Effects of chronic ozone exposure on growth, root respiration and nutrient uptake of rice plants

Em'ironmental Pollution 74 ( 1991 ) 149-164

Effects of Chronic Ozone Exposure on Growth, Root Respiration and Nutrient Uptake of Rice Plants lsamu Nouchi, Osamu / t o , a Yoshinobu Harazono & Kazuhiko Kobayashi National Institute of Agro-Environmental Sciences, Kannondai, Tsukuba, lbaraki 305, Japan (Received 16 October 1990; revised version received 4 January 1991: accepted 20 March 1991 t

.4 B S T R A C T

To clar{[y the response of growth and rootJimctions to low concentrations o / ozone (03), riee plants ( O r y z a sativa L.) were exposed to 03 at 0.0 (control). 0"05 a,td O'lOppm .['or 8 weeks .[~'om t'egetative to ear O' heading stages. Exposure to O'05 ppm 03 tended to slightly stimulate the dry weight of whole plants up to 5 weeks and then slightly decrease the dry weight of whole plants. However, these ef~wts were statistically sign!/icant onh' at 6 weeks. Exposure to O'lOppm 03 reduced the dry weight of whole plants hv 50% at 5 and 6 weeks, and thereafter the reduction o[" the dry weight o/ whole plants was gradually allel~iated. Those changes in dry weight can he accountedJor hv a decrease or increase in the relative growth rate ( R G R ) . The changes in the RG R caused hy 0.05 and O"10 ppm 0 3 could he mainly attributed to the ~fffect st/ 0 3 on the net assimilation rate. Root~shoot ratio was lowered by both 0"05 and O.10 ppm 0 3 throughout the exposure period. The root~shoot ratio which had .severely decreased at O.10 ppm 0 3 #~ the first half period of exposure ( 1--4 weeks) became close to the control in the latter part c~[.exposure ( 5 8 weeks). Time-course changes in NH4-N root uptake rate were simihtr to those in the root shoot ratio e.wecially /m" 0. lO ppm 03. On the other haml. root respiration mcreased /?om the middh, to htter periods. Since it is to he supposed tltat plants grown under stressed condttiotts change the ratio ¢?/plant organ weight to achieve balance between the proportion O['shoots to roots in the plant and their activity for maintaining "F'resent address: International Crops Research Institute for Semidried Tropics ~ICRISAT). Patancheru, Andhra Pradesh 502 324, India. 149

Era'iron. Pollut. 0269-7491/91/$03.50 ~ 1991 Elsevier Science Publishers Ltd. England Printed in Great Britain

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Isamu Nouchi, Osarnu lto, Yoshinobu Harazono, Kazuhiko Kobayashi plant growth, these changes in root/shoot ratio andnitrogen uptake rate under long-term exposure can be considered to be an adaptive response to maintain rice growth under 03 stress.

INTRODUCTION Ozone (03) is presently the most widespread phytotoxic air pollutant in the world (Koziol & Whatley, 1984). Studies with open-top field chambers (Heagle et al., 1979; Fuhrer et al., 1989) and controlled chambers (Walmsley et al., 1980; Reich et al., 1986; Atkinson et al., 1988) reveal that 0 3 decreases the growth and yield of crops. Studies conducted in National Crop Loss Assessment Network (NCLAN) in the USA have clearly shown that ambient 0 3 levels over a range 0"04--0"07 ppm (7 h day - ~ seasonal mean) can significantly reduce yield in many crops (Heck et al., 1982, 1984, 1988). In Japan, however, attempts to assess the effects of relatively low levels o f O 3 o n crops are still scarce. In particular, only a small number of studies have been carried out to examine the effects of low concentrations ( < 0" 10 ppm) of 0 3 on the growth and yield of rice plants ( O r y z a satit, a) (Asakawa et al., 1981; Satoh et al., 1983; Kats et al., 1985), which represent one ofthe main crops in southeast Asia. Since ambient 0 3 concentrations are fairly high levels (0.024-0.046ppm: 7h day-~ seasonal mean) in the agricultural regions around urbanized areas in Japan (Kobayashi, 1988), the growth and yield loss of rice and many other crops due to 03 must be affected. Many studies concerning the direct relationship between photosynthetic rate and growth or yield and growth analysis techniques have revealed that O3-induced reduction in photosynthesis was quantitatively related to declines in growth or yield (Oshima et al., 1978, 1979; Horsman et al., 1980; Walmsley et al., 1980; Shimizu et al., 1981; Endress & Grunwald, 1985; Reich & Amundson, 1985; Reich, 1987). On the other hand, numerous studies have documented that 0 3 reduces root growth more than shoot growth in a wide range of plant species (Cooley & Manning, 1987). The inhibition of photosynthesis by 0 3 reduces the translocation of photosynthates from leaves to roots and shifts the allocation pattern within a plant to put the highest priority on actively growing tissues (Tingey et al., 1973; Oshima et al., 1978). Accordingly, root growth should be damaged more heavily than shoot growth. The reduction in the supply of photoassimilates from shoots to roots should alter physiological functions of the root such as respiration (Hofstra et al., 1981; Ito et al., 1985) and nutrient uptake (lto et al., 1985). However, little is known about changes in those functions of roots throughout long-term exposure to relatively low concentrations of 0 3. Thus, the present study was conducted to determine if long-term exposure to low concentrations o f O 3 (0"05 and 0.10 ppm) affects dry matter production of rice plants and if the inhibition of growth is related to the reduction in

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photosynthetic activity. In addition, the impacts of ozone exposure were studied for root/shoot ratio, root respiration and nutrient uptake. MATERIALS AND METHODS

Plants 'Koshihikari' cultivars of rice (Oryza sativa L.) were used in this study. Rice seeds were immersed in tap water for three days at room temperature (24 January, 1988). Germinated rice seeds were grown on a saran net floating in a vat filled with distilled water for a week and then with Kimura's B culture solution (Baba & Takahashi, 1956) in a naturally lit, environmentally controlled glass chamber (2m (d) x 2m(w) x l.Sm (h)) at a constant temperature of 25 + 0"1 :C and a relative humidity of 75 + 5% and equipped with activated charcoal filters. At the third leaf emergence (22 February), the seedlings were transplanted with two plants per hill in a hydrophonic culture bed (1-26m (d) x 0 - 8 7 m ( w ) x 0 . 1 0 m (h)) filled with Kimura's B culture solution. Ninety-six hills with a spacing of 10cm x 10cm were planted in each hydroponic culture bed in the three naturally lit glass chambers. The culture solution was renewed once a week and adjusted to pH 5.5 with l y HCI and IN NaOH. In each chamber, plants were rotated (seven times per hour) on a turntable to minimize possible position effects. Under short-day conditions in winter to spring, rice plants attained the flowering stage rather early, 45 days after sowing, and the panicle was in the early heading stage at the end of the experiment.

Ozone exposure The 0 3 exposures were initiated on 3 March with 34-days-old plants (lburth leaf) and terminated on 28 April. Ozone was generated by a silent electrical discharge in dry air, passed through a water trap, and introduced into the air stream entering the two naturally lit chambers in which the plants were placed. The water trap should have eliminated nitrogen by-products such as NzO~ produced by ozone generators with electrical discharge in air (Brown & Roberts, 1988). The concentration of 0 3 in each chamber was continuously monitored with UV absorption ozone analysers (Dasibi (California), model 1003AH) and regulated by a controlling system based on PID logic (Dylec Co. (Tokyo), model DACS- 1000). Plants were continuously exposed to 03 for 8h per day (from 0800 through 1600h) for 8 weeks.

Experimental design Exposure to three levels of 0 3 concentration was performed using the three naturally lit, environmentally controlled glass chambers. The 03 con-

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lsamu Nouchi, Osamu lto, Yoshinobu Harazono, Kazuhiko Kobayashi

centrations in each chamber were maintained at 0+0-01 ppm (control), 0.05 + 0"005 p p m and 0.10 + 0.005 ppm. Since only three chambers were available, the experiment was conducted without replicates. For reducing differences among the chambers in the microclimate, e.g. temperature, humidity and radiation, plants were rotated among the three chambers at one week intervals. This procedure must have reduced the chamber effects significantly, though not perfectly, and the hill-to-hill variation within a chamber was used as a surrogate of true error variation. Thus, the statistical tests in this paper tend to underestimate the error variance, and the results of the tests should be regarded as an approximate measure. It should also be noted that the temporal changes that happened over less than three weeks might have been caused by the chamber effects.

Sampling for growth analysis Nine hills (two plants per hill) were selected at random from each 03 treatment for weekly monitoring of plant response. Transpiration rates were measured and the number of injured leaves per hill was counted. Leaf area was measured for individual leaf blades (minus leaf sheaths), and their area determined using a leaf area meter (Hayashi Denkoh Co. Ltd, (Tokyo), AAC-400). Leaf blades, leaf sheaths and roots were then dried at 70°C for 72h. Total dry weights for leaves, leaf sheaths and roots were then determined for each hill. At the seventh and eighth harvest, panicles were included with leaf sheaths.

The growth analysis parameters The relative growth rate (RGR, g g - 1 day-~) and net assimilation rate (NAR, mg c m - 2 day- 1) were calculated using standard equations (Radford, 1967). Leaf area ratio (LAR, cm 2 g- 1) and root/shoot ratio (RSR, g g- 1) were also calculated.

Transpiration and nutrient uptake A hill was placed in an Erlenmeyer flask (100 or 200ml) with the same solution as used for cultivation except for the usage of demineralized water. Eight hills were put in a controlled chamber illuminated with 15 metal halide lamps (seven 400W Yoko lamps and eight 400W BOC lamps: Toshiba) for 2-5 h, depending on the size of the plants. Environmental conditions were constant at 25°C temperature, 75 +__5% relative humidity and approximately 700/~mol m - 2 s- ~ light intensity at the middle position of the leaves. Weight and concentrations of NH4-N and P in the culture solution were

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measured at the beginning and end of the incubation. The differences between the two measurements were used for calculation of the transpiration rate and the nutrient uptake rates. NH4-N was determined by the indophenol colorimetric method (Chaykin, 1969) and P by the molybdenum blue colorimetric method (Murphy & Riley, 1962).

Root respiration Part of a detached root was placed in a serum vial (33 ml)with 10 ml o1" the cultivation solution and incubated for 3 h at 30'C. The solution was injected into a gas chromatograph equipped with thermal conductivity detectors (Shimadzu (Kyoto), GC-4A) and a catalytic device for conversion of all organic carbon to CO 2 (Sumitomo Chemical Co. Ltd, (Osaka) GCT-12N). Separations of CO 2 in the medium were carried out at 60';C using a stainless steel column (3 mm in diameter and 100cm in length) packed with activated carbon (60--80 mesh). The CO2 evolved during the incubation represented the total of the amount in the solution and in the head space gas in the serum vial. The amount of CO 2 in the head space gas was calculated from the liquid-gas phase equilibrium of CO 2 in the solution quantified with the gas chromatograph.

Chlorophyll The top leaf of each plant at the beginning of the 0 3 exposure was marked with clips, and the marked four leaves of two hills were sampled every week throughout the experiment. Each leaf was homogenated with 80% acetone, and chlorophyll concentrations were determined for each sample according to the method of Arnon (1949) and expressed on a leaf area basis.

Statistical analysis Plant data in the harvests and physiological responses were subjected to analysis of variance (ANOVA) and significant differences between treatment means were identified with a Dunnett t-test.

RESULTS

Foliar injury and total number of leaves Eoliar injury (reddish-brown stipplings) occurred on the adaxial surface of the oldest leaves at 4 and 12 days after the beginning of 0"10 and 0"05 ppm 03

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exposure, respectively. The number of injured leaves per hill increased with the duration o f O 3 exposure. The percentage of the number of injured leaves relative to the total number of leaves per hill ranged from 10 to 48% and 23 to 72% for 0'05 and 0"10ppm 03, respectively (Fig. 1). The percentage of injured leaves per hill reached a maximum of 48 and 72% at 5 weeks after the beginning of exposure to 0"05 and 0-10 ppm, respectively. Total number of leaves per hill decreased significantly with exposure to 0" 10 ppm 03 from 3 to 7 weeks, while little or no change was observed throughout exposure to 0.05 ppm 03 (Fig. 1).

Chlorophyll Changes in chlorophyll concentration paralleled the development of leaf injury (data not shown). During the exposure to 0-10ppm O3, the chlorophyll content in the leaves rapidly decreased at 2 weeks after the

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beginning of the exposure and then continuously decreased. On the other hand, the chlorophyll content at 0.05 ppm 0 3 remained unchanged except for 5 weeks after the beginning of the exposure. Growth

Since rice plants were grown under short-day conditions throughout the experiment, heading was initiated on the main stem at 45 days after the start o f O 3 exposure. Although there was no difference in the early heading stage between control and 0-05 ppm 0 3, rice plants exposed to 0.10 ppm failed to complete heading by the end of the experiment (56days). Thus, the 0 3 exposure delayed the developmental growth of rice plants more than I week. Changes in dry weight of whole plants per hill exposed to 03 for 8 weeks are shown in Fig. 2(a)). The reduction in the dry weight of whole plants caused by 0.10 ppm 0 3 gradually became more severe up to 5 weeks after the beginning of the exposure. Thereafter the growth reduction due to 03 was gradually alleviated. Dry weight ofwhole plants exposed to 0-10 ppm 0 3 was 12 and 21% smaller than that of the control plants at 1 and 2 weeks after the beginning of the exposure, respectively, although these differences were not statistically significant. Dry weight of whole plants was significantly reduced bv 37, 33, 52, 51, 47 and 38% compared to that o f t h e control at 3.4, 5, 6, 7 and 8 weeks. Exposure to 0-10 ppm 03 caused a significant reduction in the dry weights of roots, leaf sheaths and leaves per hill. Initially (up to 3 weeks' exposure), leaf dry weight was 6-25% smaller than for controls, while the dry weights of roots and leaf sheaths were reduced by 12--52 and 13-42% compared to those of the control plants, respectively. The reduction in root dry weight was greater than that of other organs. Consequently, the root/'shoot ratio decreased significantly with exposure to 03 at 0" I 0 ppm and root growth was remarkably inhibited during initial exposure IFig. 2tb)). The leaf area per hill was also reduced by 0"10ppm 0 3 similarly to the leaf dry weight (data not presented here). In contrast, the effects of 0"05 ppm 0 3 on the dry weight of whole plants were much less than those of 0"10 ppm 03. The dry weight of whole plants exposed to 0-05 ppm 0 3 was 8-20% greater than that of the control at 2 and 3 weeks (Fig, 2(a)), although the difference was not statistically significant. Thereafter, the dry weight of whole plants became almost the same as that of the control at 4 and 5 weeks and then became approximately 5% smaller than that of the control at 7 and 8 weeks (insignificant) except for a 23% reduction at 6 weeks (significant). The changes in the dry weight of the leaf sheaths and the leaves were similar to those ofthe dry weight of whole plants throughout 8 weeks. However, the root dry weight, in particular, was

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reduced by 8-31% compared to that ofthe control from 5 to 7 weeks, which is the same as in the case of 0.10 ppm 0 3. The root/shoot ratio was decreased even by exposure to 0.05 ppm 0 3 (Fig. 2(b)).

Growth parameters The data obtained from the rice plants exposed to O a were examined in more detail through growth analysis. Figure 3 shows the changes in the relationship between the RGR and the NAR over time. Over the first and the second weeks of exposure, the RGR of rice plants was reduced by 12 17% with 0-10ppm 03 compared to the controls, while it increased by 9 14°/,, with 0"05 ppm 0 3. Over the third and fourth weeks, the RGR was reduced by 6-19% with 0.05 and 32-36% with 0.10ppm O a. The RGR for the fifth week, however, was almost the same among all O 3 exposures. By the sixth, seventh and eighth weeks, exposures to 0.05 and 0.10ppm O a tended to increase the RGR. For control plants, both RGR and NAR decreased with the progress of growth until the seventh week and then began to reverse. The increase in the NAR was much greater than that in the RGR. Although the physiological implication of the turning point, which is the time when the RGR and NAR began to the increase together in course of growth, for the RGR and NAR is unclear, the same turning point can be observed in 0-05 and 0"10ppm 0 3 (Fig. 3). 1.20

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The changes in the LAR of the plants exposed to 03 were nearly equal to those of the control plants except for the initial periods (2-4 weeks) at 0.10 ppm (Fig. 4).

Nutrient uptake Transpiration rate increased for the first 2 weeks and then decreased up to 4 weeks (Fig. 5). It then stayed relatively constant until the end of the experiment. Ozone exposure tended to lower the transpiration rate for 0.10ppm 03, in particular, although the difference was not statistically significant except at 4 weeks. This tendency became obscure by the latter part of the exposure period. The NH4-N uptake rate decreased promptly after the first sampling and then stayed constant (Fig. 6). Ozone exposure at 0.10 ppm tended to inhibit NH4-N uptake except at 1 and 6 weeks when considerable promotion was observed. Exposure to 0"05 ppm 03 did not produce a significant difference from the control. The rate of P uptake showed a temporal change similar to that of NH4-N. However, the P uptake was unaffected by 03 exposure (data not shown).

Root respiration rate Figure 7 shows root respiration rate (root dry weight basis) relative to the control. There were significant changes in root respiration during O 3

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exposure, especially for 0" 10 ppm 0 3. R o o t respiration rate was reduced by 7% at 0"05 ppm 03 and 16% at 0" 10 ppm after 1 week and recovered or even accelerated compared to the control up to 3 weeks. In the later period, root respiration was remarkably increased compared to that of the control. In particular, root respiration of plants exposed to 0.10 ppm 03 was 26 86% greater than that of the control. "7¢ 8 0

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DISCUSSION This study revealed significant suppression of growth of rice plants with continuous exposure to O a at 0.10 ppm for 8 weeks. This result confirmed other reports that near ambient concentrations of O3 suppressed the growth and yield of many plant species, e.g. soybean (Glycine max) (Tingey et al., 1971; Heagle el al., 1979), radish (Raphanus sativus) (Walmsley et al., 1980), wheat (Triticurn aestivum) (Amundson et al., 1987), parsley (Petroselinum crispum) (Oshima et al., 1978), poplar (Populus deltoides × trichocarpa) (Reich & Lassoie, 1985), and rice (Satoh et al., 1983; Kats et al., 1985). Exposure to 0.10ppm 0 3 progressively decreased significantly the dry weight of whole plants up to 6 weeks, when the reduction amounted to 50% compared to that of the control (Fig. 2(a)). After six weeks, the growth reduction was gradually alleviated (Fig. 2(a)). On the other hand, exposure to 0.05 ppm 03 slightly increased the dry weight of whole plants up to 5 weeks and then slightly decreased in the latter part ofexposure (Fig. 2(a)). However, the 0.05 ppm O a effects were not statistically significant except for a 23% reduction at 6 weeks. As mentioned in the 'Experimental design' section in Materials and Methods, a temporal change in 0.05 ppm 0 3 might be due to chamber effects. It was thus assumed that there was no significant effect of 0-05 ppm Oa on growth such as the dry weight of whole plant and leaf area except for the root/shoot ratio. Therefore, there could be a distinct difference

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in the mode of the growth response of rice to 03 between a low concentration (0.05 ppm) and a relatively high concentration (0-10 ppm). The R G R positively correlated with the N A R when compared within each sampling period (Fig. 3). This indicates that increases in the NAR directly reflect the increases in the R G R before the turning point, which is the time when the R G R and the N A R began to increase together in the course of growth, under both control and 0 3 exposures. A positive (0"05ppm) or a negative effect (0"10ppm) was also in line with this relationship between the R G R and the NAR. On the other hand, a disorder occurred in the relationship between the R G R and the NAR after the turning point (0.0 ppm: week 8, 0.05 ppm: week 6 and 0.I0 ppm: week 41 as a border line. That is, the great increase in the N A R was not accompanied by an increase in the RGR. This disorder indicates that an increase in the NAR does not directly reflect an increase in the RGR. After the turning point, a considerable change occurred in the effect o f O 3 o n the relationship between R G R and NAR. in many crop species, NAR is decreased by air pollutants such as 0 3, SO2 and NO2; and RGR, which is the product o f t h e NAR and LAR, is initially lowered, but thereafter, R G R in the exposed plants recovered due to a larger LA R (Oshima et aL, 1978, 1979: Walmsley et al., 1980; Shimizu et al., 1981: Okano & Totsuka, 1985). This means an increase in LAR. in part, compensated the reduction of photosynthetic etficiency by air pollutants. Therefore, the increase in the LAR in the later 03 exposure period can be considered as an adaptive growth response of plants to air pollutant stresses (Walsmley ~,t aL, 1980; Okano & Totsuka, 1985). In the present study, however, the L A R was not increased in the later part of exposure to 0"10 ppm 0 3 (5-8 weeks), although the LAR increased in the first half period of exposure (1-4 weeks) (Fig. 4). This result suggests that adaptive growth responses of rice plants may differ from those of many other crop specics. The root/shoot ratio in rice plants was significantly decreased by exposure to 0-05 and 0"10 ppm 0 3 (Fig. 2(b)), indicating that the root dry weight was reduced more than the shoot dry weight. This result was confirmed in many other plant species such as radish (Tingey et al., 1971 : Reinert & Gray, 1980; Walmsley et aL, 1980; Reinert & Sanders, 1982), parsley IOshima et al., 1978~, c~rrot (Dat,'u,~ ~'arola)(Bennett & Oshima, 1976), sunflower (Helianthu.~ ¢znnzzus) (Shimizu et ~ll., 1981) and grass (Horsman et al., 1980). Thus, these results also suggest that 0 3 decreases root growth through alteration of photosynthate partitioning between root and shoot and the inhibition ot" photosynthate translocation. Okano and Totsuka (1985) pointed out that the preferential partitioning of photoassimilates to developing leaves might be a genetic adaptation to accomplish as much growth as possible under stressed conditions or limited periods of photosynthate production. The

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lsamu Nouchi, Osamu lto, Yoshinobu Harazono, Kazuhiko Kobayashi

increase in root respiration in the later period (Fig. 7) suggests that the proportion of photosynthates used by root respiration to total photosynthates from shoots is very high. On the other hand, NH4-N uptake rates tended to be inhibited by 0 3, especially by 0.10 ppm 03 (Fig. 6). This result suggests that the energy for nutrient uptake is less. Since nutrient uptake depends upon the root respiration, these results indicate that the energy produced by the increase in root respiration might be used for other purposes such as a repair process for the damage caused by ozone. It is well known that there is a certain balance between the ratio of shoots to roots in plants and their activity (Davidson, 1969). Therefore, the malfunction of roots in the initial exposure will be reflected in shoot growth sooner or later, and the root/shoot ratio will recover to the same level in the later exposure period. In the present study, rice plants exhibited a lowered function of the shoots and roots in the initial period of exposure, and thereafter realtered the ratio ofplant organ weight to maintain plant growth. This change in the root/shoot ratio and nutrient uptake under long-term O3 exposure can be considered to be an adaptation response to maintain rice growth. However, the energy which would be used for growth may be greatly directed to consumption for the maintenance and repair of 0 3 damage. CONCLUSION Continuous exposure of rice plants to 0"10 ppm 0 3 for 8 weeks suppressed the total number of leaves and total dry weight, decreased the root/shoot ratio, and retarded development for more than 1 week. This suppression of rice growth and development could be attributed to depression of photosynthetic activity. In addition, 0"10 ppm 0 3 tended to inhibit nutrient uptake and accelerate root respiration. However, rice plants exposed to 0" 10 ppm 0 3 recovered their root/shoot ratio which had been lowered in the initial period of the exposure. On the other hand, although 0"05 ppm 03 caused visible foliar injury, the injury did not lead to significant differences in the growth and physiological functions such as root respiration and nutrient uptake except for the root/shoot ratio. Overall, the changes in weight ratio of plant organs during long-term exposure suggested that rice plants may adapt to 03 stress. REFERENCES Amundson, R. G., Kohut, R. J. & Schottle, A. W. (1987). Correlative reductions in whole-plant photosynthesis and yield of winter wheat caused by ozone. PhytopathoL, 77, 75-9.

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