Adaptation of radish Raphanus sativus L. in response to continuous exposure to ozone

Environmental Pollution(SeriesA) 23 (1980)165-177

A D A P T A T I O N OF RADISH R A P H A N U S S A T I V U S L. IN RESPONSE TO C O N T I N U O U S EXPOSURE TO OZONE

L. WALMSLEY,~ M. R. ASHMORE~ & J. N. B. BELL

Department of Botany, Imperial College Field Station, Silwood Park, Ascot, Berkshire, SL5 7PY, Great Britain

ABSTRACT

A comparison was made between the development of radish Raphanus sativus L. cv. Cherry Belle in clean air and under continuous exposure to O"17ppm (340 #g m - 3) of ozone. The area and dry weight of the individual leaves, and the dry weight of other plant organs, were determined at jrequent, regular intervals throughout the experiment. AIthough the commercial yieM of the ozone-treatedplants, expressed as hypocotyl dry weight, was significantly reduced, changes in the pattern of development of these plants were observed which were of adaptive value. The pattern of assimilate distribution was altered so that new leaves were produeed more rapidly in the ozone-treatedplants. These later leaves were more resistant to ozone, showing a slower rate of senescence than the cotyledons or first leaves. Measurements of stomatal resistance suggested that this was an acquired, rather than an inherent, characteristic of the later leaves. By the end of the experiment, the relative growth rates in the two treatments did not differ significantly.

INTRODUCTION

The effect of ozone on the growth of radish Raphanus sativus L. has been described by a number of workers. A consistent feature of the results obtained is that the impact of ozone on the leaves is much less than its impact on the hypocotyl, the economically important part of the plant. Tingey et al. (1971), for instance, found that exposure of Raphanus sativus cv. Cherry Belle to 0.05 ppm ozone for a total of 2 h produced a 50 ~ depression in hypocotyl dry weight, but only a 10 ~o reduction in t Present address: Department of Metabolism, Huntingdon Research Centre, Alconbury, Huntingdon, Cambridgeshire, Great Britain. :~ To whom correspondence should be addressed.

165 Environ. Pollut. Ser. A. 0143-1471/80/0023-0165/$02.25 © Applied Science Publishers Ltd, England, 1980 Printed in Great Britain

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L. WALMSLEY, M. R. ASHMORE, J. N. B. BELL

leaf dry weight. Adedipe & Ormrod (1974) found that exposure to 0-25 ppm ozone for 3h at different temperatures consistently caused a greater depression in hypocotyl dry weight than in leaf dry weight, while Tingey et al. (1973a) reported that exposure to 0.4 ppm ozone for 1.5 h at different stages of growth consistently produced the same result. (All these authors refer to leaf and 'root' dry weight. We assume that the major part of this 'root' was the edible portion of the plant which is, in fact, a swollen hypocotyl.) Tingey et al. (1971) suggested that ozone initially depressed the photosynthetic rate and that this then caused an alteration in the pattern of assimilate distribution in favour of the leaves. No direct effect of ozone on translocation need be postulated since a reduction in the amount of available assimilate, produced in various ways, is itself known to cause such a change in assimilate distribution (Wardlaw, 1968). This hypothesis could not be verified, however, since leaf and hypocotyl dry weight were only measured at the end of the experiment. Recently, Oshima et al. (1978, 1979) have demonstrated, by carrying out a series of harvests through time, that this sequence of events can indeed occur. The growth rate of both Gossypium hirsutum and Petroselinum crispum was initially reduced by exposure to ozone. Subsequently, the pattern oftranslocation was altered so that more leaf tissue was produced by the ozone-treated plants and, at final harvest, ozone depression of leaf dry weight was less than that of other plant organs. The use of regular harvests, accompanied by growth analysis, may thus allow the elucidation of the dynamic response of a plant to ozone stress. However, if the response of a plant to a continuous ozone stress does change through time, it is possible that the response of the successive leaves produced by the plant also changes. In almost all previous studies, only total leaf area and dry weight, sometimes divided into live and dead components, have been measured. This measurement may disguise important changes in the behaviour of individual leaves. We describe here the development of Raphanus sativus cv. Cherry Belle subjected to a continuous ozone stress; since only a few leaf pairs are produced by this plant before it is harvested, it is a convenient species on which to study effects on the development of individual leaves.

MATERIALS AND METHODS

The plants were grown in two perspex chambers, l-0m x 0-5m x 0.5m in size, which were placed within a controlled environment cabinet (Fisons 480PG). The base of each chamber was lined with black polythene and filled with sand to a depth of 3 cm. An extensive system of interconnecting muslin wicks, attached to a water reservoir, was placed in the sand to keep it moist. The sand was covered by a sheet of expanded polystyrene in which holes were cut to accommodate the plant pots. The plants were subjected to a 16-h light period at 20 °C and an 8 h dark period at

ADAPTATION OF RADISH TO CONTINUOUS OZONE EXPOSURE

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15 °C. The relative humidity was maintained at 70 +_ 2 ~ , i.e. the vapour pressure deficit was 7.0 Pa during the light period and 5.1 Pa during the dark period. The photon flux density at plant height, measured at different positions within the chambers, was 250 _+ 10/aEm-2 s -1. Air was passed into each chamber through an activated charcoal filter at a flow rate of 0.020m 3 s-1 from a pump situated within the controlled environment cabinet. Ozone, generated from a bank of ultra-violet bulbs (Phillips 0Z4W), was introduced into the air stream entering one chamber, while the other chamber received only filtered air. The ozone concentration in the experimental chamber was monitored continuously using an amperometric analyser (Mast 724-2). The calibration of this instrument was checked periodically against an analyser using ultra-violet absorption (Dasibi 1003-AH). The concentration varied between 0. i 5 ppm (300/~g m - 3) and 0-20 ppm (400/ag m - a), the mean value being 0.17 ppm. The ozone concentration in the control chamber was determined regularly and was never found to exceed 0.02 ppm (40 ~tgm-3). Seeds of Raphanus sativus cv. Cherry Belle were sown in John Innes No. 2 potting compost in 7-cm diameter plastic cups, in the base of which holes had been cut. In addition to the water supplied through the sand, the plants were watered from above every three days. The pots were numbered, with those pots in equivalent positions in the two chambers being assigned the same number. Six plants from each chamber were randomly selected for harvest seven days after germination and at three-day intervals thereafter. The area of each pair of leaves was measured using an electronic planimeter (Paton Industries Ltd) and, after drying at 70 °C, the weights of each leaf pair, the hypocotyl and the roots were determined. A standard growth analysis was carried out on the data using the computer program described by Hunt & Parsons (1973). This uses a stepwise regression procedure to fit curves to the logarithmic values of leaf areas and dry weights plotted against time. The growth analysis parameters--and the errors attached to t h e m - are then derived from the fitted curves. The values of any parameters not included in the growth analysis were compared by means of paired 't' tests at each harvest. The effect of ozone on stomatal resistance was determined using a diffusive resistance porometer (Lambda Instruments, LI-60). Pairs of plants from equivalent positions in the two chambers were selected on day 28. For a period of 48h thereafter, half of these plants were kept in their original chamber while the other half had their treatment reversed, i.e. they were transferred from the ozonated chamber to the clean air chamber and vice versa. At the end of this 48-h period, the stomatal resistance of all the selected plants was measured. It was thus possible to assess the effects of both long- and short-term ozone exposure on stomatal resistance. It was not possible to replicate the two treatment chambers because of limitations of space. Therefore, a second experiment was carried out with the positions of the ozone and clean air chambers reversed. The effects of ozone observed in this second

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L. WALMSLEY, M. R. ASHMORE, J. N. B. BELL

experiment were very similar to those observed in the first experiment, indicating that the effect of position within the controlled environment cabinet was small. For ease of presentation, we shall describe here only the results obtained in the first experiment.

RESULTS

The change in hypocotyl and total leaf dry weight through time is shown in Fig. 1. The dry weight of the hypocotyl was lower in the ozone-treated plants throughout the experiment. Leaf dry weight, although initially lower in the ozone treatment, was similar in the two treatments by 30 days after planting. Figure 2 shows the development of the individual leaf pairs, expressed in terms of area. There was clearly a very great difference in the effect of ozone on successive leaf pairs. Both the cotyledons and the first leaves of the ozone-treated plants developed more slowly, reached a lower maximum leaf area and senesced more rapidly. However, the second leaves developed more rapidly--and reached a higher leaf area - - i n ozone than in clean air, while the third leaves not only developed more rapidly in ozone, but also emerged several days earlier. These differences between successive leaves are summarised in Table 1 as the leaf area duration, i.e. the area under each fitted curve. The changes in the development of the successive leaves were accompanied by changes in their visible appearance. The cotyledons of the ozone-treated plants did not develop normally so as to present a horizontal surface, but were rolled tightly inwards. This effect was not apparent on the later leaves. In addition to the chlorosis which accompanied senescence, necrotic injury developed in the form of interveinal tan-coloured lesions, initially on the adaxial surface, but later on both surfaces of the leaf. The time required for the development of this injury increased in successive leaf pairs. The cotyledons showed injury almost from the day of emergence, while injury appeared on the first leaves 9 days after emergence and on the second leaves only 15 days after emergence. No injury had appeared on the third leaves by the end of the experiment, which was 18 days after their emergence. The decreased sensitivity of the later leaf pairs to ozone may be an inherent characteristic of these leaves, or might have been acquired in the course of the experiment. It is well known that exposure to ozone causes an increase in stomatal resistance (Hill & Littlefield, 1969; Rich & Turner, 1972). Table 2 shows the values of stomatal resistance measured on day 30, after plants had been transferred between treatment chambers for a 48-h period. At this time, the second leaf pair was fully expanded in both treatments. Both the first and second leaves of plants transferred from clean air to ozone showed a significantly higher stomatal resistance than the leaves of plants kept in clean air. The stomatal resistance of the second leaf of plants which had received ozone continuously, however, did not differ significantly from that of plants which had received clean air continuously. No measurements were

169

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LEAF AREA D U R A T I O N

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On day 28, plants were transferred from the pre-treatment chamber to the treatment chamber. The stomatal resistance was measured 48 h later. Values of mean stomatal resistance followed by different letters were found to differ significantly at p = 0.05 using Duncan's multiple range test. CA, Clean air: 03, Ozone. made on the first leaves o f the ozonated plants, which were senescent at this time. These results would suggest that the second leaves o f the continuously o z o n a t e d plants had adapted physiologically so that the sensitivity o f the stomata to ozone observed in the non-fumigated plants was no longer present. The effect of the developmental changes on the overall growth of the plant can be assessed using growth analysis. Figure 3 shows the values o f three parameters, leafarea ratio ( L A R dm 2 g-~), net assimilation rate ( N A R g d m - 2 d a y - 1 ) and relative growth rate (g g - 1 d a y - 1) t h r o u g h time. These three parameters are related by the equation R G R = L A R x N A R . The values and the 95 ~o confidence limits shown in Figs 3 and 4 are those of the curves fitted to the original data; they are not the measured values. Since the curves can be fitted with less accuracy at the beginning and end o f the experiment, the confidence limits at these times tend to be wider. The leaf area ratio (LAR) was initially higher in the clean air plants. At this time, the hypocotyl had not begun to swell and the difference was due to the effect o f ozone on the specific leaf area ratio (i.e. the leaf area per unit leaf dry weight). This was significantly lower in the ozone-treated plants t h r o u g h o u t the course o f the experiment. Ozone is k n o w n to cause a decrease in the rate o f leaf expansion (Evans, 1973) and thus its effect on area m a y be greater than on dry weight. The L A R o f the clean air plants began to decline after day 12, whereas that o f the ozonated plants did not start to decline until day 21. These dates coincide with the times at which the

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hypocotyl began to swell in the two treatments (cf. Fig. I). The significantly higher LAR of the ozonated plants after day 18 may be attributed to the smaller size of the hypocotyl. The net assimilation rate (NAR) did not initially differ in the two treatments. However, it was very difficult to measure the area of the ozone-treated cotyledons, which were rigid and tightly rolled. It is probable that their leaf area was underestimated, leading to an overestimation of NAR and an underestimation of LAR. The NAR of the ozone-treated plants was significantly lower from day 19 to day 28. During this period, the cotyledons were dead, the first leaves were senescing and the second leaves were developing (cf. Fig. 2). Once the second leaves had fully expanded, there was no significant difference between the two treatments. This suggests that the photosynthetic rates of the second and third leaves, unlike those of the first leaves, were not affected by ozone. It is clear from Fig. 3 that, of the two components of relative growth rate (RGR), changes in LAR had a greater effect than changes in NAR. For a short period, the ozonated plants were able to maintain a higher growth rate because a higher proportion of their assimilate had been used to produce new leaf tissue. The production of new leaves in the ozone treatment occurred at the expense of hypocotyl development. This change in the pattern of assimilate distribution is quantified in Fig. 4. The NAR has been split into two components: leaf net assimilation rate (LNAR) and hypocotyl net assimilation rate (HNAR). These values represent the amount of assimilate used for the production of leaf or hypocotyl tissue in unit time. The value of HNAR was consistently lower in the ozonated plants until the last few days of the experiment. However, for a considerable period (day 19 to day 28) the value of LNAR was significantly higher in the ozonated plants. This implies that the accelerated senescence of the earlier leaves induced physiological changes in the plant which caused more of the available assimilate to be used for the production of new leaf tissue. At the end of the experiment, there were no significant differences between the values of RGR and NAR in the two treatments. The pattern of assimilate distribution, as shown by the values of LNAR and HNAR, was also similar. Hence, after five weeks of continuous exposure to a high concentration of ozone, the ozonated plants, although considerably smaller, had adapted to the presence of ozone, in the sense that they were growing at the same rate and distributing assimilate in the same way as plants which had received ozone continuously. This had occurred because of the production of new leaves which were more resistant to ozone.

DISCUSSION

The process of adaptation to continuous ozone fumigation shown by Raphanus sativus in this experiment may conveniently be divided into two components. The

ADAPTATION OF RADISH TO CONTINUOUS OZONE EXPOSURE

175

first component is an alteration in the pattern of assimilate distribution so that new leaves are produced more rapidly in the ozone-treated plants at the expense of hypocotyl development. The second component is the progressive decrease in sensitivity to ozone shown by successive leaf pairs. Exposure to ozone has been found to have a greater effect on root growth than on shoot growth in a wide range of species, including Raphanus sativus itself (Adedipe & Ormrod, 1974; Tingey et ah, 1971,1973a), Phaseolus vulgaris (Engle & Gabelman, 1967), Pinus ponderosa (Parmeter & Miller, 1968), Beta vulgaris (Ogata & Maas, 1973)~ Glycine max (Tingey et ah, 1973b), Daucus carota (Bennett & Oshima, 1976), Trifolium incarnatum and Lolium multiflorum (Bennett & Runeckles, 1977) and Petroselinum crispum (Oshima et al., 1978). It is thought that this occurs because ozone reduces leaf photosynthetic efficiency and thence the amount of assimilate translocated to the organs. Blum & Tingey (1977) demonstrated experimentally that ozone only affected the root growth of Glycine max when applied to the leaves; when the leaves were protected, and ozone only reached the plant via the soil, there was no effect. Miller et al. (1968) found that the decline in root growth of Pinusponderosa caused by ozone was associated with a reduction in the concentration of soluble sugars in the phloem. Tingey et ah (1976) found that ozone increased the concentrations of soluble sugars and starch of the tops of Pinusponderosa seedlings but decreased the root concentrations, suggesting that a decreased amount of sugar was translocated. Ozone may also cause a decreased rate of translocation to other organs. Bennett et ah (1979) reported that exposure of Capsicum annuum to ozone for 9 h a week for 15 weeks had no effect on the dry weight of leaf, stem or root, but significantly reduced the dry weight of the fruit. Oshima et ah (1979) reported that exposure of cotton Gossypium hirsutum to 0.25 ppm ozone for 12 h a week for 19 weeks reduced root, stem and boll dry weight by 40-70 ~ , but leaf dry weight by only ! 7 ~o. This work is of particular interest since it is the only study of which we are aware to describe a detailed growth analysis of the effect of ozone. Initially, the relative growth rate (RGR) of the ozonated plants was reduced, but it then recovered and for a period exceeded that of the control plants. The net assimilation rate (NAR) of the ozonated plants remained lower throughout the experiment and the increase in their RGR was due to an increase in leaf area ratio (LAR). New leaves were produced at a more rapid rate in the ozone-treated plants, but at the expense of the cotton bolls, which emerged later and expanded more slowly. Similar results were obtained by Oshima et al. (1978) using Petroselinum crispum. An initial decrease in the RGR of ozone-treated plants was followed by a period of recovery, in which these plants produced more new leaf tissue than the control plants but less root tissue. Thus the first component of the adaptive process which we identified above is one that has been observed in a number of other studies. This, however, is not the case for the second component, i.e. the increase in ozone resistance of later leaf pairs. For instance, in the study of Gossypium hirsutum by Oshima et al. (1979), large numbers of injured leaves were present throughout the experiment on the ozonated plants.

176

L. WALMSLEY, M. R. ASHMORE, J. N. B. BELL

The NAR of the ozonated plants, in contrast to our results, remained lower than that of the control plants throughout the experiment. A number of studies of the effects of successive exposures to ozone, or of pretreatment with different concentrations of ozone, on plant sensitivity have been carried out, with conflicting results. However, these experiments differ from ours in two important respects. First, they have not involved continuous exposure to ozone, but have usually employed intermittent periods of ozone fumigation (e.g. 8 h a day for 5 days a week ?) interrupted by periods in which the plants received filtered air. Clearly, the constant ozone concentration to which our plants were subjected is not typical of ambient air, in which the ozone concentration fluctuates within and between days, but neither is exposure to ozone in the field immediately preceded and followed by a period of exposure to no ozone. It is possible, however, that resistance to ozone is more likely to develop when plants are continuously exposed to this stress than when it is imposed and withdrawn intermittently. The second difference in our work is that we have examined the response of individual leaves, whereas earlier workers have described effects on total leaf injury or growth. It has been suggested that resistance to ozone induced by a previous ozone exposure is caused by an increase in stomatal resistance (Macdowell, 1965; Hill & Littlefield, 1969) which may occur within minutes. In contrast, the resistance to ozone in our experiment developed over a period of weeks and was associated with the emergence of new leaves whose stomatal resistance did not differ significantly from those in the clean air treatment. Clearly, some change in the metabolism, or hormonal balance, of the plant occurred as a result of continuous exposure to ozone which caused the new leaves to be more resistant. The nature of this change cannot be deduced from our results. The ozone concentration of0.17 ppm used in this experiment is not exceptionally high; the maximum hourly mean concentration recorded in the U K is 0.24ppm (Apling et a/., 1977). However, such ambient concentrations occur infrequently and only last for a few hours. It is unknown for concentrations of this magnitude to be maintained for 2 4 h d a y -1 over a period of several weeks; even in the severely polluted Los Angeles basin, the average daily mean ozone concentration in summertime is only 0.06-0.08 ppm (Dimitriades, 1976). We have only demonstrated this adaptive response with one species under an unrealistically severe ozone stress. It is not clear that it is of any importance in the response of plants to ozone under field conditions. However, our results do indicate that the effects of ozone on the development of plants may be complex and that detailed studies of development through time may be of considerable value in understanding the effects of ozone on plant growth. ACKNOWLEDGEMENTS

This work was supported by the Natural Environment Research Council, who supplied a research grant to J.N.B.B., and an MSc studentship to L.W. We thank Dr

ADAPTATION OF RADISH TO CONTINUOUS OZONE EXPOSURE

177

R. Hunt, of Sheffield University, for his assistance in carrying out the growth analysis, and E. E. Green and C. Dalpra for technical assistance. REFERENCES ADEDIPE, N. O. & ORMROD,D. P. (1974). Ozone-induced growth suppression in radish plants in relation to pre- and post-fumigation temperatures. Z. Pflanzenphysiol., 71,281 7. APLING, A. J., SULLIVAN,E. J., WILLIAMS, M. L., BALL, D. J., BERNARD,R. E., DERWENT,R. G., EGGLETON, A. E. J., HAMPTON, L. & WALLER, R. E. (1977). Ozone concentrations in south-east England during the summer of 1976. Nature, Lond., 269, 569 73. BENNETT,J. P. ~g OSH1MA,R. J. (1976). Carrot injury and yield response to ozone. J. Am. Soc. hort. Sci., 101,638 9. BENNETT, J. P. & RUNECKLES,V. C. (1977). Effects of low levels of ozone on growth of crimson clover and annual ryegrass. Crop Sci., 1% 443-5. BENNETT, J. P., OSHIMA, R. J. ~ LIPPERT, L. F. (1979). Effects of ozone on injury and dry matter partitioning in pepper plants. Environ. exp. Bot., 19, 33-9. BLUM, U. ~z.TINGEY,D. T. (1977). A study of the potential ways in which ozone could reduce root growth and nodulation in soybean. Atmos. Environ., I1, 737 9, DIMITRIADES,B. (1976). Photochemical oxidants in the ambient air of the United States. Report No. EPA600/3-76-017, Environmental Protection Agency. Springfield, Virginia, National Technical Information Service. ENGLE, R. L. & GAnELMAN, W. H. (1967). The effects of low levels of ozone on pinto beans, Phaseolus vulgaris L. Proc. Am. Soe. hort. Sci., 91,304-9. EVANS, L. S. (1973), Bean leaf growth response to moderate ozone levels. Environ. Pollut., 4, 17-26. HILL, A. C. & LIT'rLEFIELD,N. (1969). Ozone. Effect on apparent photosynthesis, rate of transpiration and stomatal closure in plants. Environ. Sci. & Technol., 3, 52 6. HUNT, R. & PARSONS,I. T. (1973). A computer program for deriving growth functions in plant growth analysis. J. appl. Ecol., 10, 297 307. MACDOWALL, F. D. H. (1965). Predisposition of tobacco to ozone damage. Can. J. PI. Sci., 45, 1 12. MIt, LER, P. R., Corm, JR., F. W. & ZAVARIN, E. (1968). Photochemical oxidant injury and bark beetle (Coleoptera: Solytidae) infestation of ponderosa pine, 111, Effect of injury on oleoresin composition, phloem carbohydrates and phloem pH. Hilgardia, 39, 135-40. OGATA, G. & MAAS, E. V. (1973). Interactive effects of salinity and ozone on growth and yield of garden beet. J. environ. Qual., 2, 518 20. OSHIMA, R. J., BENNETT, J. P. & BRAEGELMANN,P. K. (1978). Effect of ozone on growth and assimilate partitioning in parsley. J. Am. Soc. hort. Sci., 103, 348-50. OSHIMA,R. J., BRAEGELMANN,P. K., FLAGLER,R. B. & TESO, R. R. (1979). The effects of ozone on the growth, yield and partitioning of dry matter in cotton. J. environ. Qual., 8, 474-9. PARMETER,J. R. JR. t~, MILLER, P. R. (1968). Studies relating to the cause of decline and death of ponderosa pine in southern California. P/. Dis. Reptr, 52, 707-11. RICH, S. & TURNER, N. C. (1972). Importance of moisture on stomatal behaviour of plants subjected to ozone. J. Air Pollut. Control Ass., 22, 718-21. TINGEY, D. T., HECK, W. W. ,g" REINERT, R. A. (1971 ). Effect of low concentrations of ozone and sulfur dioxide on foliage, growth and yield of radish. J. Am. Soc. hort. Sci., 96, 369 71. TINGEY, D. T., DUNNING, J. A. & JIVIDEN, G. M. (1973a). Radish root growth reduced by acute ozone exposures. Proe. int. Clean air Congr., 3rd, A154-A156. Dusseldorf, Verein Deutscher lngenieur. T1NGEY, D. T., REINERT, R. A., WICKLIFF, C. & HECK, W. W, (1973b). Chronic ozone or sulfur dioxide exposures, or both, affect the early vegetative growth of soybeans. Can. J. PI. Sei., 53, 875-9. TINGEY, D. T., WILHOUR, R. G. ~/: STANDLEY,C. (1976). The effect of chronic ozone exposures on the metabolite content of ponderosa pine seedlings. For. Sci., 22, 234-41. WARDLAW,I. F. (1968). The control and pattern of movement of carbohydrates in plants. Bot. Rev., 34, 79-105.