©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 (1) (3)(§) Paul D. Sima (2)

From the  (1)Medical and (2)Research Services, Edward Hines, Jr., Department of Veterans Affairs Hospital, Hines, Illinois 60141 and the (3)Departments of Medicine and of Molecular and Cellular Biochemistry, Loyola University, Stritch School of Medicine, Maywood, Illinois 60153

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Aqueous extracts of the intercellular fluid from Sedum album L. leaves generated singlet oxygen chemiluminescence at 1270 nm when exposed to a nitrogen gas stream containing ozone at 21 ± 2 ppm. The concentration of ascorbic acid in the intercellular fluid extracts was 310 ± 40 µM. The intensity of the singlet oxygen chemiluminescence from the intercellular fluid extracts was comparable with the chemiluminescence from a control solution containing 300 µM ascorbic acid. The intensity of the singlet oxygen emission from intercellular fluid treated with ascorbate oxidase was 0.19 ± 0.07 of the intensity of the singlet oxygen chemiluminescence from untreated samples of intercellular fluid extract. The simplest explanation for the effect of ascorbate oxidase is that ascorbic acid is the major ozone target generating singlet oxygen. Much weaker singlet oxygen chemiluminescence was detected at 1270 nm when intact S. album L. plant tips were exposed to a nitrogen gas stream containing ozone at 22 ± 5 ppm. Various explanations for the relatively low intensity of the singlet oxygen chemiluminescence from intact S. album L. plant tips are discussed.


INTRODUCTION

Exposure of plants to low levels of ozone can result in significant plant damage, but the biochemistry of the ozone-mediated toxicity has yet to be fully characterized(1) . Based on theoretical calculations, Chameides (2) has proposed that ascorbic acid is the major target of ozone within plant leaves. If this is true, then substantial quantities of singlet oxygen should be generated within plant leaves, because ozone reacts with ascorbic acid to generate 0.61 mol of singlet oxygen/mol of ozone consumed(3, 4) .

The detection of chemiluminescence at 1270 nm has proven to be one of the most specific tests for the demonstration of singlet oxygen generation in biological systems(5) . Normally it is very difficult to detect singlet oxygen emission from intact tissues, because the singlet oxygen lifetime within cells is very short (on the order of 0.1 µs) (6, 7, 8, 9) . Consequently, the steady-state concentration of singlet oxygen is low, and any singlet oxygen chemiluminescence would be expected to be very weak. However, recent work from this laboratory has shown that the intensity of the singlet oxygen chemiluminescence should be significantly enhanced when singlet oxygen is generated at the surface of a tissue in contact with air(4, 10) . This is the case for plant leaves exposed to ozone. Thus, one would expect to detect singlet oxygen chemiluminescence from plant leaves exposed to ozone.


EXPERIMENTAL PROCEDURES

Plants

Sedum album L. plants were selected for these studies because prior work by Castillo and Greppin (11) had shown that the intercellular fluid of these plants contains high levels of ascorbic acid. S. album L. plants were obtained from commercial growers in Northern Illinois.

Preparation of Intercellular Fluid Extracts

Intercellular fluid from the S. album L. leaves was obtained with the vacuum infiltration technique of Castillo and Greppin(11) . The extraction buffer used contained 40 mM sodium acetate (pH 4.5), 100 mM potassium chloride, and 25 µM diethylenetriamine pentaacetic acid (DTAC). (^1)The DTAC was included to inhibit metal-catalyzed oxidation of ascorbic acid. Prior to the measurement of chemiluminescence, the pH of the leaf extracts was adjusted to 7.0 by the addition of 0.5 M tribasic sodium phosphate. The ascorbic acid content of the intercellular fluid extracts was calculated from the reduction in absorbance at 265 nm caused by the addition of ascorbate oxidase (activity, 1.7 units ml) to the extract as described by Castillo and Greppin(11) .

Measurement of Singlet Oxygen Chemiluminescence

The chemiluminescence spectrometer used to measure the singlet oxygen emission has been described previously(4, 10, 12, 13) . Near-infrared chemiluminescence from leaf extracts was studied by placing 0.75 ml of the extract in a 13-mm diameter cuvette. A 5 times 2-mm Teflon-covered magnetic stirring bar rapidly mixed the solution during the chemiluminescence measurements(4, 10) . For most experiments, the background light emission was integrated for 1 min. The flow of carrier gas containing ozone was then started, and light emission was integrated for 3 min. The background emission was then subtracted from the experimental emission (divided by 3) to obtain the net chemiluminescence.

Ozone was generated and diluted with carrier gas as described previously(4, 10) . A 17-gauge stainless steel tube brought the ozone into the cuvette. The flow of carrier gas through the cuvette was 1.25 ± 0.01 ml s. The amount of ozone exiting the cuvette was measured iodometrically(4, 10, 14) . The iodometric assay used agreed well with the indigo method of Bader and Hoigné(15) . The difference between the ozone concentration exiting from an empty cuvette and the ozone concentration exiting from a cuvette with a sample was used to calculate the amount of ozone consumed by the sample.

For the measurement of chemiluminescence from S. album L. plants, the tips of actively growing plants were cut and then placed in the chemiluminescence spectrometer cuvette, a 13-mm diameter glass tube. The plant tips were 1 cm long and typically had 5 or 6 leaves. The tops of the cut plant tips were oriented toward the infrared detector.

Reagents

Ascorbate oxidase (activity, 230 units mg) was obtained from Sigma. Aldrich Chemical Co. was the source of the DTAC. All other chemicals were reagent grade. Oxygen (99%) and nitrogen (99.5%) gases were obtained from Puritan Bennett, La Grange, IL.

Statistical Analysis

Unless specified otherwise, results are reported as a mean ± S.E. The number of measurements is given in parentheses.


RESULTS

We chose to use a two-phase system to assay for singlet oxygen generation from the reaction of ozone with S. album L. intercellular extracts rather than a homogeneous system because prior work has shown that the chemistry of some ozone-biomolecule reactions at gas-liquid interfaces may be different than the chemistry in bulk solution(16) . We wanted to model the processes that occur within intact plant leaves, and these ozone reactions are believed to occur very close to plant cell wall surfaces that are in contact with air (2) . Fig. 1shows the time course of the 1270-nm emission from the reaction of ozone with the intercellular extract of the S. album L. plants. Table 1shows that the maximum emission is near 1270 nm. Table 2shows that the intensity of the chemiluminescence is decreased by the substitution of oxygen carrier gas for nitrogen carrier gas. This result is consistent with gas-phase singlet oxygen emission and is due to the more efficient quenching of singlet oxygen by oxygen than by nitrogen(17) . This effect has been reported previously for the reaction of ozone with other biomolecules at gas-liquid interfaces(4, 10) .


Figure 1: Time course of 1270-nm emission from the reaction of ozone with an aqueous extract of the intercellular fluid from S. album L. leaves. The ozone concentration was 21 ppm in nitrogen carrier gas. The flow rate of the carrier gas was 1.25 ml s. The ascorbic acid concentration in the extract was 324 µM. The pH of the extract was adjusted to 7.0.







The concentration of ascorbic acid in the intercellular extracts was 310 ± 40 µM (n = 5). This confirms the prior work of Castillo and Greppin(11) , showing that the concentration of ascorbic acid in the intercellular fluid of S. album L. is high.

As shown in Table 2, the intensity of the chemiluminescence and the ozone flux into the intercellular solution were comparable with a standard solution containing 300 µM ascorbic acid. As also shown in Table 2, treatment of the intercellular extract with ascorbate oxidase greatly decreased the intensity of the 1270-nm emission and the ozone flux. The simplest explanation for the effect of ascorbate oxidase is that most of the singlet oxygen is generated from the reaction of ozone with ascorbic acid. Alternatively, ascorbate oxidase may initiate a more complex oxidation process in which other ozone targets are oxidized in secondary reactions. Ascorbate oxidase-treated extracts still produced much greater 1270-nm emission and consumed more ozone than did the control buffer. This is an expected result because many biomolecules, including proteins and glutathione, have been shown to react with ozone to generate singlet oxygen(3, 4) .

Fig. 2shows the time course of the 1270-nm emission of S. album L. plant tips exposed to ozone. The assignment of this chemiluminescence to singlet oxygen is supported by the spectral analysis shown in Table 1. There is an obvious emission maximum at 1270 nm. Also, as shown in Table 2, substitution of oxygen carrier gas for the nitrogen carrier gas resulted in a large decrease in the 1270-nm emission. This decrease in emission is likely caused by the more efficient quenching of singlet oxygen by oxygen gas compared with nitrogen gas (17) and suggests that the singlet oxygen is generated close to the surface of plant cells in contact with the carrier gas.


Figure 2: Time course of 1270-nm emission from the reaction of ozone with S. album plant tips. The curve is an average of 10 experiments. The S. album L. plant tips weighed 64 ± 7 mg. The ozone concentration was 22 ± 5 ppm. The flow rate of the nitrogen carrier gas was 1.25 ml s. The unit of intensity on the ordinate scale is the same as the unit of intensity used in Fig. 1.



As also shown in Table 2, the intensity of the emission from the S. album L. plant tips was small compared with the intercellular extracts. This was true even when the emission intensity was corrected for the small flux of ozone into the plant tips compared with the flux of ozone into the intercellular fluid.


DISCUSSION

This study demonstrates the production of substantial quantities of singlet oxygen when ozone reacts with aqueous extracts of the intercellular fluid of S. album L. leaves. Using diffusion theory, Chameides (2) has calculated that almost all of the ozone entering plant leaves through stomata will react with ascorbic acid in the walls of plant cells without reaching the cell membranes. In this sense, ascorbic acid functions as a sacrificial antioxidant. Because ascorbic acid reacts more rapidly with singlet oxygen than with ozone (18, 19) , almost all of the singlet oxygen generated would be expected to react with ascorbic acid in the cell wall or be quenched by water before reaching the cell membrane. The increased tolerance of ozone for some plant leaves having high ascorbic acid contents (20, 21) is consistent with this model of ozone inactivation.

Singlet oxygen chemiluminescence was also detected when ozone reacted with the tips of S. album L. plants, but the intensity of the chemiluminescence was much lower than the intensity of the chemiluminescence from the S. album L. leaf extracts. One cause for the relatively low singlet oxygen chemiluminescence is the relatively small ozone consumption by the plant tips. It is generally believed that almost all of the ozone consumption by plant leaves occurs by diffusion of ozone through stomatal openings and not by diffusion into the plant cuticle(22, 23) . The relatively small surface area of the open stomata in the leaves compared with the surface area of the stirred intercellular extract accounts for the low ozone consumption. Further, in our experiments the relatively large ozone concentration that is needed to generate a signal may have caused many of the plant stomata to close.

When the intensity of the chemiluminescence is divided by the rate of ozone consumption, however, the plant tips still have a much lower ratio than the leaf extracts. One factor contributing to the low chemiluminescence from the plant tips is the location of the singlet oxygen generation. The singlet oxygen is produced within leaves on the surfaces of small air passages and not on the outer surface of the leaves(2, 22, 23) . Thus, some of the light generated within the leaves will be scattered and absorbed and will not exit from the leaves. In fact, the transmission of 1270-nm light through intact S. album L. leaves, which have an oval cross-section and are roughly 2 mm thick, is only 5 ± 1%.

A major reason for the large chemiluminescence from the leaf extracts is that the two-phase assay system used to measure the chemiluminescence greatly enhances the intensity of the chemiluminescence relative to a homogeneous aqueous system(10) . Using equations derived from diffusion theory(10) , the intensity of the singlet oxygen chemiluminescence from a two-phase system with 300 µM ascorbic acid in the aqueous phase will be 34 times that of a homogeneous aqueous system containing 300 µM ascorbic acid. The theory used to calculate this enhancement of chemiluminescence assumes that the air space above the aqueous phase is very large. For the small air passages within the leaves, this theory may greatly overestimate the intensity of chemiluminescence. Additional theoretical and experimental work will be required to more accurately predict the intensity of singlet oxygen chemiluminescence from complex geometric structures with small air passages.


FOOTNOTES

*
This work was supported by the Department of Veterans Affairs 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 article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Box 278, Hines VA Hospital, Hines, IL 60141. Tel.: 708-343-7200, ext. 1429; Fax: 708-216-2319.

(^1)
The abbreviation used is: DTAC, diethylenetriamine pentaacetic acid.


ACKNOWLEDGEMENTS

We thank Brian Dunlap and John Schaefer for help with the construction of apparatus.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.