Materials and methods Directory UMM :Data Elmu:jurnal:A:Agriculture, Ecosystems and Environment:Vol80.Issue1-2.Aug2000:

160 A. Donnelly et al. Agriculture, Ecosystems and Environment 80 2000 159–168 Elevated concentrations of O 3 in the atmosphere tends to decrease the photosynthetic rate of C 3 plants Amundson et al., 1987; Lehnherr et al., 1988 either by inducing stomatal closure Heath, 1994 and restricting CO 2 entry to the leaf or by entering the intercellular spaces of the leaf mesophyll and disrupt- ing cell metabolism Heath, 1980. Cell metabolism is disrupted by the activated oxygen species which are produced as O 3 instantaneously dismutases on entry into the leaf intercellular spaces. Subsequent metabolism of O 3 -derived activated oxygen species is dependent on the activities of several antioxidant enzymes Rao et al., 1995. The effect of elevated O 3 on cell metabolism is frequently associated with increased rates of leaf senescence and the loss of chlorophyll Grandjean and Fuhrer, 1989; Fangmeier et al., 1994; Finnan et al., 1998; Ojanperä et al., 1998, thus reducing the duration for photosynthetic activity and resulting in a reduction in growth and yield Soja and Soja, 1995; Finnan et al., 1996. The mechanisms which explain the interactive effects of both elevated CO 2 and elevated O 3 on pho- tosynthetic processes in leaves are, as yet, unclear. One proposal suggests that elevated CO 2 induces partial stomatal closure and so reduces the effective dose of O 3 reaching the photosynthetic apparatus within the plant Allen, 1990; McKee et al., 1995. A second proposal, suggested by McKee et al. 1995 and Mulholland et al. 1997, is that an increase in the amount of in vivo active Rubisco at elevated CO 2 may provide some compensation for damage caused by moderately elevated O 3 . A third proposal from Rao et al. 1995, suggests that increased production of antioxidants in the leaf mesophyll at elevated lev- els of CO 2 , which increase the rate of destruction of active O 3 as it enters the leaf, may account for the apparent protection from ozone damage. Experiments were carried out as part of the Envi- ronmental Stress Physiology and Climate Change Ex- periment on wheat ESPACE-wheat project of the EU Environment and Climate Programme to test whether elevated CO 2 protects spring wheat flag leaves from a ozone induced chlorophyll loss and b ozone in- duced damage to the photosynthetic apparatus, and that this is primarily the result of partial stomatal closure at elevated CO 2 which reduces O 3 flux into the leaf. In this experiment, spring wheat Triticum asetivum L., cv. ‘Minaret’ was grown in open-top Table 1 Timetable of events during the 1994 and 1996 growing seasons Event 1994 1996 Planting 6 April 1 April Fertilisation NPK 5 April 14 March Fertilisation CAN 3 June 30 April Insecticide application a 28 May, 11 July 3 May, 7 June Fungicide application b 28 May, 24 June 7 and 18 June Final harvest 25 August 12 August a Insecticide: oxydemeton-methyl 420 mls ha − 1 . b Fungicide: flutriafolchlorothalonil 1.5 l ha − 1 . chambers at a site in Carlow, Ireland and exposed to combinations of ambient and elevated CO 2 and O 3 . The chlorophyll content, photosynthetic activity and stomatal conductance of flag leaves of spring wheat plants exposed to elevated CO 2 and elevated O 3 , singly and in combination, were examined over two growing seasons.

2. Materials and methods

2.1. Open top chambers and carbon dioxide and ozone treatments Spring wheat Triticum aestivum L., cv. ‘Minaret’ was grown in open-top chambers OTC’s, for two growing seasons 1994 and 1996, at Oak Park Re- search Centre, Carlow, Ireland which is located 80 km south-west of Dublin at latitude 50 ◦ 51 ′ 12 ′′ N and lon- gitude 6 ◦ 54 ′ 15 ′′ W. The chambers were 2.8 m in height and 3 m in diameter. Sowing conditions and crop man- agement have been described in detail in Donnelly et al. 1999. Briefly, seeds were sown directly into the OTCs at a density of 385 m 2 and irrigated to main- tain field capacity. Approximately 90 of the plants emerged from the sown seed, resulting in an average density of 350 plants m 2 . The timing of events during the 1994 and 1996 growing seasons are shown in Table 1. Fertiliser was applied in accordance with best local practice and pesticides were applied when necessary. In 1994, plants were exposed to two carbon diox- ide CO 2 concentrations, consisting of ambient ∼350 ppmv and elevated 680 ppmv, and two ozone O 3 concentrations consisting of ambient ∼20 ppbv and elevated ambient +50 ppbv. Eight OTCs were used, allowing for all combinations of CO 2 and O 3 with two replicates. The experiment expanded in A. Donnelly et al. Agriculture, Ecosystems and Environment 80 2000 159–168 161 1996 to include 12 OTCs and an additional inter- mediate CO 2 treatment was added 510 ppmv. In 1996 the elevated O 3 concentration was increased to ambient +90 ppbv. In 1996 there were three CO 2 concentrations and two O 3 concentrations allowing all combinations of CO 2 and O 3 with two replicates. Fu- migation with additional CO 2 and O 3 began at anthesis in 1994 and was season-long in 1996. The exposure index, AOT40 Accumulated Ozone exposure above a Threshold of 40 ppbv was calculated for each grow- ing season for both ambient and elevated conditions Table 1. The AOT40, advanced by Fuhrer 1994, is an indication of accumulated exposure to levels above a threshold which causes damage. An AOT group is the censored sum of all hourly concentrations above a certain threshold value. In this case, the threshold value is 40 ppbv which is subtracted from the value of each hourly concentration which exceeds this thresh- old before summation. However, one disadvantage of this parameter is that it masks peak O 3 concentrations which may have a larger effect than the AOT40 value indicates. That is, the effect of O 3 is not linearly related to concentration above the threshold. The CO 2 was supplied continuously under feed- back computer control via mass flow controllers. An air sample from each OTC requiring CO 2 control was drawn via a diaphragm pump into an infrared gas analyser Model WMA-2, PP Systems, Hitchin, Herts, UK and the CO 2 supply was regulated to achieve the required concentration. The O 3 in the elevated O 3 treatment was supplied for 7 h per day and 5 days per week. O 3 was generated from industrial grade oxygen by electrical discharge ABB-Ozone generator Type LN 103, Asea Brown Boveri, Baden, Switzerland and supplied to the OTCs under manual feedback con- trol via needle valves. Air samples were taken from the chamber requiring O 3 control, via a Teflon mem- brane pump Model No. N.726, KNF Neuberger, Ox- ford, UK, and lead through an ozone analyser Model 8810, Monitor Laboratories, San Diego, CA, USA. The analyser was connected to a Campbell Scientific 21×data logger which recorded evesry 60 s and logged and averaged every 30 min. 2.2. Chlorophyll content The chlorophyll content of 10 flag leaves, on ran- domly selected plants, was measured on a weekly basis from full anthesis until senescence in the 1996 growing season only. Measurements were taken in the middle of the leaf using a Minolta chlorophyll meter, model SPAD-502 Minolta Camera, 3-13, 2-Chrome, Azuchi-Machi, Chuo-Ku, Osaka 541, Japan. SPAD values were recorded from the meter and used to estimate the chlorophyll content of the leaves non-destructively. The SPAD values were con- verted to units of mg of chlorophyll per cm 2 of leaf using the following quadratic equation which best describes the relationship; Y=5.65+0.39X+0.018X 2 where, Y is the extractable chlorophyll content of the leaf mg cm − 2 and X is the SPAD reading Finnan et al., 1997. 2.3. Gas exchange measurements The light saturated rates of photosynthesis, stom- atal conductance, transpiration and the internal in- tercellular concentration of CO 2 were determined in vivo on 10 main stem flag leaves on four occasions in 1994 and on two occasions in 1996 at times between anthesis and grain filling. Measurements were made in the middle section of leaves that did not show any visible signs of senescence. An LCA4 photosyn- thesis system Analytical Developments Company was used in 1994 to determine the gas exchange pa- rameters and a LI-Cor 6200 LI-COR, Lincoln, NE portable photosynthesis system was used in 1996. Photosynthesis measurements were carried out on dry and sunny days between 11.00 and 15.00 hours to ensure light saturation in 1994, as an artificial light source was not used during this growing season. PAR Photosynthetically Active Radiation 400–700 nm was in excess of 1800 mmol m − 2 s − 1 in 1994 and 2000 mmol m − 2 s − 1 in 1996 using an artificial light source Li-Cor QB-1205LI Hybrid Lamp. There was no direct temperature control of leaves, but it was found that when using the artificial light source the leaf temperature did not fluctuate significantly during a set of measurements. 2.4. Statistical analysis The experiment was based on a randomised 2-factorial design with two replicates. All parame- ters were statistically analysed by multiway analysis 162 A. Donnelly et al. Agriculture, Ecosystems and Environment 80 2000 159–168 Table 2 Seasonal climate data, CO 2 and O 3 concentrations, exposure duration period together with AOT40 values for the 1994 and 1996 growing seasons Year Mean daily Mean daily CO 2 24 h O 3 24h AOT40 a Start of End of temperature radiation seasonal mean seasonal mean fumigation fumigation ◦ C kW m − 2 in ppmv in ppbv period period 1994 13.1 0.14 360 ambient 17 ambient 3356 ambient 27 June 25 August 678 680 ppmv 24 ambient +50 24281 b elevated 1996 12.6 0.16 362 ambient 27 ambient 33321 ambient 12 April 12 August 675 680 ppmv 53 ambient +90 95930 elevated 20 year average 13.1 0.15 a Accumulated ozone exposure above a threshold of 40 ppbv. b Fumigation started at anthesis. of variance ANOVA using the statistical package Minitab, version 8.3 extended, for personal comput- ers. When the multiway ANOVA showed a statis- tically significant effect of one of the parameters a oneway analysis of variance was carried out to re- veal at what level the effect was significant. In such cases a two-sided confidence interval for the differ- ences between the treatment means and the control or for all pairwise differences between level means, was constructed. Data for the intermediate CO 2 con- centration 510 ppmv in 1996 have been omitted from the results in order to make direct comparisons with the 1994 data as there was no intermediate CO 2 concentration used in that year.

3. Results