Atmospherrc Enuironment Vol 11. pp 273-275 Pergamon Press 1977 PrInted I” Great Brttaln

EVIDENCE FOR REPAIR OF OZONE INDUCED MEMBRANE INJURY: ALTERATION IN

SUGAR UPTAKE

ROSEMARY SUTTON and IRWIN P. TING

Department of Biology, University of California Riverside, CA 92502, U.S.A.

(First received I June 1976 and in jnal form 18 August 1976)

Abstract-After short term ozone exposure (0.60.8 ppm 0, for 1 h) of pinto bean plants, leaf discs punched from the primary leaves show an enhanced uptake of 2-deoxyglucose. We assume the latter is the result of an alteration in membrane selectivity or permeability. In leaves which are too young to show visible ozone induced necrosis, after 5 days in a normal light regime the increased uptake returned to the control, an indication of natural repair of ozone injury. Continuous light or glucose treatment enhances the rate of repair whereas continuous dark delays the repair. We assume these observations indicate the presence of an energy dependent repair process for ozone induced injury.

Short term exposure of plants to ozone doses compar- able to those found in the Los Angeles basin is known to induce a variety of injurious effects. Reduction in net photosynthesis, changes in cellular levels of many different organic compounds and certain enzymes, as well as disturbance of intracellular structure have been reported (Dugger and Ting, 1970; Ting and Heath, 1975). There is also considerable evidence to support the idea that ozone alters permeability of cells. Our group and others have demonstrated changes in uptake or efflux of potassium, amino acids, and sugars, occurring at different times after ozone fumigation (Ting and Heath, 1975). Little is known, however, about natural repair processes. We propose that the data presented herein are a direct measure of endogenous membrane repair. Furthermore, our experiments indicate that the rate of repair can be altered by various treatments opening up possibilities for therapeutic treatment of ozone injury.

Eight- or twelve-day-old Pinto bean plants grown in a growth chamber (12 h light at 27°C; 12 h dark at 15°C; radiant energy, 3.4 x lo4 ergs crn2 s-l), were exposed to 0.408 ppm ozone for 1 h in a fumi- gation chamber as previously described (Perchoro- wicz and Ting, 1974). Plants were returned to the growth chamber for 24 h prior to measurement of solute uptake, the assay for injury and repair. To fol- low the increased uptake of organic solutes that is observed 24 hours after ozonation, we chose to use t4C-UL-2-deoxy-d-glucose since it is readily available and not metabolized. In a previous study, its uptake was shown to be similar to that of glucose (Perchoro- wicz and Ting, 1974).

The method for assaying 2-deoxyglucose uptake was adapted from that used by Perchorowicz and Ting (1974). Ten discs were punched with a number 2 cork borer from the periphery of primary leaves and incubated at 25°C in 5 ml of a 0.2 M mannitol solution containing 0.4mM CaS04 and 17 mM Tris-MES buffer pH 6.4. After 45 min, approx. 0.5 PCi

of 14C-UL-2-deoxy-glucose was added with sufficient carrier to give a final concentration of 100 PM. After one hour the discs were washed and post-incubated for half an hour in 5 ml of the same buffer containing 1 mM unlabeled 2-deoxyglucose. The discs were then extracted by grinding in 2ml of SOo/, ethanol, and 1 ml aliquots were counted by liquid scintillation.

The primary leaves of Pinto bean plants grown un- der our conditions appear to be maximally sensitive to ozone 12 days after planting. At this age, maximum leaf necrosis is seen as evidenced by 75100% flecking of the upper surface. Older plants show less or no injury and with younger 8-day-old plants there is no visible injury even when doses of up to 1 ppm for 1 h are used (see Fig. 3 of Ting and Heath, 1975). Previously, it has been shown that 12-day-old ozone exposed leaf tissue accumulates considerably more 2-deoxyglucose than controls (Perchorowicz and Ting, 1974). We have now demonstrated that 8-day- old ozonated plants also show an enhanced uptake of 2-deoxyglucose, though of a lower magnitude (see Fig. 1). Since these plants do not develop necrosis, we suspected that the ozone induced injury, as mani- fested by the change in accumulation of 2-deoxyglu- case, might be repaired in the days following ozona- tion.

Figure 2(A) shows the time course of this repair process over the 5 day period following ozonation. After fumigation these plants were returned to their normal 12 hour photoperiod and assayed at daily in- tervals. After 5 days there was no significant difference in uptake between control and ozone treated plants. If plants were kept in continuous light following fumi- gation, repair was complete within 72 h (Fig. 2B), but fumigated plants kept in the dark still showed an uptake of more than twice the controls after 5 days (Fig. 2C). These data have led us to conclude that the repair of ozone injury, as measured by 2-deoxy- glucose uptake, is certainly light influenced and per- haps energy dependent. In another set of experiments

273

274 ROSEMARY SUTTON and IRWIN P. TING

*-

a, E c

Ozone ( 32 12 day

:one Control 8 day

Fig. 1. Eight and twelve day old pinto bean plants were fumigated at 0.6 ppm for 1 h, 7 h into the light period. Twenty-four hours later, discs from the primary leaves of these plants and non-ozonated controls were tested for 14C 2-deoxyglucose uptake. Each point represents the mean of 6 determinations of 10 leaf discs each and standard

ations given.

were in solutions after fumigation. shown Table ozonated which dipped 0.2 glucose

a to control of when the day. leaves 0.2 mannitol, or M acid little, any, This suggest glucose

enhancing is simply as osmoticum washing

Our are with report Dug- et al. (1962) that glucose treatment of leaves

applied before ozone fumigation protected sensitive plants against visible injury, and that high levels of

endogenous soluble carbohydrates were associated with the normal periods of ozone resistance. It was speculated that during the period of maximum leaf susceptibility, normal repair processes were inhibited by the low levels of soluble carbohydrate. Such plants are probably unable to provide sufficient energy quickly enough for the repair process.

Further evidence for energy dependent repair comes from experiments in which plants were

A- normal 12L /I20

C-contmwus dork

0 I 2 3 4 5

Days after ozone fumlgatlon

Fig. 2. Eight day old pmto bean plants were fumigated with ozone at 0.8 ppm for 1 h, 7 h into the light cycle. Immediately after fumigation, Group A plants were returned to the normal growth chamber; Group B plants were placed with their controls in another chamber which differed only in that the lights were on continuously; Group C plants were placed with their controls in a box covered with black cloth m the Group A growth chamber. On successive days after fumigation, ozone and control plants were tested for 2-deoxyglucose uptake. Each point represents the mean of 4 plants plus the standard devl-

ation. A = ozone treated, l = control.

returned for 24 h to a cold growth chamber after O3 exposure (Table 1). Here, there was 23% greater uptake of 2-deoxyglucose in the cold (17°C) treated, than in normal temperature (26”) regime.

Table 1. Effect of various treatments on the ozone enhanced uptake of “C-UL-2-deoxyglucose

Treatment

Exp. 1 Normal Dipped in 2M Mannitolt Dipped m HzOt

Control

0.613 + O.lOO* 0.706 * 0.017 0.633 k 0.052

Ozone

1.010 * 0.055 1.163 k 0.160 1.008 & 0.255

“; of normal ozone values

115 100

Exp. 2 Normal 0.666 * 0.050 1.771 f 0.127 Cold$ 2.173 f 0.104 Dipped in 0.2M Glucose? 0.699 + 0.194 Dipped in 0.05M Ascorbic Acid? 1.482 f 0.363

* Data are given as nmoles f std. devlatlon 2-deoxyglucose uptake per leaf disc per hour. t Plants were dipped twice immediately following fumigation. $ In cold treatment the day temperature was 17°C instead of 26°C.

123 39 84

Evidence for repair of ozone induced membrane injury 275

There is now much evidence in the literature that ozone affects membrane selectivity by lipid peroxida- tion of the unsaturated fatty acids and oxidation of sulfhydryl groups of membranes (Heath et al., 1974). We suspect that our data reflect a repair of these deleterious oxidative processes. Using our assay method, it may be possible to !ind other simple treat- ments which would enhance this repair and thus be of value in preventing necrosis of sensitive plant tis- sue.

Acknowledgements-Supported in part by funds from the Environmental Protection Agency under grant No. 801311.

REFERENCES

Dugger W. M., Taylor 0. C., Cardiff E. and Thompson C. R. (1962) Relationship between carbohydrate content and susceptibility of pinto bean plants to ozone damage. Proc. Am. Sot. Hart. Sci. 81, 304315.

Dugger W. M. and Ting I. P. (1970) Air pollution oxi- dants-their effects on metabolic processes in plants. Ann. Rev. Plant Physiol. 21, 215-234.

Heath R. L., Chimiklis P. and Frederick P. (1974) Role of potassium and lipids in ozone injury to plant mem- branes. In Air PoUution EfSects on Plant Growth (Edited by Dugger M.), Series 3, pp. 58-75. Amer. Chem. Sot. Symp.

Perchorowicz J. T. and Ting I. P. (1974) Ozone effects on plant cell permeability. Am. J. Bot. 61, 787-793.

Ting I. P. and Heath R. L. (1975) Responses of plants to air pollutant oxidants. Agronomy 27, 89-121.