In order to study senescence, we selected leaves which emerged early in the experiment and observed performance throughout development. At the Pennsylvania site, when black cherry seedlings and hybrid poplar ramets were approximately 18 cm in height, and when the second flush of sugar maple and northern red oak were initiated, a newly emergent leaf on each plant in every chamber was tagged for gas exchange and destructive harvest. Once every 1 to 2 weeks throughout the growing seasons, leaves were sampled from two plants per chamber. At the Norris, TN site, leaves of the second flush of northern red oak were destructively harvested following the same protocol as at the Pennsylvania site. Gas exchange analysis for this tissue was performed on fully expanded foliage.
In situ net photosynthesis and leaf conductance were measured at full sunlight by non-destructive gas exchange analysis with a Li-Cor 6200 closed-loop photosynthesis system (Li-Cor, Inc., Lincoln, NE) (Pell, Eckardt & Enyedi 1992; Hanson et al. 1994). Measurements were taken early in the day when conductance should have been at a maximum. After the gas exchange analysis, leaves were harvested in the field, frozen in liquid nitrogen and stored at –80 °C.
Cumulative O3 uptake was estimated for each sample for the duration of exposure to the pollutant. For the Pennsylvania site, daily stomatal conductance was estimated based on a linear interpolation of periodic stomatal conductance measurements of water vapour conductance. These values were converted to O3 conductance utilizing the molecular diffusivity of O3, and assuming leaf intracellular O3 concentrations close to zero (Laisk, Kull & Moldau 1989). The dose was calculated by multiplying the O3 concentration by the duration of treatment, for each day, from the measurements made in the open-top chambers every 30 min throughout the 8 h exposure period. The daily O3 uptake rate (mmol m–2 d–1) was calculated as the product of daily O3 conductance and daily O3 dose. Uptake rate was summed over time to give an estimate of the cumulative O3 uptake at the time of leaf harvest. At the Norris, TN site fewer stomatal conductance values were available. Analysis of stomatal conductance measurements from studies at this site in 1991–94 (Samuelson & Edwards 1993; Edwards et al. 1994; Hanson et al. 1994) revealed minimal variation, within or among seasons. Consequently, a water vapour conductance of 0·114 mol m–2 s–1, representing a multiple year average, was selected to estimate O3 uptake as described above. The daily O3 dose was determined using the monitoring data provided by Tennessee Valley Authority and described by Edwards et al. (1994).
Tissue extraction and analysis
Reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted, and solutions were treated with Chelex 100 (Biorad Corp., Hercules, CA) to remove potentially interfering metals. Total soluble protein (TSP) was extracted by grinding 0·2 g frozen tissue to a powder under liquid nitrogen in a mortar. The frozen powder was transferred to a glass vial and 10 volumes of cold extraction buffer were added. Extraction buffer (pH 7·8) for hybrid poplar and black cherry contained 90 mM potassium phosphate, 1 mM EDTA, 3% PVPP, 5 mM ascorbate, and 8% glycerol. For northern red oak and sugar maple extractions, the buffer was optimized by increasing the PVPP to 4% and including 1·5% PVP-40T. Tissue was homogenized on ice for 30 s with a Tissue-tearor (Biospec Products, Bartlesville, OK) and the homogenate subsequently centrifuged for 15 min at 15 850 g. Aliquots were immediately frozen in liquid nitrogen and stored at –80 °C until analysis. Total soluble protein was determined by the method of Bradford (1976), using BioRad protein dye concentrate and BSA as the standard. Rubisco quantity was determined as described by Eckardt & Pell (1994).
Enzyme assays were performed with a Beckman DU-64 spectrophotometer (Beckman Instruments, Inc., Fullerton, CA), using the Kinetics Soft-pac module to calculate rates of absorbance change over time. Enzyme activity measurements were run in triplicate, and TSP in duplicate.
Ascorbate peroxidase (APX) activity was measured by monitoring the consumption of ascorbate at 290 nm as described by Torsethaugen et al. (1997). The APX activity was measured during the first 60 s of the reaction. Final APX activity was calculated after correcting for non-enzymatic degradation of ascorbate (background) and ascorbate oxidase activity.
Glutathione reductase (GR) activity was measured using a method based on Smith, Vierheller & Thorne (1988). The reaction mixture contained 100 mM potassium phosphate, pH 7·8, 0·5 mM DTNB, 0·2 mM NADPH, 0·2 mM GSSG, and 100 μL crude plant extract in a final volume of 1000 μL. The change in absorbance of DTNB at 412 nm was monitored for 2 min. The reaction remained linear for more than 5 min. Each set of assays included measurements of a GR standard from wheat germ (Sigma Chemical Co.); periodic tests of a mix of all reagents except extract or extract plus all reagents except GSSG revealed no interfering ‘background’ reaction. On the basis of preliminary data we found that APX and GR activities were not affected by filtration of extracts with a Sephadex G-25 gel exclusion column (Pharmacia Biotech, Piscataway, NJ; PD-10).
The ORAC assay was adapted from the method of Cao et al. (1993). The assay measures the antioxidant potential of tissue samples. It is based on the capacity of free radicals generated by 2, 2’-azobis (d-amidinopropane) dihydrochloride (AAPH) to oxidize β-phycoerythrin (BPE), an algal pigment which fluoresces at 565 nm (excitation at 540 nm). In the presence of free radical scavengers, the free radical-induced loss of fluorescence of BPE is reduced. The assay was performed with a soluble leaf extract derived by the method described above, but using an argon purged 75 mM sodium phosphate buffer, pH 7·0; all extraction steps were performed under argon to further minimize oxidation. After centrifugation, the extracts were diluted to 0·2% concentration for immediate use in the ORAC assay. Leaf extract of all species did not absorb light at 540 or 565 nm; similarly there was no fluorescence when extracts were excited at 540 nm.
The sample reaction mixture contained 75 mM sodium phosphate, pH 7·0, 16·7 nM BPE (CyanoTech Corp., Hawaii, USA), 3 mM AAPH (Wako Chemicals USA, Richmond, VA), and 20 μL dilute plant extract in a final volume of 200 μL. Blanks were comprised of BPE and AAPH in the absence of extract, to measure free radical-induced decay in fluorescence of the pigment. Standards contained no plant extract, but instead had the water-soluble antioxidant Trolox (Boehringer Mannheim, Indianapolis, IN) added to final concentrations of 1–3 μM. Triplicates of each blank, sample and standard were loaded onto 96 well untreated Costar microtitre plates; fluorescence was measured with a PerSeptives Cytofluor II fluorescence plate reader (PE Biosystems, Perkin Elmer, Inc., Norwalk, CT). The assay was conducted at 37 °C and followed to completion. ORAC values were calculated using the area under the fluorescence decay curves as in Cao et al. (1993):
ORAC value = (Fluorescencesample– Fluorescenceblank) / (Fluorescence1 μMtrolox– Fluorescence blank)
One ORAC unit equals the amount of protection provided to BPE by the 1 μM Trolox standard.
The reliability of the method was verified by conducting recovery assays in which 2 μM Trolox were added to the plant extract; recovery of Trolox protection was always between 80 and 90%. In addition we determined that in the absence of AAPH, plant extract did not interfere with fluorescence of BPE.
Enzymatic activities were analyzed on specific activity, dry weight and fresh weight bases. The ORAC data were calculated on both dry and fresh weight bases.