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Singlet Oxygen Generation from the Reaction of Ozone with Plant Leaves

Singlet Oxygen Generation from the Reaction of Ozone with Plant Leaves THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270, No. 14, Issue of April 7, pp. 7850-7852, 1995 Printed in U.SA Singlet Oxygen Generation from the Reaction of Ozone with Plant Leaves* (Received for publication, December 13, 1994, and in revised form, January 27, 1995) Jeffrey R. Kanofsky*§ll and Paul D. Simall From the +.Medical and IlResearch Services, Edward Hines, Jr., Department of Veterans Affairs Hospital, Hines, Illinois 60141 and the §Departments of Medicine and of Molecular and Cellular Biochemistry, Loyola University, Stritch School of Medicine, Maywood, Illinois 60153 Aqueous extracts of the intercellular fluid from Se- plant leaves exposed to ozone. Thus, one would expect to detect dum album L. leaves generated singlet oxygen chemilu- singlet oxygen chemiluminescence from plant leaves exposed to minescence at 1270 nm when exposed to a nitrogen gas ozone. stream containing ozone at 21 ± 2 ppm. The concentra- EXPERIMENTAL PROCEDURES tion of ascorbic acid in the intercellular fluid extracts Plants--8edum album 1. plants were selected for these studies be- was 310 ± 40 tJ-M. The intensity of the singlet oxygen cause prior work by Castillo and Greppin (11) had shown that the chemiluminescence from the intercellular fluid extracts intercellular fluid of these plants contains high levels of ascorbic acid. S. was comparable with the chemiluminescence from a album 1. plants were obtained from commercial growers in Northern control solution containing 300 tJ-M ascorbic acid. The Illinois. intensity of the singlet oxygen emission from intercellu- Preparation of Intercellular Fluid Extracts-Intercellular fluid from lar fluid treated with ascorbate oxidase was 0.19 ± 0.07 the S. album 1. leaves was obtained with the vacuum infiltration technique of Castillo and Greppin (11). The extraction buffer used of the intensity of the singlet oxygen chemilumines- contained 40 mM sodium acetate (pH 4.5), 100 ma potassium chloride, cence from untreated samples of intercellular fluid ex- and 25 p.M diethylenetriamine pentaacetic acid (DTAC).' The DTAC tract. The simplest explanation for the effect of ascor- was included to inhibit metal-catalyzed oxidation of ascorbic acid. Prior bate oxidase is that ascorbic acid is the major ozone to the measurement of chemiluminescence, the pH of the leaf extracts target generating singlet oxygen. Much weaker singlet was adjusted to 7.0 by the addition of 0.5 Mtribasic sodium phosphate. oxygen chemiluminescence was detected at 1270 nm The ascorbic acid content of the intercellular fluid extracts was calcu- when intact S. album L. plant tips were exposed to a lated from the reduction in absorbance at 265 nm caused by the addi- nitrogen gas stream containing ozone at 22 ± 5 ppm. tion of ascorbate oxidase (activity, 1.7 units ml t ') to the extract as Various explanations for the relatively low intensity of described by Castillo and Greppin (11). Measurement of Singlet Oxygen Chemiluminescence-The chemilu- the singlet oxygen chemiluminescence from intact S. minescence spectrometer used to measure the singlet oxygen emission album L. plant tips are discussed. has been described previously (4, 10, 12, 13). Near-infrared chemilumi- nescence from leaf extracts was studied by placing 0.75 ml of the extract in a 13-mm diameter cuvette. A 5 x 2-mm Teflon"'-covered magnetic Exposure of plants to low levels of ozone can result in signif- stirring bar rapidly mixed the solution during the chemiluminescence icant plant damage, but the biochemistry of the ozone-medi- measurements (4, 10). For most experiments, the background light ated toxicity has yet to be fully characterized (1). Based on emission was integrated for 1 min. The flow of carrier gas containing ozone was then started, and light emission was integrated for 3 min. theoretical calculations, Chameides (2) has proposed that The background emission was then subtracted from the experimental ascorbic acid is the major target of ozone within plant leaves. If emission (divided by 3) to obtain the net chemiluminescence. this is true, then substantial quantities of singlet oxygen Ozone was generated and diluted with carrier gas as described pre- should be generated within plant leaves, because ozone reacts viously (4, 10). A 17-gauge stainless steel tube brought the ozone into with ascorbic acid to generate 0.61 mol of singlet oxygen/mol of the cuvette. The flow of carrier gas through the cuvette was 1.25 :!: 0.01 ozone consumed (3, 4). ml S-I. The amount of ozone exiting the cuvette was measured iodo- metrically (4, 10, 14). The iodometric assay used agreed well with the The detection of chemiluminescence at 1270 nm has proven indigo method of Bader and Hoigne (15). The difference between the to be one of the most specific tests for the demonstration of ozone concentration exiting from an empty cuvette and the ozone con- singlet oxygen generation in biological systems (5). Normally it centration exiting from a cuvette with a sample was used to calculate is very difficult to detect singlet oxygen emission from intact the amount of ozone consumed by the sample. tissues, because the singlet oxygen lifetime within cells is very For the measurement of chemiluminescence from S. album L. plants, short (on the order of 0.1 JLs) (6-9). Consequently, the steady- the tips of actively growing plants were cut and then placed in the chemiluminescence spectrometer cuvette, a 13-mm diameter glass tube. state concentration of singlet oxygen is low, and any singlet The plant tips were 1 cm long and typically had 5 or 6 leaves. The tops oxygen chemiluminescence would be expected to be very weak. of the cut plant tips were oriented toward the infrared detector. However, recent work from this laboratory has shown that the Reagents-Ascorbate oxidase (activity, 230 units mg"") was obtained intensity of the singlet oxygen chemiluminescence should be from Sigma. Aldrich Chemical Co. was the source of the DTAC. All significantly enhanced when singlet oxygen is generated at the other chemicals were reagent grade. Oxygen (99%) and nitrogen surface ofa tissue in contact with air (4,10). This is the case for (99.5%) gases were obtained from Puritan Bennett, La Grange, IL. Statistical Analysis-Unless specified otherwise, results are reported as a mean z S.E. The number of measurements is given in parentheses. * This work was supported by the Department of Veterans Affairs RESULTS Research Service and by the Potts Estate. The costs of publication of this article were defrayed in part by the payment of page charges. This We chose to use a two-phase system to assay for singlet article must therefore be hereby marked "advertisement" in accordance oxygen generation from the reaction of ozone with S. album L. with 18 U.S.C. Section 1734 solely to indicate this fact. 11 To whom correspondence should be addressed: Box 278, Hines VA Hospital, Hines, IL 60141. Tel.: 708-343-7200, ext. 1429; Fax: 708-216- I The abbreviation used is: DTAC, diethylenetriamine pentaacetic 2319. acid. This is an Open Access article under the CC BY license. 7851 Singlet Oxygen Generation TABLE I (j) +-' Spectral analysis of the near-infrared emission from the reaction of 'c ozone with S. album L. leaf extract and with S. album L. plant tips ::J Relative chemiluminescence" ... Interference filter" «l Plant tips" ... Leaf extract" +-' :.0 40 nrn ... 1070 -0.02 ± 0.02 (n = 5) 0.10 ± 0.05 (n = 6) 1170 -0.Dl ± 0.01 (n = 5) -0.02 ± 0.07 (n = 6) Q) CJ 1268 1.00 ± 0.05 (n = 15) 1.00 ± 0.19 (n = 7) 1370 0.Dl ± 0.02 (n = 5) 0.00 ± 0.05 (n = 6) Q) 20 CJ 1475 -0.02 ± 0.02 (n = 5) -0.06 ± 0.09 (n = 6) (fl Q) a The interference filters had bandwidths of 50 nm. b The relative chemiluminescence has been corrected for the wave- 'f ;:j length dependence of the detector and for the transmission through each interference filter. 'f Q) C The leaf extracts had ascorbate concentrations between 300 and 470 -10 s: I-£M. The reagents in the extraction buffer were 90 mM potassium chlo- U 0 100 -100 ride, 36 mx sodium acetate, 14.6 mMsodium phosphate (pH 7.0), and 23 I-£M DTAC. The ozone concentration in the nitrogen gas was 23 ± 3 ppm. Time (s) Because we did not try to make the ozone concentrations identical for all experiments, the error reported is the standard deviation and not FIG. 1. Time course of 1270-nm emission from the reaction of the standard error. The ozone consumption was 0.37 ± 0.03 nmol/min. ozone with an aqueous extract of the intercellular fluid from S. d The plant tips contained 5 or 6 leaves and weighed 54 ± 2 mg. The album L. leaves. The ozone concentration was 21 ppm in nitrogen ozone concentration in the nitrogen carrier gas was 27 ± 4 ppm. The carrier gas. The flow rate of the carrier gas was 1.25 ml S-1. The error reported is the standard deviation and not the standard error. ascorbic acid concentration in the extract was 324 I-£M. The pH of the extract was adjusted to 7.0. of oxygen carrier gas for the nitrogen carrier gas resulted in a large decrease in the 1270-nm emission. This decrease in emis- intercellular extracts rather than a homogeneous system be- sion is likely caused by the more efficient quenching of singlet cause prior work has shown that the chemistry of some ozone- oxygen by oxygen gas compared with nitrogen gas (17) and biomolecule reactions at gas-liquid interfaces may be different suggests that the singlet oxygen is generated close to the sur- than the chemistry in bulk solution (16). We wanted to model face of plant cells in contact with the carrier gas. the processes that occur within intact plant leaves, and these As also shown in Table II, the intensity of the emission from ozone reactions are believed to occur very close to plant cell the S. album L. plant tips was small compared with the inter- wall surfaces that are in contact with air (2). Fig. 1 shows the cellular extracts. This was true even when the emission inten- time course of the 1270-nm emission from the reaction of ozone sity was corrected for the small flux of ozone into the plant tips with the intercellular extract of the S. album L. plants. Table I compared with the flux of ozone into the intercellular fluid. shows that the maximum emission is near 1270 nm. Table II DISCUSSION shows that the intensity of the chemiluminescence is decreased This study demonstrates the production of substantial quan- by the substitution of oxygen carrier gas for nitrogen carrier tities of singlet oxygen when ozone reacts with aqueous ex- gas. This result is consistent with gas-phase singlet oxygen emission and is due to the more efficient quenching of singlet tracts of the intercellular fluid of S. album L. leaves. Using diffusion theory, Chameides (2) has calculated that almost all oxygen by oxygen than by nitrogen (17). This effect has been reported previously for the reaction of ozone with other biomol- of the ozone entering plant leaves through stomata will react ecules at gas-liquid interfaces (4, 10). with ascorbic acid in the walls of plant cells without reaching the cell membranes. In this sense, ascorbic acid functions as a The concentration of ascorbic acid in the intercellular ex- tracts was 310 ± 40 JLM (n = 5). This confirms the prior work of sacrificial antioxidant. Because ascorbic acid reacts more rap- idly with singlet oxygen than with ozone (18, 19), almost all of Castillo and Greppin (11), showing that the concentration of ascorbic acid in the intercellular fluid of S. album L. is high. the singlet oxygen generated would be expected to react with As shown in Table II, the intensity of the chemiluminescence ascorbic acid in the cell wall or be quenched by water before and the ozone flux into the intercellular solution were compa- reaching the cell membrane. The increased tolerance of ozone rable with a standard solution containing 300 JLM ascorbic acid. for some plant leaves having high ascorbic acid contents (20, As also shown in Table II, treatment of the intercellular extract 21) is consistent with this model of ozone inactivation. with ascorbate oxidase greatly decreased the intensity of the Singlet oxygen chemiluminescence was also detected when 1270-nm emission and the ozone flux. The simplest explanation ozone reacted with the tips of S. album L. plants, but the for the effect of ascorbate oxidase is that most of the singlet intensity of the chemiluminescence was much lower than the oxygen is generated from the reaction of ozone with ascorbic intensity of the chemiluminescence from the S. album L. leaf acid. Alternatively, ascorbate oxidase may initiate a more com- extracts. One cause for the relatively low singlet oxygen chemi- plex oxidation process in which other ozone targets are oxidized luminescence is the relatively small ozone consumption by the in secondary reactions. Ascorbate oxidase-treated extracts still plant tips. It is generally believed that almost all of the ozone produced much greater 1270-nm emission and consumed more consumption by plant leaves occurs by diffusion of ozone ozone than did the control buffer. This is an expected result through stomatal openings and not by diffusion into the plant because many biomolecules, including proteins and glutathi- cuticle (22, 23). The relatively small surface area of the open one, have been shown to react with ozone to generate singlet stomata in the leaves compared with the surface area of the oxygen (3, 4). stirred intercellular extract accounts for the low ozone con- Fig. 2 shows the time course of the 1270-nm emission of S. sumption. Further, in our experiments the relatively large album L. plant tips exposed to ozone. The assignment of this ozone concentration that is needed to generate a signal may chemiluminescence to singlet oxygen is supported by the spec- have caused many of the plant stomata to close. tral analysis shown in Table I. There is an obvious emission When the intensity of the chemiluminescence is divided by maximum at 1270 nm. Also, as shown in Table II, substitution the rate of ozone consumption, however, the plant tips still 7852 Singlet Oxygen Generation TABLE II Effect of ascorbate oxidase and the type of carrier gas on ozone consumption and singlet oxygen chemiluminescence Relative emission" at Sample Carrier gas Ozone concentration" Ozone consume db 1268 nm ppm nmol5- BufferI Nitrogen 22 :±: 1 7) 0.04:±: 0.02 0.007 :±: 0.002 (n = 300 I-tM ascorbate" Nitrogen 25:±: 1 0.35 :±: 0.03 1.00:±: 0.09 (n = 3) Leaf extract Nitrogen 21 :±: 2 0.29 :±: 0.03 0.83 :±: 0.14 (n = 3) Leaf extract + ascorbate oxidase" Nitrogen 21:±: 2 0.16 :±: 0.02 0.16 :±: 0.03 (n = 3) BufferI Oxygen 23:±: 4 0.05 :±: 0.01 0.012 :±: 0.004 (n = 5) 300 }.LM ascorbate" Oxygen 27:±: 1 3) 0.31 :±: 0.03 0.17 :±: 0.01 (n = Leaf extract Oxygen 19:±: 2 0.24 :±: om 0.13 :±: 0.01 (n = 3) Empty cuvette Nitrogen 23:±: 5 (n = 16) or 0.0026 :±: 0.0005 Plant tips" Nitrogen 22:±: 5 0.D7 :±: 0.01 0.017 :±: 0.002 (n = 10) Empty cuvette Oxygen 23:±: 4 or 0.001 :±: 0.0005 (n = 25) Plant tips" Oxygen 23:±: 4 0.06:±: 0.01 0.001 :±: 0.0005 (n = 20) a Because we did not try to make the ozone concentration identical for each experiment, the standard deviation rather than the standard error is reported. b The reaction period studied was 3 min. c The emission from a 300 I-tM solution of ascorbic acid was given a value of 1.00. d The buffer contained 90 mM potassium chloride, 36 rnx sodium acetate, 14.6 mM sodium phosphate (pH 7.0), and 23 }.LM DTAC. e The leaf extract was treated with 1.7 units ml "! of ascorbate oxidase for 5 min. f The amount of ozone flowing through an empty cuvette was used as a reference for zero ozone consumption. g The weight of the plant samples was 64 :±: 7 mg. h The weight of the plant samples was 61 :±: 1 mg. tem (10). Using equations derived from diffusion theory (10), en :t= the intensity of the singlet oxygen chemiluminescence from a 2.0 :::> two-phase system with 300 J.LM ascorbic acid in the aqueous phase will be 34 times that of a homogeneous aqueous system .... co 1.5 .... containing 300 p.,M ascorbic acid. The theory used to calculate :t= .D this enhancement of chemiluminescence assumes that the air .... space above the aqueous phase is very large. For the small air 1.0 Q) passages within the leaves, this theory may greatly overesti- mate the intensity of chemiluminescence. Additional theoreti- c:: Q) cal and experimental work will be required to more accurately 0.5 (/l predict the intensity of singlet oxygen chemiluminescence from Q) complex geometric structures with small air passages. 'E 0.0 ::J Acknowledgments-We thank Brian Dunlap and John Schaefer for 'E help with the construction of apparatus. Q) -0.5 ..c: REFERENCES -100 0 100 200 1. Kangasjarvi, J., Talvinen, J., Utriainen, M., and Karjalainen, R (1994) Plant Cell Environ. 17,783-794 Time (s) 2. Chameides, W. L. (1989) Environ. Sci. & Technol. 23,595-600 3. Kanofsky, J. R, and Sima, P. (1991) J. Bioi. Chem. 266,9039-9042 FIG. 2. Time course of 1270-nm emission from the reaction of 4. Kanofsky, J. R, and Sima, P. D. (1993) Photochem. Photobiol. 58, 335-340 ozone with S. album plant tips. The curve is an average of 10 5. Kanofsky, J. R (1989) Chem.-Biol. Interactions 70, 1-28 experiments. The S. album L. plant tips weighed 64 :±: 7 mg. The ozone 6. Matheson, 1. B. C., Etheridge, R. D., Kratowich, N. R., and Lee, J. (1975) Photochem. Photobiol. 21, 165-171 concentration was 22 :±: 5 ppm. The flow rate of the nitrogen carrier gas 7. Krasnovsky, A. A., Jr. (1988) in Molecular Mechanisms of Biological Action of was 1.25 ml S-l. The unit of intensity on the ordinate scale is the same Optical Radiation (Rubin, A. B., ed) pp. 23-41, Nauka, Moscow as the unit of intensity used in Fig. 1. 8. Moan, J., and Berg, K. (1991) Photochem. Photobiol. 53,549-553 9. Baker, A., and Kanofsky, J. R. (1992) Photochem. Photobiol. 55,523-528 have a much lower ratio than the leaf extracts. One factor 10. Kanofsky, J. R., and Sima, P. D. (1994) Arch. Biochem. Biophys. 312,244-253 11. Castillo, F. J., and Greppin, H. (1988) Environ. Exp. Bot. 28, 231-238 contributing to the low chemiluminescence from the plant tips 12. Kanofsky, J. R. (1988) J. Bioi. Chem. 263, 14171-14175 is the location of the singlet oxygen generation. The singlet 13. Kanofsky, J. R. (1983) J. BioI. Chem. 258,5991-5993 14. Saltzman, B. E., and Gilbert, N. (1959) Anal. Chem. 31, 1914-1920 oxygen is produced within leaves on the surfaces of small air 15. Bader, H., and Hoigne, J. (1981) Water Res. 15,449-456 passages and not on the outer surface of the leaves (2, 22, 23). 16. Kanofsky, J. R., and Sima, P. D. (1995) Arch. Biochem. Biophys. 316,52-62 Thus, some of the light generated within the leaves will be 17. Wayne, R. P. (1985) in Singlet O (Erimer, A. A., ed) Vol. 1, pp. 81-175, CRC Press, Inc., Boca Raton, FL scattered and absorbed and will not exit from the leaves. In 18. Giamalva, D., Church, D. F., and Pryor, W. A. (1985) Biochem. Biophys. Res. fact, the transmission of 1270-nm light through intact S. album Commun. 133,773-779 L. leaves, which have an oval cross-section and are roughly 2 19. Rougee, M., and Bensasson, R. (1986) C. R. Acad. Sci. Paris Ser. II 302, 1223-1226 mm thick, is only 5 :t 1%. 20. Lee, E. H., Jersey, J. A., Gifford, C., and Bennett, J. (1984) Environ. Exp. Bot. A major reason for the large chemiluminescence from the 24,331-341 21. Luwe, M. W. F., Takahama, D., and Heber, U. (1993) Plant Physiol. 101, leaf extracts is that the two-phase assay system used to meas- 969-976 ure the chemiluminescence greatly enhances the intensity of 22. Kerstiens, G., and Lendzian, K. J. (1989) New Phytol. 112, 13-19 23. Laisk, A., Kul!, 0., and Moldau, H. (1989) Plant Physiol. 90, 1163-1167 the chemiluminescence relative to a homogeneous aqueous sys- http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Singlet Oxygen Generation from the Reaction of Ozone with Plant Leaves

Kanofsky, Jeffrey R.; Sima, Paul D.
Journal of Biological Chemistry – Apr 1, 1995
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270, No. 14, Issue of April 7, pp. 7850-7852, 1995 Printed in U.SA Singlet Oxygen Generation from the Reaction of Ozone with Plant Leaves* (Received for publication, December 13, 1994, and in revised form, January 27, 1995) Jeffrey R. Kanofsky*§ll and Paul D. Simall From the +.Medical and IlResearch Services, Edward Hines, Jr., Department of Veterans Affairs Hospital, Hines, Illinois 60141 and the §Departments of Medicine and of Molecular and Cellular Biochemistry, Loyola University, Stritch School of Medicine, Maywood, Illinois 60153 Aqueous extracts of the intercellular fluid from Se- plant leaves exposed to ozone. Thus, one would expect to detect dum album L. leaves generated singlet oxygen chemilu- singlet oxygen chemiluminescence from plant leaves exposed to minescence at 1270 nm when exposed to a nitrogen gas ozone. stream containing ozone at 21 ± 2 ppm. The concentra- EXPERIMENTAL PROCEDURES tion of ascorbic acid in the intercellular fluid extracts Plants--8edum album 1. plants were selected for these studies be- was 310 ± 40 tJ-M. The intensity of the singlet oxygen cause prior work by Castillo and Greppin (11) had shown that the chemiluminescence from the intercellular fluid extracts intercellular fluid of these plants contains high levels of ascorbic acid. S. was comparable with the chemiluminescence from a album 1. plants were obtained from commercial growers in Northern control solution containing 300 tJ-M ascorbic acid. The Illinois. intensity of the singlet oxygen emission from intercellu- Preparation of Intercellular Fluid Extracts-Intercellular fluid from lar fluid treated with ascorbate oxidase was 0.19 ± 0.07 the S. album 1. leaves was obtained with the vacuum infiltration technique of Castillo and Greppin (11). The extraction buffer used of the intensity of the singlet oxygen chemilumines- contained 40 mM sodium acetate (pH 4.5), 100 ma potassium chloride, cence from untreated samples of intercellular fluid ex- and 25 p.M diethylenetriamine pentaacetic acid (DTAC).' The DTAC tract. The simplest explanation for the effect of ascor- was included to inhibit metal-catalyzed oxidation of ascorbic acid. Prior bate oxidase is that ascorbic acid is the major ozone to the measurement of chemiluminescence, the pH of the leaf extracts target generating singlet oxygen. Much weaker singlet was adjusted to 7.0 by the addition of 0.5 Mtribasic sodium phosphate. oxygen chemiluminescence was detected at 1270 nm The ascorbic acid content of the intercellular fluid extracts was calcu- when intact S. album L. plant tips were exposed to a lated from the reduction in absorbance at 265 nm caused by the addi- nitrogen gas stream containing ozone at 22 ± 5 ppm. tion of ascorbate oxidase (activity, 1.7 units ml t ') to the extract as Various explanations for the relatively low intensity of described by Castillo and Greppin (11). Measurement of Singlet Oxygen Chemiluminescence-The chemilu- the singlet oxygen chemiluminescence from intact S. minescence spectrometer used to measure the singlet oxygen emission album L. plant tips are discussed. has been described previously (4, 10, 12, 13). Near-infrared chemilumi- nescence from leaf extracts was studied by placing 0.75 ml of the extract in a 13-mm diameter cuvette. A 5 x 2-mm Teflon"'-covered magnetic Exposure of plants to low levels of ozone can result in signif- stirring bar rapidly mixed the solution during the chemiluminescence icant plant damage, but the biochemistry of the ozone-medi- measurements (4, 10). For most experiments, the background light ated toxicity has yet to be fully characterized (1). Based on emission was integrated for 1 min. The flow of carrier gas containing ozone was then started, and light emission was integrated for 3 min. theoretical calculations, Chameides (2) has proposed that The background emission was then subtracted from the experimental ascorbic acid is the major target of ozone within plant leaves. If emission (divided by 3) to obtain the net chemiluminescence. this is true, then substantial quantities of singlet oxygen Ozone was generated and diluted with carrier gas as described pre- should be generated within plant leaves, because ozone reacts viously (4, 10). A 17-gauge stainless steel tube brought the ozone into with ascorbic acid to generate 0.61 mol of singlet oxygen/mol of the cuvette. The flow of carrier gas through the cuvette was 1.25 :!: 0.01 ozone consumed (3, 4). ml S-I. The amount of ozone exiting the cuvette was measured iodo- metrically (4, 10, 14). The iodometric assay used agreed well with the The detection of chemiluminescence at 1270 nm has proven indigo method of Bader and Hoigne (15). The difference between the to be one of the most specific tests for the demonstration of ozone concentration exiting from an empty cuvette and the ozone con- singlet oxygen generation in biological systems (5). Normally it centration exiting from a cuvette with a sample was used to calculate is very difficult to detect singlet oxygen emission from intact the amount of ozone consumed by the sample. tissues, because the singlet oxygen lifetime within cells is very For the measurement of chemiluminescence from S. album L. plants, short (on the order of 0.1 JLs) (6-9). Consequently, the steady- the tips of actively growing plants were cut and then placed in the chemiluminescence spectrometer cuvette, a 13-mm diameter glass tube. state concentration of singlet oxygen is low, and any singlet The plant tips were 1 cm long and typically had 5 or 6 leaves. The tops oxygen chemiluminescence would be expected to be very weak. of the cut plant tips were oriented toward the infrared detector. However, recent work from this laboratory has shown that the Reagents-Ascorbate oxidase (activity, 230 units mg"") was obtained intensity of the singlet oxygen chemiluminescence should be from Sigma. Aldrich Chemical Co. was the source of the DTAC. All significantly enhanced when singlet oxygen is generated at the other chemicals were reagent grade. Oxygen (99%) and nitrogen surface ofa tissue in contact with air (4,10). This is the case for (99.5%) gases were obtained from Puritan Bennett, La Grange, IL. Statistical Analysis-Unless specified otherwise, results are reported as a mean z S.E. The number of measurements is given in parentheses. * This work was supported by the Department of Veterans Affairs RESULTS Research Service and by the Potts Estate. The costs of publication of this article were defrayed in part by the payment of page charges. This We chose to use a two-phase system to assay for singlet article must therefore be hereby marked "advertisement" in accordance oxygen generation from the reaction of ozone with S. album L. with 18 U.S.C. Section 1734 solely to indicate this fact. 11 To whom correspondence should be addressed: Box 278, Hines VA Hospital, Hines, IL 60141. Tel.: 708-343-7200, ext. 1429; Fax: 708-216- I The abbreviation used is: DTAC, diethylenetriamine pentaacetic 2319. acid. This is an Open Access article under the CC BY license. 7851 Singlet Oxygen Generation TABLE I (j) +-' Spectral analysis of the near-infrared emission from the reaction of 'c ozone with S. album L. leaf extract and with S. album L. plant tips ::J Relative chemiluminescence" ... Interference filter" «l Plant tips" ... Leaf extract" +-' :.0 40 nrn ... 1070 -0.02 ± 0.02 (n = 5) 0.10 ± 0.05 (n = 6) 1170 -0.Dl ± 0.01 (n = 5) -0.02 ± 0.07 (n = 6) Q) CJ 1268 1.00 ± 0.05 (n = 15) 1.00 ± 0.19 (n = 7) 1370 0.Dl ± 0.02 (n = 5) 0.00 ± 0.05 (n = 6) Q) 20 CJ 1475 -0.02 ± 0.02 (n = 5) -0.06 ± 0.09 (n = 6) (fl Q) a The interference filters had bandwidths of 50 nm. b The relative chemiluminescence has been corrected for the wave- 'f ;:j length dependence of the detector and for the transmission through each interference filter. 'f Q) C The leaf extracts had ascorbate concentrations between 300 and 470 -10 s: I-£M. The reagents in the extraction buffer were 90 mM potassium chlo- U 0 100 -100 ride, 36 mx sodium acetate, 14.6 mMsodium phosphate (pH 7.0), and 23 I-£M DTAC. The ozone concentration in the nitrogen gas was 23 ± 3 ppm. Time (s) Because we did not try to make the ozone concentrations identical for all experiments, the error reported is the standard deviation and not FIG. 1. Time course of 1270-nm emission from the reaction of the standard error. The ozone consumption was 0.37 ± 0.03 nmol/min. ozone with an aqueous extract of the intercellular fluid from S. d The plant tips contained 5 or 6 leaves and weighed 54 ± 2 mg. The album L. leaves. The ozone concentration was 21 ppm in nitrogen ozone concentration in the nitrogen carrier gas was 27 ± 4 ppm. The carrier gas. The flow rate of the carrier gas was 1.25 ml S-1. The error reported is the standard deviation and not the standard error. ascorbic acid concentration in the extract was 324 I-£M. The pH of the extract was adjusted to 7.0. of oxygen carrier gas for the nitrogen carrier gas resulted in a large decrease in the 1270-nm emission. This decrease in emis- intercellular extracts rather than a homogeneous system be- sion is likely caused by the more efficient quenching of singlet cause prior work has shown that the chemistry of some ozone- oxygen by oxygen gas compared with nitrogen gas (17) and biomolecule reactions at gas-liquid interfaces may be different suggests that the singlet oxygen is generated close to the sur- than the chemistry in bulk solution (16). We wanted to model face of plant cells in contact with the carrier gas. the processes that occur within intact plant leaves, and these As also shown in Table II, the intensity of the emission from ozone reactions are believed to occur very close to plant cell the S. album L. plant tips was small compared with the inter- wall surfaces that are in contact with air (2). Fig. 1 shows the cellular extracts. This was true even when the emission inten- time course of the 1270-nm emission from the reaction of ozone sity was corrected for the small flux of ozone into the plant tips with the intercellular extract of the S. album L. plants. Table I compared with the flux of ozone into the intercellular fluid. shows that the maximum emission is near 1270 nm. Table II DISCUSSION shows that the intensity of the chemiluminescence is decreased This study demonstrates the production of substantial quan- by the substitution of oxygen carrier gas for nitrogen carrier tities of singlet oxygen when ozone reacts with aqueous ex- gas. This result is consistent with gas-phase singlet oxygen emission and is due to the more efficient quenching of singlet tracts of the intercellular fluid of S. album L. leaves. Using diffusion theory, Chameides (2) has calculated that almost all oxygen by oxygen than by nitrogen (17). This effect has been reported previously for the reaction of ozone with other biomol- of the ozone entering plant leaves through stomata will react ecules at gas-liquid interfaces (4, 10). with ascorbic acid in the walls of plant cells without reaching the cell membranes. In this sense, ascorbic acid functions as a The concentration of ascorbic acid in the intercellular ex- tracts was 310 ± 40 JLM (n = 5). This confirms the prior work of sacrificial antioxidant. Because ascorbic acid reacts more rap- idly with singlet oxygen than with ozone (18, 19), almost all of Castillo and Greppin (11), showing that the concentration of ascorbic acid in the intercellular fluid of S. album L. is high. the singlet oxygen generated would be expected to react with As shown in Table II, the intensity of the chemiluminescence ascorbic acid in the cell wall or be quenched by water before and the ozone flux into the intercellular solution were compa- reaching the cell membrane. The increased tolerance of ozone rable with a standard solution containing 300 JLM ascorbic acid. for some plant leaves having high ascorbic acid contents (20, As also shown in Table II, treatment of the intercellular extract 21) is consistent with this model of ozone inactivation. with ascorbate oxidase greatly decreased the intensity of the Singlet oxygen chemiluminescence was also detected when 1270-nm emission and the ozone flux. The simplest explanation ozone reacted with the tips of S. album L. plants, but the for the effect of ascorbate oxidase is that most of the singlet intensity of the chemiluminescence was much lower than the oxygen is generated from the reaction of ozone with ascorbic intensity of the chemiluminescence from the S. album L. leaf acid. Alternatively, ascorbate oxidase may initiate a more com- extracts. One cause for the relatively low singlet oxygen chemi- plex oxidation process in which other ozone targets are oxidized luminescence is the relatively small ozone consumption by the in secondary reactions. Ascorbate oxidase-treated extracts still plant tips. It is generally believed that almost all of the ozone produced much greater 1270-nm emission and consumed more consumption by plant leaves occurs by diffusion of ozone ozone than did the control buffer. This is an expected result through stomatal openings and not by diffusion into the plant because many biomolecules, including proteins and glutathi- cuticle (22, 23). The relatively small surface area of the open one, have been shown to react with ozone to generate singlet stomata in the leaves compared with the surface area of the oxygen (3, 4). stirred intercellular extract accounts for the low ozone con- Fig. 2 shows the time course of the 1270-nm emission of S. sumption. Further, in our experiments the relatively large album L. plant tips exposed to ozone. The assignment of this ozone concentration that is needed to generate a signal may chemiluminescence to singlet oxygen is supported by the spec- have caused many of the plant stomata to close. tral analysis shown in Table I. There is an obvious emission When the intensity of the chemiluminescence is divided by maximum at 1270 nm. Also, as shown in Table II, substitution the rate of ozone consumption, however, the plant tips still 7852 Singlet Oxygen Generation TABLE II Effect of ascorbate oxidase and the type of carrier gas on ozone consumption and singlet oxygen chemiluminescence Relative emission" at Sample Carrier gas Ozone concentration" Ozone consume db 1268 nm ppm nmol5- BufferI Nitrogen 22 :±: 1 7) 0.04:±: 0.02 0.007 :±: 0.002 (n = 300 I-tM ascorbate" Nitrogen 25:±: 1 0.35 :±: 0.03 1.00:±: 0.09 (n = 3) Leaf extract Nitrogen 21 :±: 2 0.29 :±: 0.03 0.83 :±: 0.14 (n = 3) Leaf extract + ascorbate oxidase" Nitrogen 21:±: 2 0.16 :±: 0.02 0.16 :±: 0.03 (n = 3) BufferI Oxygen 23:±: 4 0.05 :±: 0.01 0.012 :±: 0.004 (n = 5) 300 }.LM ascorbate" Oxygen 27:±: 1 3) 0.31 :±: 0.03 0.17 :±: 0.01 (n = Leaf extract Oxygen 19:±: 2 0.24 :±: om 0.13 :±: 0.01 (n = 3) Empty cuvette Nitrogen 23:±: 5 (n = 16) or 0.0026 :±: 0.0005 Plant tips" Nitrogen 22:±: 5 0.D7 :±: 0.01 0.017 :±: 0.002 (n = 10) Empty cuvette Oxygen 23:±: 4 or 0.001 :±: 0.0005 (n = 25) Plant tips" Oxygen 23:±: 4 0.06:±: 0.01 0.001 :±: 0.0005 (n = 20) a Because we did not try to make the ozone concentration identical for each experiment, the standard deviation rather than the standard error is reported. b The reaction period studied was 3 min. c The emission from a 300 I-tM solution of ascorbic acid was given a value of 1.00. d The buffer contained 90 mM potassium chloride, 36 rnx sodium acetate, 14.6 mM sodium phosphate (pH 7.0), and 23 }.LM DTAC. e The leaf extract was treated with 1.7 units ml "! of ascorbate oxidase for 5 min. f The amount of ozone flowing through an empty cuvette was used as a reference for zero ozone consumption. g The weight of the plant samples was 64 :±: 7 mg. h The weight of the plant samples was 61 :±: 1 mg. tem (10). Using equations derived from diffusion theory (10), en :t= the intensity of the singlet oxygen chemiluminescence from a 2.0 :::> two-phase system with 300 J.LM ascorbic acid in the aqueous phase will be 34 times that of a homogeneous aqueous system .... co 1.5 .... containing 300 p.,M ascorbic acid. The theory used to calculate :t= .D this enhancement of chemiluminescence assumes that the air .... space above the aqueous phase is very large. For the small air 1.0 Q) passages within the leaves, this theory may greatly overesti- mate the intensity of chemiluminescence. Additional theoreti- c:: Q) cal and experimental work will be required to more accurately 0.5 (/l predict the intensity of singlet oxygen chemiluminescence from Q) complex geometric structures with small air passages. 'E 0.0 ::J Acknowledgments-We thank Brian Dunlap and John Schaefer for 'E help with the construction of apparatus. Q) -0.5 ..c: REFERENCES -100 0 100 200 1. Kangasjarvi, J., Talvinen, J., Utriainen, M., and Karjalainen, R (1994) Plant Cell Environ. 17,783-794 Time (s) 2. Chameides, W. L. (1989) Environ. Sci. & Technol. 23,595-600 3. Kanofsky, J. R, and Sima, P. (1991) J. Bioi. Chem. 266,9039-9042 FIG. 2. Time course of 1270-nm emission from the reaction of 4. Kanofsky, J. R, and Sima, P. D. (1993) Photochem. Photobiol. 58, 335-340 ozone with S. album plant tips. The curve is an average of 10 5. Kanofsky, J. R (1989) Chem.-Biol. Interactions 70, 1-28 experiments. The S. album L. plant tips weighed 64 :±: 7 mg. The ozone 6. Matheson, 1. B. C., Etheridge, R. D., Kratowich, N. R., and Lee, J. (1975) Photochem. Photobiol. 21, 165-171 concentration was 22 :±: 5 ppm. The flow rate of the nitrogen carrier gas 7. Krasnovsky, A. A., Jr. (1988) in Molecular Mechanisms of Biological Action of was 1.25 ml S-l. The unit of intensity on the ordinate scale is the same Optical Radiation (Rubin, A. B., ed) pp. 23-41, Nauka, Moscow as the unit of intensity used in Fig. 1. 8. Moan, J., and Berg, K. (1991) Photochem. Photobiol. 53,549-553 9. Baker, A., and Kanofsky, J. R. (1992) Photochem. Photobiol. 55,523-528 have a much lower ratio than the leaf extracts. One factor 10. Kanofsky, J. R., and Sima, P. D. (1994) Arch. Biochem. Biophys. 312,244-253 11. Castillo, F. J., and Greppin, H. (1988) Environ. Exp. Bot. 28, 231-238 contributing to the low chemiluminescence from the plant tips 12. Kanofsky, J. R. (1988) J. Bioi. Chem. 263, 14171-14175 is the location of the singlet oxygen generation. The singlet 13. Kanofsky, J. R. (1983) J. BioI. Chem. 258,5991-5993 14. Saltzman, B. E., and Gilbert, N. (1959) Anal. Chem. 31, 1914-1920 oxygen is produced within leaves on the surfaces of small air 15. Bader, H., and Hoigne, J. (1981) Water Res. 15,449-456 passages and not on the outer surface of the leaves (2, 22, 23). 16. Kanofsky, J. R., and Sima, P. D. (1995) Arch. Biochem. Biophys. 316,52-62 Thus, some of the light generated within the leaves will be 17. Wayne, R. P. (1985) in Singlet O (Erimer, A. A., ed) Vol. 1, pp. 81-175, CRC Press, Inc., Boca Raton, FL scattered and absorbed and will not exit from the leaves. In 18. Giamalva, D., Church, D. F., and Pryor, W. A. (1985) Biochem. Biophys. Res. fact, the transmission of 1270-nm light through intact S. album Commun. 133,773-779 L. leaves, which have an oval cross-section and are roughly 2 19. Rougee, M., and Bensasson, R. (1986) C. R. Acad. Sci. Paris Ser. II 302, 1223-1226 mm thick, is only 5 :t 1%. 20. Lee, E. H., Jersey, J. A., Gifford, C., and Bennett, J. (1984) Environ. Exp. Bot. A major reason for the large chemiluminescence from the 24,331-341 21. Luwe, M. W. F., Takahama, D., and Heber, U. (1993) Plant Physiol. 101, leaf extracts is that the two-phase assay system used to meas- 969-976 ure the chemiluminescence greatly enhances the intensity of 22. Kerstiens, G., and Lendzian, K. J. (1989) New Phytol. 112, 13-19 23. Laisk, A., Kul!, 0., and Moldau, H. (1989) Plant Physiol. 90, 1163-1167 the chemiluminescence relative to a homogeneous aqueous sys-

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