Duration: 10/01/1995 to 09/30/2000

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Tropospheric ozone resulting from man's activities is the major air pollutant effecting crops and native vegetation in the eastern US. A strong oxidant, ozone causes foliar injury, accelerates leaf senescence, and reduces growth and yield of many species. While the effects of ozone have been well documented, several aspects of its toxicity are poorly understood. A more complete understanding of plant responses to ozone in the Northeast would enable more effective development of ozone resistant varieties , as well as provide additional information for developing ambient air quality standards.

Related, Current and Previous Work

Numerous boos, chapters and review articles have been written about the effects of O, on plants (Alscher and Wellburn, 1994; Runeckles and Krupa, 1994; USEPA Criteria Document on Oxidants, 1993; Lefohn, 1992; and Manning, 1990). These references should be consulted for detailed information concerning our existing knowledge..



In the following section, research conducted by scientists in NE-176 is summarized and schematically presented in Figure 1. Whole plant experiments have led to the characterization of O, tolerant and sensitive cultivars, growth and yield performance of genotypes in field conditions, and interactions of 03 and C02 on plant growth and physiology. Mechanistic studies have focused on biochemical/molecular attributes of genotypes that may be associated with 03 tolerance. A major accomplishment has been the production of specific genotypes that can serve as model experimental systems. Transgenic tobacco plants that overexpress antioxidant enzymes have been developed by Zilinskas at NJ and Oh, tolerant and sensitive white clover clones have been selected by Heagle and Miller at USDA/ Raleigh, NC. Research proposed by cooperators in this Regional Project revision make extensive use of these model plants. Whole plant experiments:



Characterizing plant responses to nonce



In MA, Manning continued characterization of plant response to 03 using a number of species important to the economy of the Northeast, such as strawberry, cranberry, turfgrass, tomato, morning glory, and browailia and native plants, such as white pine, several species of aster, and blackberry. For most species studied, a wide range of 03 sensitivity among cultivars and within populations was observed.



At USDA/Beltsville, Lee identified several 03 tolerant and sensitive soybean cvs. and near isolines (Lee et. al., 1994; Foy et al., 1995). Some cvs. showing high tolerance to O, (Biloxi, Bossier, Lee, and Perry) were also acid soil (AL) tolerant, but two AL tolerant cvs. (Aurora and Brunatna) were 03 sensitive. Some cvs. (Chief, Salute 216 and Smena) were sensitive to both AL and 03.



In NY, McGrath examined the effects of natural 03 exposures on symptom development, yield and powdery mildew infection in curcubits. Plant developmental stage was the most important determinant of injury occurrence. Both 03 symptoms and powdery mildew infections were first observed near the start of fruit production in six successive plantings. Younger plants were not sensitive to the high 03 concentrations, that caused extensive injury to plants with fruit. Open-top chamber studies.



Whole plant, field experiments were conducted at MD, USDA/Beltsville and Raleigh using open-top chambers to examine the interactive effects of 03 and elevated CO, on the growth, yield and physiology of several crop species, including soybean, corn, cotton and wheat. At USDA. Beltsville (Lee) and MD (Mulchi), high 03 levels (60 nl L-) reduced yields up to 2U0o, however, increased CO, alleviated the negative impact of 03 Mulchi el a!, 1992, Rudorff, 1993; Zakaria et al., 1994a,b). At USDA/Raleigh (Miller),- a similar protective effect of elevated CO= against U; exposure was observed for soybean cv. Essex and white clover (Heagle el al., 1993).
The physiological basis for CO,-enhanced defense against 03 does not appear to be limited to stomatal closure alone. Rates of photosynthesis were stimulated when plants were grown in CO, enriched atmospheres, even in the presence of moderate levels of O, (Slaughter et al., 1993). Use of antioxidant chemicals



The application of antioxidant compounds, such as ethylene diurea (EDU), to vegetation is known to prevent leaf damage mediated by03 . However, the mechanism(s) of protection is poorly understood. Studies with EDU, and other compounds, were continued at several participating stations to further document dose-response relationships, chemical effects on plants and the extent of the protective capacity against 03 exposures.



At MA (Manning), the urea portion of EDU was compared to the whole molecule for efficacy against 03 injury using Bel-W3 and Bel-B tobacco seedlings. EDU protected all plants in all experiments. Urea did not provide any protection and urea-treated Bel-B plants, when exposed to 03, sustained extensive injury. EDU dose-response studies with tobacco cvs. Bel-W3, Bel-B, MD59 and MD872 and clover clones NC-R and NC-S demonstrated that a single foliar spray application at 300 mg L-' a.i. protected leaves from injury for a 7-day period at peak 03 concentrations of 50 to 100.



At PA, Pell conducted to assess the role of antisenescent properties of EDU in foliar protection using an 03 sensitive potato cv., Norland. EDU, at 15 mg L-' soil treatment, was sufficient to provide 03 protection (100 n( L-' ) with no signs of chemical toxicity. Net photosynthesis, chlorophyll content, Rubisco quantity and relative levels of mRNA for the Rubisco large and small subunits showed 03-induced decreases (Reddy et al., 1993; Pell et al., 1994). EDU provided complete protection from effects caused by 03 alone, but did not affect changes resulting from senescence processes.



At TX, Flagler found that both EDU and Ozoban (sodium erythorbate) prevented 03 injury on field-grown shortleaf pine over experimental periods of two to four years (Flagler and Toups, 1991; Flagler et al., 19946). Influence of environmental factors and plant physiological status on the ozone response.



At AL, Chappelka investigated the interaction of 03 with other environmental stresses (low temperature) on loblolly pine (Chappelka et al., 1990), 03 effects on assimilate allocation between roots and shoots (Qui et al., 1993), and the occurrence of 03 injury on native
vegetation in wilderness areas (Chappelka et al., 1994). Ozone can enhance the low temperature sensitivity of loblolly pine new needle growth and restrict carbon partitioning to the roots. Visible foliar injury, attributed to ambient 0, was observed on seedling, sapling and mature trees in the Great Smokey Mountains National Park.



At TX, Flagler investigated the interaction of 03 and drought stress on growth and physiology of loblolly and shortleaf pine. In loblolly pine, 03 decreased diameter and biomass (Elsik et al., 1992), and inhibited photosynthesis and needle conductance (Elsik et al., 1993; Flagler et al., 1994). Similar results were observed with shortleaf pine and no interaction between O, exposure and drought stress occurred (Flagler et al., 1993).



Characterizing the relationship between natural 03 exposures and the development of foliar symptoms is an important aspect in assessing the impact of pollutant concentrations on plant growth and yield Environmental conditions, edaphic factors, plant growth stage, and biotic stresses are all known to influence O, effects on plants (Krupa and Kickert, 1990; Runeckles, 1992). While the uptake of 03 through open stomata is of primary consideration in correlating exposure concentrations to plant response, the physiological status and developmental stage of the vegetation also influence the degree of injury.



A cooperative study between MA (Manning) and MN (Krupa) has been initiated to assess the relative importance of 03 and other environmental factors in the development of foliar injury on Bel-W3 tobacco (Krupa et al., 1993). An integrated model is being developed to predict the timing and severity of 03 injury on this bioindicator species. Mechanistic studies:



Mechanisms of ozone effects on roots.



At USEPA /Corvallis, Andersen has focused on understanding the mechanistic effects of 03 on the root and rhizosphere systems of plants. Labelling studies were used to construct a carbon budget for a ponderosa pine seedling growing in association with an ectomycorrhizal fungus (Andersen and Rygiewicz, 1991; Rygiewicz and Andersen, 1994) and to follow the effects of 03 on the carbon budget from all major pools (Andersen and Rygiewicz, 1994, in review). Mycorrhizal and nonmycorrhizal plants generally responded in a similar fashion to 03; the presence of 03 made mycorrhizal plants more similar to non-mycorrhizal plants in carbohydrate allocation patterns. The results suggest that 03 exposure may influence the vigor of the symbiosis and diminish the beneficial effects of mycorrhiza. Ozone-induced modifications of Rubisco.



At PA, Pell has characterized 03-induced changes in the Rubisco protein that could influence plant vulnerability to accelerated senescence. The total activity of Rubisco was contrasted in leaves of potato genotypes exposed to 0, from leaf emergence until senescence. An accelerated loss of enzyme activity was observed in sensitive cvs., Norland and Cherokee, but not in tolerant cvs., Superior and Norgold Russet (Enyedi, et al., 1992). Rubisco protein, in both genotypes, contained a similar number of available and total sulfhydryl groups, chemical moieties that are susceptible to oxidation (Enyedi et al., 1992), and the nucleotide sequence
and deduced amino acid sequence for the large subunit of the protein was identical (Enyedi and Pell, 1992). These studies demonstrated that Rubisco structure was probably not responsible for the variation in 03-induced loss of activity between the genotypes.



Rubisco activity, in purified protein exposed to 03 in vitro, declined in conjunction with a loss in sulfhydryl groups, formation of carbonyl groups and formation of aggregates of the protein (Eckardt, 1995). However, when intact plants were exposed to 03 and Rubisco was subsequently purified, no structurally modified protein could be detected. _ Leaf discs of 03-treated plants incubated at 00C exhibited aggregates similar to those formed when the protein was treated with oxidant in vitro, whereas in discs incubated at 300C, no aggregates of Rubisco were formed and the protein was present at much lower concentrations than in control tissue (Eckardt, 1993). These results indicate that 03 induces structural modifications in Rubisco which can lead to accelerated proteolysis. The basis for accelerated loss of the enzyme me in sensitive potato cvs. in comparison to resistant cvs. still must be determined.



Ozone exposure of potato resulted in a decrease in the mRNA levels of both the small and large subunit of Rubisco, rbcS and rbcL, respectively (Reddy et al., 1993). Immature leaves sustained a large loss in rbcS, which was not noted in mature leaves. When plants were
incubated in the dark, levels of rbcS declined regardless of 03 treatment. Small decreases in Rubisco quantity, were detected in both immature and mature foliage incubated in the dark, regardless of treatment. when mature leaves were incubated in the dark following 03
treatment, large reductions in Rubisco were detected (Eckardt and Pell, 1994). The latter response is a further indication that 03 predisposes Rubisco to degradation. Antioxidant activity and gene expression.



At USDA/ Raleigh, Miller and Heagle selected ten clones of white clover on the basis of visible injury in response to 0,. Stomatal conductance (gs) of a resistant (NC-R) and a sensitive (NC-S) clone, measured in field and greenhouse low 03 environments, was not different. The differential sensitivity of the clones, therefore, could not be attributed to variation in 03 uptake rate.



The basal activities of the antioxidant enzymes SOD, catalase, APX, DHAR, monodehydroascorbate reductase (MDHAR), GR and polyphenol oxidase were measured in the resistant and sensitive clones. All enzyme activities were similar between clones. However, further research is required to examine enzyme response under conditions of oxidative stress to assess the role of these antioxidants in 03 tolerance.



Induction by 03 of plant defense responses involving phenylpropanoid metabolism was examined in soybean (Booker). In greenhouse studies, the activities of phenylalanine ammonia-lyase and 4-coumarate CoA:ligase were stimulated within 3 h of treatment with 100 nl L-' 0, (Booker , 1993). Activities of cinnamyl alcohol dehydrogenase and peroxidase increased after 1.5 d of 03 treatment, along with the appearance of foliar injury. In field studies, soluble and cell wall-bound hydroxycinnamic acid content rose 14-57% in 03-treated leaves. The level of polymeric phenolics was increased 2X by 03, although increased biosynthesis of core lignin was not evident (Booker et al., 1991). In this case, membrane degradation caused by 03 could allow pre-formed phenolics and polyphenol oxidases to react
to initiate formation of phenolic polymers. However, pressure-bomb experiments indicated that cell wall flexibility decreased in leaf tissue from 03-treated plants (Fiscus et al., 1995). Phenylpropanoid metabolism was apparently stimulated by 03 and may be related to changes in cell wall structure.



In VA (Chevone), the response of the antioxidant system to 03 in tolerant and sensitive soybean cvs. was investigated in laboratory studies. In a sensitive cv., 'Dare', stomatal closure induced by 0, was delayed compared to tolerant cvs. resulting in a 10% greater internal flux of pollutant after a 4 h fumigation (200 nl L-' 03). Isozyme activities of SOD were not altered by 03 treatment in any cv. (Sheng el al., 1993). Endogenous activities of GR, APX and
substrate concentrations of ascorbate and glutathione were similar among cvs. and were not affected by 03 exposure (Sheng, 1992). The general lack of response of the antioxidant system could have resulted from excessive oxidative pressure.



Also at VA, antioxidants in eastern white pine genotypes, susceptible or tolerant to needle tipburn, were characterized over three years in field specimens. Enzyme activity of GR, SOD
and APX was lowest during the summer months and highest during the winter (Anderson et al., 1992). Substrate concentrations of ascorbate and glutathione increased 4X to 5X from summer to winter. No differences in antioxidants were observed among the white pine
genotypes, precluding endogenous activity as a mechanism of 0, tolerance. In a preliminary study, moisture stress, high temperatures and 03 exposure resulted in an increase in the foliar
activity of SOD and GR (Anderson, 1991).



At NJ (Zilinskas), molecular tools were developed to elucidate the function of antioxidant enzymes in plant response to 0, Proteins from pea were purified and characterized, cDNAs encoding these enzymes were isolated and analyzed, antibodies to these enzymes were
produced as molecular probes, and transgenic tobacco and potato plants were constructed that overexpress these enzymes. The enzymes and cDNAs analyzed included the plastidic and cytosolic Cu/Zn-SOD (White and Zilinskas, 1992), mitochondrial Mn-SOD (White et al.,
1991; Altornare, 1992), cytosolic APX (Mittler and Zilinskas, 1991 a,b,c; Mittler and Zilinskas, 1992), and cytosolic MDHAR (Murthy and Zilinskas, 1994). Collectively, these enzymes are capable of scavenging superoxide radicals and hydrogen peroxide and regenerating reduced ascorbate. Some of the genes encoding pea antioxidant enzymes were very responsive to 03, most notably the genes encoding cytosolic proteins (Zilinskas et al., 1993b; Pitcher et al., 1992); however, for most enzymes, increased activity paralleled the development of visible foliar symptoms (Pitcher et al., 1991; Pitcher et al., 1992). Detailed studies of the response of the gene encoding cytosolic APX showed that the regulation of this gene is very complex, involving transcriptional and post-transcriptional control (Mittler and
Zilinskas, 1994; Zilinskas et al., 1993a). No EDU-associated increase in any of the SOD isozymes was found in soluble leaf extracts of pea or snapbean, contrary to the prevailing hypothesis for EDU's protective role against 03 damage (Pitcher et al., 1992).



Transgenic tobacco plants (Nicotiana tabacum Bel W3 and W38), which overproduce pea SOD and APX under control of the CaMV35S promoter, were constructed and propagated (Zilinskas et al., 1993; Pitcher et al., 1994). These plants included those that overproduce
Cu/Zn-SOD, Mn-SOD, and APX in the cytosol and Mn-SOD and APX in the chloroplast. Transgenic potato (Russet Burbank) were constructed which overproduce pea Cu/Zn-SOD or APX in the cytosol. The tolerance of these transgenic plants to oxidative stress conditions, relative to nontransformed controls, remains to be evaluated.



RELATIONSHIP TO OTHER REGIONAL PROJECTS:



NE-176 is an unique regional research project. No other regional project focuses on characterization and mechanisms of plant responses to 0, in the Northeast, or elsewhere in the U. S. A search of the CRIS data base did not reveal any projects with similar research
objectives. The National Atmospheric Deposition Program (NADP) represent a NATIONAL RESEARCH SUPPORT PROJECT (NRSP-3) with primary emphasis on precipitation chemistry and effects on terrestrial systems. The research support objectives of NRSP-3 and the research objectives of NE-176 complement each other. A number of stations participate in both NE-176 and NRSP-3, enabling effective communication between the two programs.

Objectives

1. Characterize whole plant responses to O3 including carbon assimilation and allocation, growth and productivity
   
2. Identify and delineate primary factors, both biotic and environmental, that determine plant responses to O3
   
3. and Determine mechanisms of O3 action and plant defense systems, using cultivars and genotypes characterized in whole experiments.
   
4. 
   

Methods

A. Regional Research Approach.

The characterization and development of 03 tolerant cultivars requires an understanding of biological processes that range from whole plant carbon assimilation and allocation patterns, to gas flux from the atmosphere into the leaf, to biochemical and genetic regulation of carbohydrate metabolism and oxidative defense mechanisms. The influence of environmental factors on the 03 response also must be known to assess the full productive capacity of selected, or genetically enhanced, plant material. The integration of these various components is represented in Figure 1, which also demonstrates interactive aspects of this regional research project. A unifying element of the project is common plant material available to station scientists; 03 tolerant and sensitive white clover clones from USDA/Raleigh and transgenic tobacco (overexpressors of antioxidant enzymes) from NJ.

Scientists at participating experiment stations and laboratories have developed their own unique expertise and facilities that can be applied to investigate specific hypothesis within the project objectives. The primary cooperative effort is the coordinated research approach to investigate O; effects at different levels of plant function, from the leaf canopy to gene expression No individual station has the personnel or resources available to accomplish all objectives of this regional project. General research areas among the stations are briefly summarized as follows: Field fumigation/exclusion facilities for growth and yield, canopy gas exchange rates, community structure (USDA/Raleigh, Beltsville/MD, MA, AL, TX, BTI, USEPA/Corvallis); 03 and C0, interactions (USDA/ Raleigh, Beltsville/MD); CSTR/greenhouse systems for laboratory 03 fumigations (BTI, NJ, MA. PA, VA , USDA/ Raleigh, Beltsville-, USEPA/Corvallis); plant disease interactions/mycorrhizal associations (MA, NY, USEPA/Corvallis); leaf gas exchange rates, photosynthesis (VA, MD, BTI, USDA/ Raleigh, TX); antioxidant biochemistry/enzyme activity (NJ, VA, PA, USDA/Beltsville, Raleigh); gene regulation/molecular biology (NJ, PA); plant transformation (NJ, PA); modelling pollutant uptake and plant growth responses (MN, BTI, USDA/Raleigh, USEPA/Corvallis).

A primary consideration to attain the objectives of this revised regional project is to enable the effective flow of information between the different levels of research. This interchange will be accomplished between whole plant studies and mechanistic studies by the exchange of unique plant material. Whole plant experiments will characterize growth and yield responses, examine the influence of environmental conditions on 03 effects, and further identify genotypes that are sensitive or tolerant to 03. Plant material identified by such research will be used by other scientists to test one or more of the hypotheses in Figure 1, which are related to the mechanism of 03 toxicity or defense.

Information derived from mechanistic studies will identify specific proteins and genes that respond to 03 and enable
characterization of enzyme activity and gene expression. Such 'marker' genes can be incorporated into a biochemical screening procedure to assess the relative tolerance of important plant species and genotypes in the northeast. These genes also will be utilized in biotechnology experiments to produce genotypes with enhanced 03 tolerance. Transgenic plants will be tested for field performance to verify 03 resistance.

The information obtained from field research and the influence of environmental variables on plant response will increase our understanding of 03 effects under natural conditions. This knowledge is necessary to develop process-oriented models of 03 impact on plant productivity which can be utilized to estimate growth and productivity losses under a number of ambient pollutant concentration regimes. Results from all aspects of the project's research will be incorporated into USEPA Criteria Assessment Documents that constitute the scientific bases for establishing Ambient Air Quality Standards.

1. Characterizing Plow Responses to Ozone

At MD (Mulchi), studies will be conducted on physiological and morphological processes by which CO, enrichment alleviates the negative impact of low 03 concentrations on plant yield. Soybean will be grown to maturity in open-top chambers aspirated with charcoal filtered air (CF) containing two levels of CO, (350 or 500 ul L-) and three levels of ozone (25, 40 or 55 nl L') under well-watered conditions. The plants will be grown to the third trifoliolate stage of growth before initiating the collection of data.

Specific studies will examine the effects of treatments on the morphology of cells (light microscopy) and membranes (HPLC analysis of lipid fraction) of the vascular system in leaves. Carbohydrate translocation activities and partitioning in plants will be investigated by pulse labeling with highly enriched isotopes of carbon( '3C or "C) as *CO, These studies will be conducted on leaf tissue of the same age and location on the plants and at different nodes to span both the vegetative and fruiting phases of growth.

In NY (Long Island), McGrath has reported that sensitivity to high ambient 03 concentrations and infection by powdery mildew was related to plant developmental stage in cucurbits; fruiting coincided with increased sensitivity to both. Proposed research is to further characterize whole plant responses to 03 and foliar pathogens, emphasizing alterations in sensitivity to either, in relation to carbon allocation shifts that occur during fruiting. The impact of 03 on yield will be examined by using foliar applications of EDU to protect plants from natural exposures. Cultivars varying in their sensitivity to 03 will be compared for foliar injury under natural field conditions and in greenhouse exposure chambers (cooperative study with Zilinskas, NJ). Changes in susceptibility to 03 injury and to infection by powdery mildew, associated with fruit production in cucurbits, will be investigated by using successive plantings of cultivars that differ in 03 sensitivity and comparing foliar symptomatology and yield during the growing season. A second approach to be utilized is a comparison among concurrent plantings of cultivars at various sites in the northeast.

At MA, Manning will continue investigations examining the response of cultivated and native plant species to 03. These studies will be conducted under controlled, greenhouse conditions to identify sensitive and tolerant genotypes. Promising candidates will be further evaluated in the field in open-top chambers. EDU will be used to determine ambient 03 effects on candidate plants in the field. Potential species include: browallia, turfgrasses, asters, blackberry, viburnum, apple, and white, loblolly, and short-leaf pine. This work will be done cooperatively with researchers from VA, AL, MD and TX where EDU application procedures and data collection will follow protocols developed at MA.

The response of white clover to 03 will be studied in depth. Little is known about the effects of 03 on leaf and shoot production. Detailed growth analysis, including RGR, ULR, RSR, LAR, LWR and SLA will be conducted with the NC-R and NC-S clones to determine the morphological and physiological responses to 03 under greenhouse and ambient conditions. EDU will be used in the field to distinguish 03 effects on growth parameters.

At TX, Flagler will continue research with antioxidant chemicals in cooperation with Manning (MA). These studies will consist of a multi-field site evaluation of EDU and Ozoban as protectants against 03 for growth and biomass accumulation of three pine species, white, loblolly and short-leaf. Physiological and biochemical investigations addressing the mode(s) of action of these chemicals will be initiated. Specific measurements for these studies will include foliar injury, height and diameter incremental growth, biomass accumulation, needle gas exchange rates (LiCor Photosynthesis system) and alterations in nitrogen metabolism (total foliar N, nitrate reductase activity) and Rubisco content (PAGE and immuno-blotting). Greenhouse studies, in the absence of 03, will be conducted to assess the effect of chemical application on growth, physiology and metabolism of the pine species.

At Boyce Thompson Institute, Cornell University, NY, a group of scientists (Laurence, group leader, Weinstein, Topa, MacLean, Jacobson, Comstock) will continue studies of ecosystem and individual plant responses to 0, and other environmental stresses. At the individual level, researchers will evaluate whether the differences in growth patterns between seedlings and mature hardwood trees (red oak and sugar maple) lead to a difference in sensitivity to O,. At the community level, studies will examine the effect present and future patterns of O, have on the productivity of eight major forest tree species in four regional forest types of North America. In this project, scientists will be evaluating the degree to which nutrient of soil limitations and inter-tree competition after the 03 damage to productivity. At the regional level, researchers will assess, through modelling, the relative significance of 03 in comparison to other climate change factors in altering the productivity and composition of forests in the southeasfem U.S.

Studies with sugar maple are designed to test the central hypothesis that long-term exposure to 03 reduces allocation of photosynthate to roots of seedlings. In testing this hypothesis, scientists will examine changes in source/sink relationships and a direct inhibitory effect of 03 on the transport process (inhibition of phloem loading or sieve tube occlusion). Sugar maple seedlings will be grown for three years in open-top chambers at three 03 levels. Changes in seasonal growth and carbon source/sink relationships will be assessed by examining carbon acquisition, allocation (to various plant organs), partitioning (among various chemical fractions), in conjunction with relative growth measurements. Staggered harvests will be conducted over three growing seasons, and phenological observations, and measurements of net photosynthesis (LiCor photosynthesis system), relative growth rates, and carbon partitioning among starch, sucrose and reducing sugars, will be interfaced with short-term 14CO2,-labeling experiments. These latter experiments will assess the fate of recently assimilated 14CO2 by following partitioning of label into various chemical fractions, including respiratory losses. Ozone inhibition of photosynthate transport will be assessed using both autoradiography on leaf tissue (to determine vein loading) and micro-autoradiography on leaf and stem tissue (to examine the transport pathway). Finally, 03 effects on N retranslocation in sugar maple saplings will be examined. Foliar samples will be collected bi-weekly from mid-July to the beginning of October and analyzed for N, total protein, and soluble amino acid content. Support for the hypothesis that 03 interferes with N retranslocation would occur if decreases in foliar N content over time were slowed by high 03.

At USEPA/Corvallis (Andersen), has reported that 03 exposure of ponderosa pine seedlings reduces new growth and new root starch concentration the year following exposure (Andersen et al., 1991) and significantly increases the C02/02 ratio from the root/soil compartment (Scagel and Andersen, 1994). The hypothesis that 03 accelerates root turnover and stimulates microbial activity in these plant systems is currently being investigated. Efforts are now, under way to separate root from microbial respiration. The future direction of the root/rhizosphere research is to further characterize changes in root metabolism as a result of 03 stress, and determine how metabolic shifts may impact the plants ability to acquire both moisture and nutrient resources, and respond to other co-occurring stresses. In addition, shifts in rhizosphere biology, resulting from 03 exposure, will be further characterized.

At USDA/Raleigh, Miller and colleagues have selected ten clones of clover based on visible injury response to 03 exposure (100 nl L-', 6 h d-' for 3 days). Field and green-house studies in charcoal-filtered air indicated no differences in stomatal conductance between tolerant and sensitive clones However, the stomatal response to elevated CO, was greater in the a resistant genotype (NC-R) compared to a sensitive genotype (NC-S). Research at Raleigh will continue to investigate stomatal characteristics as a mechanism of 03 tolerance. Clones will be evaluated under a range of 0; exposure conditions, in field and laboratory studies, for stomatal response (LiCor diffusion porometer). These studies skill include 'real time' measurements of 0; flux to better quantify physiologically effective 0, doses.

At VA, Chevone will utilize the NC-R and the NC-S clover clones to continue studies of the mechanisms of 03 tolerance. Previous experiments, using high 0, exposures (150 to 200 nl L' , 4 h) and low light intensities (350 to 600 uE m-z s-'), resulted in >50% increases in stomatal resistance within 90 min., limiting internal 03 flux. In proposed studies, net photosynthesis and stomatal resistance will be measured during 03 exposures (50 to 100 nl L-' 6 h d-' for 4 or 8 days) at low and high light intensities, 500 and 1100 uE m-' s-', respectively. Changes in apparent 03 flux will be calculated to estimate the internal dose. This information will be utilized to develop fumigation regimes that minimize stomatal closure and provide a more uniform level of oxidative stress to allow for the maximum response of the antioxidant system and other protective, biochemical processes.

2. Factors that Determine Plant Response to Ozone

The importance of the ambient environment in affecting a plant's reaction to O, has been long recognized in the air pollution literature, but has been investigated primarily in controlled, experimental situations (Runekels, 1992). Much less is known about plant response in a natural field setting (Grunhage and Jager, 1994; Krupa et al., 1993a). Consequently, a research component that focuses on ambient 03 exposure dynamics and environmental interactions in relation to whole plant effects has been added to the revised proposal as a third primary objective (Fig. 1).

One of the key issues in ambient 03 exposure-plant response research is to provide a satisfactory mathematical explanation of stochastic cause-effect relationships. At MN, Krupa (in cooperation with Maiming, MA) will continue development of a predictive model of plant response to ambient 03 that incorporates the influence of other environmental variables. Plant species, such as radish, white clover, snapbean and black cherry (all species with known sensitive cvs. or clones) will be used in modeling the dynamics of ambient 03 exposures and biomass or yield responses. The comparison of tolerant and sensitive genotypes is an important consideration since the response of sensitive plants often has been found to have little relationship to ambient 03 concentrations. (Tonneijck and Bugter, 1991; Krupa et al., 1993). The approach used will be an extension of the previous work of Krupa et al. (1994a, 1995) where the daily dynamics of ambient photochemical 03 production, patterns of 03 flux from the atmosphere into plant canopies and the foliar injury or yield response per se were integrated to provide a cohesive view of cause and effect. Such an effort is required in understanding real world exposures and their adverse effects on plants.

At AL, Chappelka has conducted field surveys of follar03 injury in wilderness areas and will continue these surveys in the Great Smokey Mountains National Park. Visible injury response data will be correlated with regional 03 concentrations and environmental factors

such as temperature, soil moisture and vapor pressure deficit to develop exposure-response correlations. Initial studies will be conducted to relate visible foliar injury to growth of forest species, using radial growth of selected species as the primary indicator. Simulated ecosystem studies well be conducted in open-top chambers to investigate O, effects on an early Successional old-field community Response measures well include reproductive effort, nutrient cycling litter decomposition and shifts in species diversity.

3. effechanisimv n( 0,-one Toxicity and 0,-one Tolerance.

Several mechanisms of 0, toxicity or tolerance will be investigated by scientists at cooperating research institutions (Fig. 1). These studies will utilize plants of known 03 sensitivity, as determined in whole plant studies; or plants that have been genetically altered with respect to their o-Kidative stress capacity.

At N1, Zilinskas will continue to examine the role of antioxidant enzymes in providing 03 tolerance. The necessary molecular probes and transgenic plants have been produced and the proposed studies will provide a better understanding of the genetic and molecular basis of expression and the functional significance of these enzymes in the protection of plants against 03 and other oxidative stress conditions.

Five specific objectives will be examined during the next five years. These are:

1. to follow the expression in pea of SOD, APX and MDAR in response to 03;

2. to analyze the effects of 03 exposure (on the whole plant as well as at the physiological and molecular level) on transgenic plants which overproduce the antioxidant enzymes SOD and APX;

3. to construct transgenic plants which overproduce MDAR and to examine the tolerance of these plants to 03;

4. to construct (using antisense technology) and analyze the phenotype of transgenic plants which underexpress SOD, APX and MDAR; and

5. to construct plants (or obtain by sexual crossing) which overproduce all three enzymes (SOD, APX, MDAR) and examine the tolerance of these plants to 03.

With regard to objective 1, data obtained thus far has shown that there is clearly a response of these enzyme systems to 0, and that the overall regulation of their expression appears to be complex. Previous experiments were conducted at 03 doses (300 nl L-' for 1.5 to 6 h), which differentially affected the expression of SOD and APX genes. Proposed research will utilize these high concentrations and lower ones that are comparable to high ambient concentrations (100 to 150 nl L for 4 to 6 h). Other laboratories have shown that total APX activity increased considerably under low to moderate 03 exposures; the situation with SOD activity is not as clear, likely being influenced by the inadequacy of the indirect solution assays that have routinely been applied. With the availability of specific cDNA and antibody probes, the problem can be addressed more systematically and quantitatively, in addition to examining the more fundamental question of the level of regulation (i.e. transcription or post-translational).

The overall working hypothesis for objectives 2 through 5 is that a balanced set of enzymes is necessary to minimize oxidative damage from 0,. However, since it is not known which enzymes might be rate-limiting, the approach proposed is to overexpress each enzyme individually, as well as in concert. Likewise, it is known that an excessively high level of overproduction of SOD in the chloroplast provides no protection against 03, whereas a relatively low level of overexpression provides protection against oxidative stress. Thus it is necessary to analvze a number of independent plants where transgene expression varies from low to very high levels of overproduction of these antioxidative enzymes. As antisense technology allows one to address the same question in a converse manner, these experiments will provide an independent means to examine the role of these enzymes-in 03 tolerance.

Construction of transgenic plants will follow protocols and design previously used in Zilinskas' laboratory. Analysis for the expression and stable integration of the transgenes will be performed as in the past. Once plants have been identified that have single gene insertions and express known (low, medium and high) levels of the transgenes, their physiological response to 03 and other oxidative stress conditions will be assessed. Collaboration with other cooperating scientists (Miller,USDA/Raleigh; Lee, USDA/Beltsville and Mulchi, MD) will enable measurement of physiological responses of the genetically altered plants under field conditions and in precisely controlled fumigation chambers.

At PA, Pell will continue to explore the nature of protein oxidation by 03. Of major interest is the capacity of 03 to induce carbonyl formation on proteins and which proteins may be most susceptible. Antibodies are available for carbonyl groups to allow detection of such modifications. Another interest concernss the rapid loss of the Rubisco small subunit mRNA induced by 03 exposure. Studies will examine whether this loss in message can be detected for other important proteins, and the mechanism by which such losses occur. One critical area of this research is to determine whether 03 influences the stability of key mRNA transcripts. Also at PA, work is progressing on transforming potato plants to prevent production of 03-induced ACC synthase. Once this transformation is completed, ethylene production in response to 03 exposure will be examined to determine whether 03-induced emission of ethylene and the hypothesized acceleration of foliar senescence is a completely negative response, or perhaps partially compensatory.

A key question arising from research at PA is whether 03-induced nuclear encoded events, e.g. decline in rbcs and induction of ACC synthase leading to ethylene emission, regulate chloroplastic events. Alternatively, is the reduction in Rubisco a direct result of oxidation phenomena occurring within the chloroplast? Transformed lines of tobacco (developed by Zilinskas, NJ) which overexpress antioxidants at specific locations within the cell, viz. cytoplasm, chloroplast or both, will be used by researchers at PA to determine:

1. Whether 03-induced nuclear events, e.g. drop in rbcs or induction of ACC synthase, will occur in tobacco plants with elevated protection from oxidation vis a vis overexpres- sion of plastid AP; and whether these plants will exhibit any chloroplastic effect on concentration of Rubisco; and

2. If similar 03-induced nuclear events occur in plants exhibiting increased cytoplasmic antioxidizing potential.

At USDA/, Beltsville, Robinson will examine the role of ascorbate (AA) and natural sulfydryl reagents in ameliorating 03 damage in crop plants. In regard to the ability of AA to serve as an antioxidant, many questions remain to be answered. Among these are 1) the magnitude of carbohydrate status in relation to AA synthesis, transport and redox status, 2) the magnitude of detoxification of pollutant products in relation to AA content, 3) the mechanisms by which oxidized and reduced AA are retained within the chloroplast or transported to sites of oxidative damage, and 4) the dependence of AA synthesis, transport and redox status upon leaf starch, sucrose and hexose sugar levels, and the impact this may have on crop productivity in ambient air environments The proposed research should provide information to answer these questions and define the role of AA in plant tolerance to O,

The research approach will be to utilize genetic lines of soybean, snapbean and spinach which are sensitive or tolerant to O, and to induce increased foliar carbohydrate production via elevated CO= and nitrogen limitation Control plants will be maintained with limited or normal hexose sugars. Photosynthetically competent chloroplasts will be isolated from test and control plants, and synthesis of AA from '0C-labeled precursors, as well as the transport of oxidized and reduced AA across the chloroplast envelope will be examined. Intact leaves will be selected from 03-treated and control plants, lyophilized, subjected to the nonaqueous technique for leaf organelle separation and the fractions will be analyzed for photosynthates and 'real-time' AA synthesis, redox status, and intracellular transport patterns.

At USDA/Beltsville, Lee will continue studies on mechanisms of 03 tolerance using resistant and susceptible genotypes of soybean and snap bean to investigate the protective mechanisms) of ethylene diurea (EDU). This research will involve the development of methods and techniques (e.g. high-performance liquid chromatography-electrochemical detection [HPLC-EC], electron paramagnetic resonance [EPR], or spectrophotometry) to detect oxyradicals in plant tissues. Studies will include physiological and biochemical responses of antioxidant protective mechanisms and assessment of identified resistance components for improved crop performance.

At USDA/, Raleigh, Miller and Booker will examine mechanisms of plant tolerance to 03 using genotypes of white clover and snap bean that have a wide range of sensitivity to 03. Preliminary studies with clover clones have shown that basal levels of several antioxidant enzymes (GR, APX, DHAR, MDHAR, and SOD) are not different in sensitive and tolerant genotypes. Enzyme activities will be characterized before and after 03 exposure. Changes in lignin derivatives and phenolic polymers in response to 03 treatment will be examined in other genotypes of clover and snap beans.

At VA, Chevone will continue studies of plant antioxidant systems as a mechanism of 03 tolerance. Clover clones NC-S and NC-R will be used in CSTR fumigations in a stepfunction concentration profile that simulates ambient conditions (03 range of 40 - 120 nl L-', 8 h d-', 4 or 10 d) to allow full response of antioxidant enzymes. The activity of SOD, APX and catalase (CAT) will be characterized in the clover clones (solution assays), prior to and after 03 exposure. Isozyme activity of SOD will be analyzed using native PAGE and the NBT activity stain and mRNA levels of plastid SOD will be examined using probes from pea (B. Zilinskas, NJ). Protein concentration of enzymes and isozymes will be assayed using specific antibodies as these are available. A second area of research is the role of stressresponse proteins that include the heat-shock and chilling proteins, dehydrins and LEAS. Using specific antibody probes, the endogenous ncentrations and induction of these proteins will be examined in the NCR and NCS clover clones before and during 03 exposure.

Measurement of Progress and Results

Outputs

• Demonstrate that exposure to ozone accelerates leaf senescence.
• Determine if elevated CO2 levels helps ameliorate the severity of ozone induced stress, possibly through an increase in antioxidant enzyme activity.
• Development of more sophisticated methods of assessing ozone damage to horticultural crops and forests in North America.
• Describe the interactions of ozone stress with other environmental stressors on crop production, with the goal of developing better models for predicting the effects of prolonged episodes of ozone exposure.
• Verify the relation of ozone induced oxidative stress with the activities of antioxidant enzymes, e.g. ascorbate peroxidase and superoxide dismutase.
• Development of a database clearly implicating ozone is a major air pollutant in North America.

Outcomes or Projected Impacts

• Better understanding of the impact of ozone of agricultural production.
• Identification of ozone tolerant strains of plants.
• Explore the development of transgenic ozone tolerance in crops.
• Provide recommendations for air quality standards based on more accurate understanding of how ozone pollution causes economic losses from reduced crop productivity.

Milestones

(1999): Screen targeted crop species as whole plants.

(2000): Continue screening of targeted crop species. Conduct whole-plant-response to O3 experiments, as influenced by temperature, solar radiation, moisture, CO2, dew point an nutrients.

(2001): Characterize plant response to O3. Define levels of O3 tolerance in targeted crops.

(2002): Test changes in antioxidants, rubisco, gene expression of key enzymes, presence of stress proteins, and production of phenolics.

(2003): Describe mechanism of O3 tolerance. Begin development of O3 tolerant germplasm.

(0):0

Projected Participation

View Appendix E: Participation

Outreach Plan

Organization/Governance

The NE-176 Technical Committee is organized according to the revised 1986 Manual for Cooperative Regional Research (USDA/CSRS). Membership is in accordance with pages 18-21 of the Manual with each participating station and agency limited to one vote. All voting members are eligible for office. A Chairperson and a secretary are elected annually. Their duties are outlined on page 20 of the Manual. Meetings of the Technical Committee are held in the winter of each year in accordance with pages 21﷓21 of the Manual.

Literature Cited

Attachments

Land Grant Participating States/Institutions

AL, MA, MD, MN, NJ, NY, PA, VA

Non Land Grant Participating States/Institutions