transduction pathways that modulate ozone-induced effects. We will investigate specific mechanisms that control ozone effects on plant gene expression and how ozone interacts with signal transduction pathways activated by other biotic and abiotic stresses. These studies will provide detailed information concerning the mechanisms that control the expression of genes that play an important role in ozone-induced responses in plants and the establishment of defense mechanisms against ozone exposure. This will provide a better understanding of the cellular signaling processes involved in the induction of protective antioxidant molecules that provide tolerance against highly reactive and damaging oxygen radicals. Thus, these studies will have a direct bearing on plants’ responses to other oxidative stresses such as drought, UV-irradiation and exposure to heavy metals. Our studies will provide a sound basis for understanding the underlying mechanisms of ozone damage in plants and provide the basic knowledge necessary to rationally develop methods for the establishment of plants resistant to oxidative stress.

Specific Aims

Specific aims include 1) molecular genetic analyses of ozone-sensitive Arabidopsis ecotypes, including the cloning of the major sensitivity loci in the highly sensitive ecotype, Cvi; 2) the isolation and characterization of mutant Arabidopsis plants altered in their response to ozone; 3) detailed analyses of the ozone response of existing Arabidopsis mutants altered in the salicylic acid, jasmonic acid and ethylene response pathways; 4) identification and characterization of novel ozone-induced defense genes in hybrid poplar; 5) defining the roles of SA, JA and ethylene in modulating ozone-induced gene expression in hybrid poplar; and 6) characterization of the mechanisms of transcriptional control of defense genes induced by oxidative stress in hybrid poplar.

Key Results

The high level of ozone-sensitivity in the Arabidopsis ecotype Cvi is due to the induction of hypersensitive cell death. Sensitivity is conferred by two interacting loci; the major sensitivity locus has been mapped to the bottom of chromosome 2. Lesion formation is controlled by the interaction of at least two signaling pathways that are dependent on SA and JA respectively. Preliminary evidence indicates that ethylene also modulates the response by interacting with the SA and JA pathways.

The differential ozone sensitivity of two hybrid poplar clones has been found to be due to differences in these clones to respond to signal molecules such as JA and SA. The ozone-tolerant clone (245) exhibits high levels of defense gene expression in response to ozone and exogenous treatments with either SA or JA. The ozone-sensitive clone (388) has greatly attenuated defense gene induction in response to ozone, SA or JA treatments.

William J. Manning - University of Minnesota:

Richard Flagler and W.J. Manning began an experiment in the Stephen F. Austin Experimental Forest in Nacodoches in East Texas in 1995. The purpose was to determine the effects of ambient ozone on growth of loblolly pine (Pinus taeda L.). They planted seedlings grown from seed from a known ozone-sensitive half sib family of loblolly pine. Two randomized complete blocks of six blocks each were established in spring 1995. Seedlings in one block were treated bi-weekly with ethylenediurea (EDU) sprays, and those in the other were treated with sodium erythorbate (NaE). EDU was applied at 0, 150, 300 and 450 ppm a.i. and NaE at 515, 1030, and 1545 ppm a.i. The initial planting in each block consisted of 4 rates of the chemical x 6 blocks x 18 trees = 432/block. EDU and NaE blocks were considered to be separate experiments. Six trees per treatment (144 total) were harvested per year. The following growth parameters were determined: main stem length (tree height), main stem dry weight, basal stem diameter, total stem weight, total needle weight, and total tree weight (Biomass). Compared to the control, EDU was the most effective treatment in increasing all growth parameters. NaE 1030 was very effective in increasing growth of trees in its block. EDU 150 inhibited tree growth, as did NaE 1545. Overall, EDU 450 was the most effective treatment. These are the first results from a study designed to determine the use of protective chemicals on tree growth under natural conditions.

Sagar Krupa - University of Minnesota:

Saskatoon serviceberry or Saskatoon Amelanchier alnifolia Nutt. cv. Smoky) seedlings were planted at five study sites within a 35,000 km² airshed, that is influenced by a number of isolated stationary sources of sulfur dioxide (SO₂), oxides of nitrogen (NO_X) and hydrocarbons (HCs), among others. The locations of the five sites were based on the results of a meteorological dry deposition model for the oxides of sulfur and nitrogen. Visible foliar injury responses of Saskatoon were used as a biological indicator of SO₂ exposures, through monthly field surveys.

During late July 1998, unifacial, interveinal chlorsis was observed on some 12% of the seedlings at one study site. By September, the chlorosis had become more severe (necrosis) on some 70% of the plants at that site. Site specific ambient SO₂ levels were relatively low (maximum 5-minute concentration of 52.8 ppb). Similar data were unavailable for all, but one other site. Therefore, foliar total S and SO₄^2- concentrations were analyzed in September at four of the five study sites. Previously soil SO₄^2--S at these sites had been analyzed. There were spatial variabilities among these parameters. Based on the overall examination of these data, it is concluded that the observed visible injury symptoms were due to chronic SO₂ exposures, exacerbated by the presence of ozone (O₃). Independent of this literature-based speculation, visible foliar injury responses of Saskatoon can be used as a biological indicator for acute or chronic ambient SO₂ exposures, in the presence of other phytotoxic air pollutants.

A.H. Chappelka - Auburn University:

During 1998, a study was completed and analyzed on the response of “wild plants” with varying ozone concentrations. Species numbers, percent cover and the rate of blackberry:broomsedge litter decomposition were reduced with increasing ozone concentrations. Blackberry flowering and fruit production was affected by ozone exposure the previous year. Blackberry grown in elevated ozone exhibited increased flower production and more aborted fruits than those grown in charcoal filtered air. A study is being designed to test the effects of ozone on the competitive relationships among native vegetation. Exposure-response relationships between ambient ozone concentrations and visible injury on mature black cherry in two National Parks in the eastern U.S. were developed. The percent of injured trees increased with elevation and was positively correlated with SUM06 and W126. This relationship was strengthened significantly by combining the data from both parks. Tall milkweed has been inventoried for ozone sensitivity for 5 years in Great Smoky Mountains National Park. The data are currently being analyzed.

Bob Kohut, John Laurence, Robert Eckert and Thomas Lee -

Boyce Thompson Institute:

Identification of Bioindicators for Ozone and Assessment of Impacts at Acadia National Park

Exposures of native species of plants in open-top chambers at Acadia National Park indicated that black cherry, quaking aspen, white ash, jack pine, big-leaf aster, and spreading dogbane were sensitive to ambient levels of ozone, with red maple, pin, cherry, mountain ash, mountain holly, and flat-topped aster possibly sensitive. Species injured at levels of ozone 50% greater than ambient were gray birch, small sundrops, and bunchberry. Species uninjured at twice the ambient level of ozone were paper birch, eastern white pine, pitch pine, red spruce, northen white cedar, northern red oak, Canada bluejoint grass, wild radish, and Canada mayflower.

Clonal lines of eastern white pine derived from trees in the park considered to be responsive to ambient ozone were not affected by levels of ozone up to three times ambient concentrations in two consecutive years of exposure. There was no relationship between the type or degree of marking and the treatment level of ozone. The lack of response to three times ambient levels of ozone raises doubt that ozone is responsible for the foliar markings observed in the field.

Field surveys were conducted from 1992 through 1997 to assess the incidence and severity of foliar ozone injury on plants in the park. The primary species examined were big-leaf aster and spreading dogbane since these were the most sensitive species and it was possible to assess many individual plants in a limited period of time. Surveys were conducted on 30 permanent plots with 90 individuals of a species assessed on each plot.

Ozone or ozone-like symptoms were found each year, but their occurrence and intensity were highly variable. Although ambient levels of ozone were high in several years, when precipitation was low the plants expressed little if any injury. When Acadia experienced a high level of rainfall in July 1996, the incidence of foliar ozone injury was greater than in any other year even though the level of ozone was more moderate. These responses effectively demonstrate the interaction of ozone exposure and environment in the formation of foliar ozone injury.

Predicting the Regional Impact of Ozone on the Growth of Eastern Hardwood Forests Using Linked TREGRO and ZELIG Models

We simulated the long-term effects of ozone on eastern hardwood forests in the US by linking TREGRO, a mechanistic model of an individual tree, to ZELIG, a forest stand model, and examined the response of these forests to 5 ozone exposure regimes (0 to 90 ppm.hr SUM06 per year). Parameters for TREGRO and ZELIG were established using biological and meteorological information from three climate zones in the eastern US. TREGRO generated three-year exposure-response relationships between ozone and the growth of three dominant species, sugar maple, red oak, and black cherry. The effect of ozone on the alteration of individual tree total tree mass, leaf mass, and fine root mass were calculated and used to modify annual growth, canopy light penetration, and drought sensitivity functions in ZELIG. Ten species that typically populate the eastern hardwood forest were then simulated under canopy competition, using the ZELIG model, assuming that only the three dominant species were affected by ozone. In all climate zones, we predicated a decease in red oak abundance as ozone increased, with 10% declines under the highest ozone exposures. The major competitor of red oak in these forests, sugar maple, was predicted to increase in abundance in southerly climates as a result of the decline of red oak populations. The influence of ozone on the growth of both species was significantly decreased on infertile soils.

Eva Pell - Pennsylvania State University:

Experiments were conducted to determine the molecular biology of ozone (O₃)-induced foliar senescence. Arabidopsis plants treated with 0.15 ppm O₃ for 6-h d^-1 for 14 d and expression of 13 senescence associated genes (SAG) were determined every 2-d throughout exposure. Eight of the SAG genes were expressed earlier in O₃-treated than nontreated plants. Transgenic Arabidopsis lines that have either the SAG12 or the SAG13 promoter fused to the GUS reporter gene developed in the laboratory of R. Amasino (University of Wisconsin) were exposed to O₃. No expression of GUS was detected in the SAG12 plants. In the case of SAG13, expression of GUS was accelerated by O₃. In normal senescence GUS expression begins at the margins while in O₃-treated plants, GUS expression was distributed diffusely across the surface of the leaf. The role of ethylene in the induction of O₃-induced foliar senescence is being studied through the transformation of potato with antisense for ACS4 and ACS5, two O₃-induced ACC synthase genes. This year a successful transformation system was developed. Experiments are being conducted to determine the implications of O₃-induced foliar senescence on the nitrogen (N) economy of the plants. Hybrid poplar ramets were grown in an environment to which 0.08 ppm O₃ was supplemented 8-h d^-1 for 12 wks. Some plants received optimal N throughout the experiment while others had 80% of the N supply withdrawn in the middle of the growing season. O₃ effects on rate of leaf senescence were greatest in the latter N treatment. Redistribution of N is being assessed through measurement of total N and by observing the fate of N¹⁵ in a pulse chase experiment conducted within the larger O₃ study. In other studies we have utilized electrophysiological technique called patch clumping to determine whether O₃ might be affecting guard cells’ membranes directly. After guard cells were treated with O₃, the magnitude of inward K⁺ current was reduced, demonstrating for the first time specific regulation of an ion channel by O₃. Because the inward K⁺ channels were inhibited, these results lead to the hypothesis that O₃ acts by inhibiting stomatal opening. When isolated epidermal peels of Vicia faba were exposed to O₃, stomatal opening in response to light was significantly inhibited by O₃, thus supporting the observations of the patch clamp experiments. Whole Vicia faba plants were exposed to 0.10 or 0.18 ppm O₃ for 4-h. The exposures were initiated during darkness when stomates were closed. Subsequent light exposure stimulated stomatal opening, but O₃ inhibited that increase in stomatal conductance and decreased rates of mesophyll carbon assimilation relative to nontreated plants. A-c_i curves were identical in the 0.10 ppm O₃-treated and control plants. Therefore, the reduced rates of carbon assimilation observed could only be attributed to a reduced availability of the substrate carbon dioxide to the mesophyll, as a result of the O₃-inhibition of stomatal opening. At the higher O₃ concentration, a direct effect of O₃ on mesophyll photosynthesis could be observed as well, reflected as a depression in the A-c_i curve.

PUBLICATIONS

Ali, A.A. 1998. Ecological and Biological Studies on the Effect of Pollutants on Some Plants and Their Associated Microorganisms. Ph.D. Dissertation, Zagazig University, Zagazig, Egypt.

Ali, A.A., C.L. Mulchi, K.R. Islam, Y.A. Elzawahry and R.L. Abdelfalk. 1998. Carbon and Nitrogen States of Plants and Soils in Troposphere CO₂ or O₃ Environment Agroecosystems. Agron. Abstracts. ASA Ann. Meetings. Baltimore, MD p.17.

Ali, A.A., C.L. Mulchi, K.R. Islam, Y.A. Elzawahry and R.L. Abdelfattah. 1998. Soil Respiration Responses to Atmospheric Carbon Dioxide, Ozone and Soil Moisture Regimes in Soybean/Wheat Agroecosystems. Agron. Abstracts. ASA Ann. Meetings. Baltimore, MD p.254.

Andrews, T. 1998. Rapid Screening of Soybean Germplasm for Tolerance to Ozone Stress by Examining Growth Characteristics and Antioxidant Enzymes. MS Thesis, University of Maryland, College Park, MD.

Andrews, T., C.L. Mulchi, and T. Solomon. 1998. Rapid Screeing of Soybean Germplasm for Tolerance to Oxidative Stress. Northeastern Branch ASA Abstracts. Amherst, MA, p. 4.

Barbo, D.N., A.H. Chappelka, G.L. Somers, M.S. Miller-Goodman and K. Stolte. 1998. Diversity of an early successional plant community as influenced by ozone. New Phytol. 138:653-662.

Booker, F.L. and J.E. Miller. 1998. Phenylpropanoid metabolism and phenolic composition of soybean [Glycine Max (L.) Merr.] leaves following exposure to ozone. J. Experimental Botany 49:1119-1202.

Brendley, B. and Pell, E.J. 1998. Ozone-induced changes in biosynthesis of Rubisco and associated compensation to stress in foliage of hybrid poplar. Tree Physiology 18:81-90.

Chappelka, A.H. and L.J. Samuelson. 1998. Ambient ozone effects on forest trees of the eastern United States: A review. New Phytol. 139:91-108.

Chappelka, A., J. Skelly, G. Somers, J. Renfro and E. Hildebrand. 199_. Mature black cherry used as a bioindicator of ozone injury. Water, Air and Soil Pollut. (In press).

Chappelka, A., G. Somers and J. Renfro. 199_. Ozone effects to forest trees in Great Smoky Mountains National Park, USA. Water, Air and Soil Pollut. (In press).

Chernikova, T. 1998. Ozone Effects on Growth, Physiological Characteristics and Antioxidant Enzymes in Soybean Cultivars Exposed to Ambient and Elevated CO₂. Ph.D. Dissertation, University of Maryland, College Park, MD.

Chernikova, T., C.L. Mulchi, L. Douglass, E.H. Lee, and R. Rowland. 1998. Gas-Exchange Responses of Soybean Plants to Carbon Dioxide, Ozone and Moisture Deficit. Agron. Abstracts. ASA Ann. Meetings. Baltimore, MD p. 85.

Chevone, B., W. Manning, A. Varbanov, and S. Krupa. 1988. Relating ambient ozone concentrations to adverse biomass responses of white clover: A case study. Environ. Pollut. 103:103-108.

Heagle, A.S., J.E. Miller, and F.L. Booker. 1998. Influence of ozone stress on soybean response to carbon dioxide enrichment. I. Foliar properties. Crop Science 38:113-121.

Heagle, A.S., J.E. Miller, and W.A. Pursley. 1998. Influence of ozone stress on soybean response to carbon dioxide enrichment. III. Yield and seed quality. Crop Science 38:128-134.

Hummel, R.L., R.L. Brandenburg, A.S. Heagle and C. Arellano. 1998. Effects of ozone on reproduction of twospotted spider mite (Acari: Tetranychidae) on white clover. Environ. Entomol. 27:388-394.

Kim, M. 1999. Ambient Effects of Elevated Tropospheric Ozone, Elevated Carbon-Dioxide and Soil Moisture Deficit on Soybeans Using Fluorescence Imaging. Ph.D. Dissertation, University of Maryland, College Park, MD.

Kim, J.S., A.H. Chappelka and M.S. Miller-Goodman. 1998. Decomposition of blackberry and broomsedge bluestem as influenced by ozone. J. Environ. Qual. 27:953-960.

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