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Glutathione reductase (EC 1.6.4.2) was purified from Eastern white pine (Pinus strobus L.) needles. The purification steps included affinity chromatography using 2', 5'-ADP-Sepharose, FPLC-anion-exchange, FPLC-hydrophobic interaction, and FPLC-gel filtration. Separation of proteins by FPLC-anion-exchange resulted in the recovery of two distinct isoforms of glutathione reductase (GR(A) and GR(B)). Purified GR(A) had a specific activity of 1.81 microkatals per milligram of protein and GR(B) had a specific activity of 6.08 microkatals per milligram of protein. GR(A) accounted for 17% of the total units of glutathione reductase recovered after anion-exchange separation and GR(B) accounted for 83%. The native molecular mass for GR(A) was 103 to 104 kilodaltons and for GR(B) was 88 to 95 kilodaltons. Both isoforms of glutathione reductase were dimers composed of identical subunit molecular masses which were 53 to 54 kilodaltons for GR(A) and 57 kilodaltons for GR(B). The pH optimum for GR(A) was 7.25 to 7.75 and for GR(B) was 7.25. At 25 degrees C the K(m) for GSSG was 15.3 and 39.8 micromolar for GR(A) and GR(B), respectively. For NADPH, the K(m) was 3.7 and 8.8 micromolar for GR(A) and GR(B), respectively. Antibody produced from purified GR(B) was reactive with both native and denatured GR(B), but was cross-reactive with only native GR(A).

Ozone is known to be the most widespread phytotoxic air pollutant. Ozone causes visible injury, reductions in photosynthesis, growth, and yield. Plant response to ozone may vary with species, varieties, and physiological age. Comparison between sensitive and tolerant cultivars has a key role in assessing ozone damage, investigating the sites of cellular injury, and identifying ozone tolerance mechanism. The objectives of this study were to investigate the effects of high ozone concentration (200 ppb) as well as ambient ozone concentrations (under field conditions) on net CO2 assimilation and PSII function in plants with different sensitivities to ozone. Two species of plants, tobacco (Nicotiana tabacum L.) and black cherry (Prunus serotina) were studied. Gas exchange analysis and chlorophyll fluorometry were utilized to characterize physiological function. Two tobacco cultivars, Bel-B and Bel-W3, tolerant and sensitive to ozone, respectively, were grown in a greenhouse supplied with charcoal filtered air and then exposed to 200 ppb ozone for 4hr. Effects on chlorophyll fluorescence, net photosynthesis, and stomatal conductance are described. Quantum yield was calculated from chlorophyll fluorescence and the initial slope of the assimilation-light curve measured by the gas exchange method. Only the sensitive cultivar, Bel-W3, developed visible injury symptoms involving up to 50% of the 5th leaf. The maximum net photosynthetic rate of ozone-treated plants of the tolerant cultivar was reduced 40% compared to control plants immediately after ozone fumigation; however, photosynthesis recovered by 24 hr post fumigation and remained at the same level as control plants. In the sensitive cultivar, on the other hand, ozone exposure reduced maximum net photosynthesis up to 50%, with no recovery, apparently causing permanent damage to the photosystem. Reductions in apparent quantum efficiency, calculated from the assimilation-light curve, differed between cultivars. Bel-B showed an immediate depression of 14% compared to controls, whereas Bel-W3 showed a 27% decline. Electron transport rate (ETR), at saturating light intensity, decreased 58% and 80% immediately after ozone treatment in Bel-B and Bel-W3, respectively. Quantum yield decreased 28% and 36% in Bel-B and Bel-W3, respectively. It can be concluded that ozone caused a greater relative decrease in linear electron transport than maximum net photosynthesis, suggesting greater damage to PSII than the carbon reduction cycle. Two different sensitivity classes of black cherry, tolerant and sensitive, growing under natural environmental conditions in Giles County, VA were assessed for physiological responses to ambient ozone concentrations. Additional measurements were made at two other sites near Blacksburg. Leaf gas exchange rates and visible foliar injury were determined monthly during the growing seasons of 2000, 2001, and 2002 to characterize the relationship of injury to altered photosynthetic function. Ambient ozone levels were sufficient to induce visible symptoms which were highly correlated with a reduction in PnMAX (maximum net photosynthetic rate under saturating light conditions) and à CO2 (quantum yield for carbon reduction) only in sensitive black cherry. Electron transport rate (ETR) and quantum yield of PSII (à PSII) were also reduced in sensitive black cherry. Maximum photochemical efficiency (Fv/Fm) in sensitive trees was severely damaged by ambient ozone. There were positive correlations between increasing cumulative ozone concentration and substantial reductions in PnMAX and in à CO2 of sensitive trees compared to tolerant trees. There was a negative correlation between chlorophyll content and percent leaf injury in sensitive black cherry Ph. D.

Vitamin C (ascorbic acid, AsA), an important primary metabolite of plants, functions as an antioxidant, an enzyme cofactor, and a cell-signaling modulator in a wide array of crucial physiological processes including biosynthesis of the cell wall, secondary metabolites and phytohormones, stress resistance, photoprotection, cell division, senescence, and growth. To identify genes that may regulate vitamin C levels in plants, about 3000 activation-tagged Arabidopsis lines were treated with ozone, which is a power oxidizing agent. Two mutants were selected for identification of potential genes involved in the regulation of vitamin C synthesis. A putative F-box gene, VCF1, and a purple acid phosphatase, AtPAP15, were identified for further characterization. Two homozygous SALK T-DNA knockouts in the open reading frame (ORF) of VCF1 exhibited high tolerance to ozone when treated with 450 ppb for 3 hours and the AsA levels of these mutants were 2 to 3 fold higher than wild-type (wt) plants. Developmental studies, using RT-PCR, indicated that foliar expression of the VCF1 gene increased with plant age from 1 to 5 weeks, whereas AsA decreased during this same period. The expression of VCF1 was higher under a low-light condition in which AsA was reduced considerably. The AsA levels in two VCF1 overexpressing lines were only 50 to 70% of wt plants. These results suggested that the putative F-box gene functions as a negative regulator of leaf ascorbate content. Overexpression of AtPAP15 with the CaMV 35S promoter resulted in up to 3-fold higher AsA levels than wt plants, where two independent SALK T-DNA insertion mutants in AtPAP15 had 50% less AsA than wt plants. Enzyme activity of bacterially expressed GST:AtPAP15 was greatest with phytate as a substrate indicating that AtPAP15 is a phytase. Phytase catalyzes hydrolysis of phytate (myo-inositol hexakisphosphate) to yield myo-inositol and free phosphate. Thus, AtPAP15 may regulate AsA levels by controlling the input of myo-inositol into this branch of AsA biosynthesis in Arabidopsis. AtPAP15 was expressed in all tested organs in wt plants and suggests that the enzyme may have functions other than phytate degradation during seed germination. Ph. D.

Winter injury of bermudagrass (Cynodon spp.) continues to be a problem across the transition zone. In an attempt to delay or induce winter dormancy while maintaining cold hardiness, applications of seaweed extract (SWE) (0.54 kg ha-1), ethephon (16 L ha-1), Fe (1 kg ha-1), and N (49 kg ha-1) took place every three weeks during the fall of 2001 and 2002. Cultivars examined included 'Riviera', 'Midiron', 'Princess', and 'Tifway'. Tifway exhibited greatest fall color retention in both years of the study. Ethephon promoted early senescence and turfgrass quality during fall ratings in both years of the study while N, Fe, and SWE increased quality over the control in 2001 and only N showed better quality and color retention over the control in 2002. Samples removed from cold acclimated plots were artificially frozen as a measure of cold hardiness. Treatments did not have an effect on post freeze regrowth, however, cultivar was significant in both years. Midiron showed best regrowth followed by Riviera, Tifway, and Princess. In both years Riviera and Midiron displayed the quickest and greatest amount of spring greenup followed by Tifway and then Princess. Ethephon reduced greenup in both years and SWE, Fe, and N showed no differences from the control in 2001 and Fe showed significantly better greenup in 2002. Proline and Linolenic acid levels were highest in Midiron, followed by Riviera, Tifway, and Princess. Nitrogen, SWE, and Fe generally did not have an effect on linolenic acid and no consistent effects were noted on proline concentration. Ethephon treatments did not have an effect on linolenic acid levels, however, there was a negative effect on proline concentrations. The results of this study indicate that judicial N applications during the fall can promote color retention and do not have a negative effect on bermudagrass cold-tolerance. Linolenic acid and proline findings also help to explain differences in cold-tolerance between different bermudagrass cultivars. Ph. D.

1-substituted 1,2,4-triazoles form a class of agricultural chemicals known as sterol-inhibiting fungicides (SIFs). These fungicides function through the inhibition of sterol synthesis, which ultimately affects cell membrane fluidity and permeability. Of the two main types of sterols in plants, free sterols (FSs) are thought to be incorporated into membranes while conjugated sterols such as steryl esters (SEs), hypothetically, regulate homeostasis by inserting or removing FSs from cell membranes under changing environmental conditions. Non-target algae species possess sterol synthesis pathways that are affected by a range of SIFs. One of the main objectives of the current study was to determine the reason for the observed sensitivity of C. fusca and the tolerance of C. kessleri to SIFs relative to total lipid, FS, SE and FFA composition. These parameters were measured using gas chromatography and mass spectroscopy techniques. Both quantitative and qualitative differences in sterol number and type were noted relative to the FS and SE composition of the two species of algae over time. Notably, SEs were detected in both species of algae, although presence and amount varied with the organism. While SEs were more abundant in C. kessleri, higher amounts of FSs were found in C. fusca. The FS/SE ratios were 64/36 and 88/12 percent of the total sterol in C. kessleri and C. fusca, respectively. Treatment of C. fusca with 2, 4, and 6 ppm and C. kessleri with 6,12, and 24 ppm propiconazole caused an accumulation of methylated precursor sterols, resulting in slightly more FSs in both algae. Only 3 of the FSs produced following treatment were different from the control in C. fusca while 9 new sterols were found in C. kessleri. Treatment also altered the SE fraction in both species, with fewer SEs produced compared to the control, but more novel sterols in C. kessleri, suggesting a possible inverse relationship between FSs and SEs in both organisms. Several studies have implicated lipid/sterol concentrations with the potential for cellular bioaccumulation of lipophilic xenobiotics as they relate to membrane permeability. Cell age and environmental parameters can also affect lipid composition of algae. Although cell age did not affect the qualitative sterol composition of C. fusca and C. kessleri, quantitative differences were observed. Plants exposed to chemical and other environmental stresses accumulate free fatty acids (FFAs), which may be linked to biophysical membrane changes. SIF sensitive C. fusca, had inherently higher levels of FFAs than C. kessleri. Qualitatively, C. fusca exhibited higher percentages of 18:1 and lower ratios of 18:2/18:3 FFAs than C. kessleri. In response to increasing SIF treatment, the ratio of 18:2/18:3 FFAs increased in C. kessleri and declined in C. fusca. The amount of total lipid produced in the cells of C. fusca was higher than in C. kessleri during all growth stages. Variations were observed in lipid measured as a percent dry weight compared to lipid/cell as the cultures age. Inherent differences in FS, SE, and lipid composition of C. fusca and C. kessleri as well as age related changes could account for the differences in the susceptibility of the two algae to propiconazole. Master of Science

Spatial and temporal variation in foliar gas exchange on both a diurnal and seasonal scale was examined in 15-year-old fertilized and non-fertilized loblolly pine in the upper and lower thirds of crowns in stands located in the North Carolina sandhills. Photosynthesis rates between control and fertilized stands for both seasonal and diurnal measurement periods were different during only three months. Photosynthesis rates were consistently greater in the upper third of the crown compared to the lower third. Seasonal trends in both conductance and transpiration closely resembled trends found in seasonal photosynthesis. Foliar nitrogen concentrations were greater in fertilized stands for all months sampled. However, nitrogen content generally did not correlate with photosynthesis rates. Mean monthly water use efficiencies were significantly higher in fertilized stands during two months and were usually greater in upper crown foliage. Common empirical gas exchange models reveal that light and vapor pressure deficit (VPD) explain a majority of the variation observed in photosynthesis and transpiration, respectively. Conductance was not modeled since environmental variation did not adequately explain conductance patterns. Predicted light response curves reveal that upper crown foliage has higher maximum photosynthesis rates, respiration rates, light compensation points, and lower initial quantum yield compared to lower crown foliage. Models predict that foliage from fertilized stands is more sensitive to VPD and light during the growing season. Transpiration models predict highly variable responses to VPD depending on the treatment combination and season. Model R-square and predicted gas exchange values suggest that seasonal acclimation occurred. Master of Science

A white clover ( Trifolium repens ) system, based on one ozone-sensitive (NC-S) and one ozone-resistant (NC-R) clone, has been developed as an indicator to estimate the effects of tropospheric ozone on plant biomass production. A reduction in the vegetative biomass ratio (NC-S/NC-R) of the clones was correlated with increasing concentrations of ozone during a 28-day exposure period. However, the mechanism of ozone sensitivity or tolerance at the biochemical or molecular level is not known. Superoxide dismutase isozyme activities in the two clones did not respond differently to ozone treatment. However, catalase activity increased somewhat more in the leaf tissue of NC-R, compared to NC-S, after ozone treatment. Two-dimensional polyacrylamide gel electrophoresis (PAGE) showed the presence of two proteins (Ozone-Response-Proteins, ORPs) that were more responsive to ozone in the tolerant genotype than in the sensitive one. After ozone treatment for three days, the ORPs were four-fold higher in leaf tissue of NC-R compared to NC-S. Also, the amount of the ORPs was twenty-fold higher in leaf tissue of ozone-treated NC-R than in that of control NC-R. These proteins have apparent molecular weights of 21.5 kD and 23 kD and isoelectric points of 4.1-4.4 on SDS-PAGE gels. The filtrate of a 100 kD concentrator showed that the native molecular weights of the ORPs were less than 100 kD. The results obtained from a study of field samples demonstrated that protein content in leaf tissue of both NC-R and NC-S was positively correlated with ORP content. Master of Science

Carbon dioxide efflux rates (CER) of stems and roots of overstory and understory black cherry (Prunus serotina Ehrh., BC), red maple (Acer rubrum L., RM) and northern red oak (Quercus rubra L., RO) trees were monitored over two growing seasons at two contrasting sites in the Great Smoky Mountains to investigate diurnal and seasonal patterns in respiration and to develop prediction models based on environmental and plant parameters. CER of small roots (din situ measurements by using permanently installed flexible cuvettes. Temperature-adjusted CER of roots showed no diel variation. The moderate long-term changes occurred simultaneously in all species and size classes, suggesting that they were driven mostly by environmental factors. Mean root CER ranged from 0.5 to 4.0 nmol g⁻¹ d.w. s⁻¹. Rates were up to six times higher for fine roots (d10 cm) and twigs (dD·lnd, with D between 1.2 and 1.8. A new, scale-invariant measure of CER, based on D, facilitated comparisons across diameters. Q₁₀ varied with the method of determination, and it was higher in spring (1.8-2.5) than in autumn (1.4-1.5) for all species. Daytime bole CER often fell below temperature-based predictions, likely due to transpiration. The reduction (usually ±50%) deviations from the predictions. Deviations were higher in the canopy than in the understory. Mean bole maintenance respiration (at 20°C and d=20 cm) was 0.66, 0.43 and 0.50 μMol m⁻¹, while the volume-based growth coefficient was around 5, 6 and 8 mol cm⁻³ for BC, RM and RO, respectively. In a controlled study, BC and RM seedlings were fumigated in open-top chambers with sub-ambient, ambient and twice-ambient levels of ozone. The twice-ambient treatment reduced stem CER in BC by 50% (p=0.05) in July, but there was no treatment effect in September or in RM. Ozone reduced root/shoot ratio and diameter growth in BC, and Pmax in both species. Ph. D.

Procerum root disease (PRD) is a serious problem in eastern white pine (Pinus strobus L.) Christmas tree plantations in VIrginia. Procerum root disease is caused by Leptographium procerum (Kendr.) Wingf which is believed to be transmitted by the pales weevil Hylobius pales (Herbst). Symptoms of procerum root disease include reduced shoot elongation, reduced leaf conductance, low photosynthetic activity, low pre-dawn water potential and chlorosis of foliage. Resinous occlusion of the sapwood at the root collar is the likely cause of the suite of symptoms that resemble water stress. Increased incidence ofPRD has been associated with trees growing in poorly drained soils in low lying areas. Two studies were undertaken to explore the relationship of site factors and vascular conductivity of sapwood to the expression ofPRD symptoms in P. strobus. In the first study, plots were established in a variety of drainage classes in two Christmas tree plantations. Leaf conductance was monitored periodically in conjunction with measurements of soil factors to assess the role of abiotic factors on foliar symptom expression. At the termination of the field monitoring, trees were harvested and three vascular disease severity variables were measured: hydraulic conductivity of sapwood, percent basal occlusion and sapwood moisture content. These disease severity variables describe the permeability of sapwood to water and the relative hydration of the sapwood. Reduced leaf conductance was associated with reduced stem hydraulic conductivity, reduced sapwood moisture content and increased basal occlusion. Increased vascular disease severity and foliar symptom expression were associated with increased soil moisture content and several other factors that relate to soil moisture retention (percent slope, total porosity, textural class and bulk density). The second study was initiated to study the development of PRD in artificially inoculated P. strobus seedlings exposed to three soil water classes: droughty, optimum and saturated. Seedlings in the optimum soil water class exhibited the greatest biomass accumulation and shoot elongation, while seedlings in droughty showed the least. No negative effects of L. procerum inoculation and no symptoms ofPRD were observed eight months after inoculation regardless of the soil water class. Master of Science

Loblolly pine (Pinus taeda) occurs in mixed stands with hardwood species including red maple (Acer rubrum) and sweetgum (Liguidambar styraciflua) across much of the southern United States. Rising atmospheric CO₂ concentrations over the next several decades could influence competitive interactions between these species. This research examined the effects of increased atmospheric CO₂ concentration in combination with the availability of other resources on growth and gas exchange characteristics of these species grown in direct competition in miniature stands and compared characteristics of miniature stands with field-grown stands of similar composition. In a greenhouse study, loblolly pine and red maple were grown in monoculture and 50:50 replacement mixtures at a 2.54 x 2.54 cm spacing for two growing cycles under ambient (408 ppm) or elevated (806 ppm) CO₂ concentrations and well-watered or droughted (0.5x well-watered) conditions. Loblolly pine dominated all mixed stands and was proportionally larger while red maple was smaller in mixtures versus monocultures. This pattern was also observed in weed-free f ld-grown stands after five growing seasons. Biomass data from loblolly pine and red maple grown in monocultures and 50:50 replacement mixtures at 2.54, 5.08 and 7.62 cm2 initial spacing suggest that relative dominance of species in miniature stands is not dependent on initial spacing. Dominance of red maple over loblolly pine in all mixed stands in this experiment suggests that soil medium is important in determining relative growth of these species. Photosynthesis rates and response to elevated CO₂ differed between whole stands and individual seedlings from these stands. Individual seedlings tended to be more responsive to elevated CO₂ compared to whole stands suggesting reduced responsiveness of lower canopy leaves may dampen the effect of elevated CO₂ on canopy photosynthesis rates. Growth under elevated CO₂, alone or in combination with water did not affect relative sizes of these species. These results suggest that competitive interactions will not change between these species in response to growth in high CO₂ environments. Ph. D.

Procerum root disease of eastern white pine (Pinus strobus L.), caused by Leptographium procerum (Kendr.) Wingf., has been epidemic in Virginia Christmas tree plantations since 1990. Symptoms of chlorosis, wilt, and decreased apical growth resemble those of water stress. Resin infiltration of the xylem at the stem base may be responsible for vascular occlusion leading to severe water deficits and mortality. The pathogen has been isolated from the roots of ozone-sensitive eastern white pines in the field, although not from nearby tolerant trees, and it may be that ozone sensitivity predisposes the trees to infection. The objectives of my Studies were to investigate the physiology of diseased white pines, and to determine the effects of ozone fumigation on disease development. Impacts of vascular occlusion upon host water relations and gas exchange were investigated in 8-yr-old, plantation-grown, white pine Christmas trees. Disease severity was estimated as the proportion of resin-soaked cross-sectional area at the base of the stem. The linear response of a suite of six physiological variables to disease severity was highly significant. Individually, the variables pre-dawn water potential, daily change in pre-dawn to mid-day water potential, stomatal conductance, and photosynthetic and transpiration rates all decreased significantly with increasing disease severity. Fumigation studies were conducted on white and loblolly (P. taeda L.) pine seedlings to determine if ozone exposure increased the incidence of root disease or the amount of stem tissue colonized by L. procerum. Roots were inoculated by soil drenching with conidial suspension, and stems were wounded at the base and inoculated with mycelium. Beginning 24 h post-inoculation, and for 14 consecutive days, seedlings were fumigated in closed chambers with charcoal-filtered air or 200 ppb ozone for 5 h/day, then removed to a charcoal-filtered greenhouse. Six weeks post-inoculation, root and stem tissue were plated on a medium selective for L. procerum. Ozone treatment did not significantly affect the proportion of diseased roots per seedling or the vertical colonization of stem tissue in seedlings of either species. Ph. D.