- Email: [email protected]
Growth responses of plants to various concentrations of nitrogen dioxide
Environmental Pollution (Series A) 38 (1985) 361-373
Growth Responses of Plants to Various Concentrations of Nitrogen Dioxide K. Okano & T. Totsuka Division of Environmental Biology, National Institute for Environmental Studies, Yatabe, Tsukuba, Ibaraki 305, Japan
& T. Fukuzawa & T. Tazaki Department of Biology, Faculty of Science, Toho University, Funabashi, Chiba 274, Japan
ABSTRACT Sunflower Helianthus annuus L. and maize Zea mays L. plants in the vegetative phase were exposed to nitrogen dioxide ( NO z) at 0.0 (control), 0.2, 0.5 and l'O ppm (IA litre- 1)Jbr 2 weeks. The growth responses of the plants to NO 2 were examined by the techniques of growth analysis. The sunflower plant was more susceptible to NO z than the maize plant. Exposure to NO 2 at 0.2ppm slightly stimulated the growth of the sunflower plants. The net assimilation rate ( N A R ) was also significantly increased when the plants were exposed to 0.2ppm NO z. Exposures to NO 2 at 0.5ppm or more significantly reduced the dry weight of the sunflower plant. O f the component parts, the roots and stems were severely affected, while the leaves were less affected. This resulted in an elevated shoot~root ratio. The net assimilation rate of both species was reduced by the exposures to NO 2 at 0.5 ppm or more, while, in contrast, the leaf area ratio ( L A R ) was increased. The relative growth rate (RGR), the product of the N A R and the L A R , was therefore less affected by NO 2. The increase in the L A R was overwhelmingly the result of an increase in the leaf weight ratio ( L W R ) . These results imply that a reduction in photosynthetic efficiency induced by NO 2 could be, in part, compensated for by an increase in assimilatory area, suggesting an adaptive growth response of the plants to air pollutant stresses. 361 Environ. Pollut. Ser. A. 0143-1471/85/$03.30 (0 Elsevier Applied Science Publishers Ltd, England, 1985. Printed in Great Britain
362
K. Okano, T. Totsuka, T. Fukuzawa, T. Tazaki
INTRODUCTION Nitrogen dioxide (NO2)--an important air pollutant--has been demonstrated to be phytotoxic in a wide range of species (Taylor et al., 1975). Higher concentrations (above 3 to 4 ppm, #1 litre-1) of NO 2 can cause acute injurious effects on plant leaves, probably through the accumulation of nitrite (Kato et al., 1974; Yoneyama et al., 1979). Chronic exposure to lower concentrations of NO 2 usually reduces the growth of plants (Taylor & Eaton, 1966; Thompson et al., 1970; Ashenden & Mansfield, 1978; Totsuka et al., 1978), although no nitrite can be detected in leaf tissues. The mechanism of this type of damage by NO 2 is not known. On the other hand, several investigators (Failer, 1972; Fujiwara, 1973; Troiano & Leone, 1977; Yoneyama et al., 1980a; Marie & Ormrod, 1984) found that, in some cases, lower concentrations of NO 2 were not detrimental to plants but resulted in an increased plant weight as compared with nonexposed plants. These findings were supported by the fact that NO 2 absorbed in plant leaves was rapidly metabolized and incorporated into organic nitrogen compounds (Yoneyama & Sasakawa, 1979; Rogers & Campbell, 1979). The growth responses of plants to both ozone (03) and sulphur dioxide (SO2) have been extensively documented. A consistent feature of the results obtained is that the impact of these two pollutants on the root is more drastic than that on the shoot. This results in a lowered root/shoot ratio (Tingey et al., 1971; Bennett & Runeckles, 1977; Mejst~ik, 1980; Shimizu et al., 1980; Jensen, 1981). Techniques of growth analysis revealed that the relative growth rate (RGR) was initially lowered by exposure to 0 3 or SO 2 because of a reduction in the net assimilation rate (NAR), but that, thereafter, RGR in the fumigated plants recovered above that of the control plants due to a larger leaf area ratio (LAR) (Oshima et al., 1978, 1979; Horsman et al., 1980; Walmsley et al., 1980; Shimizu et al., 1981; Jones & Mansfield, 1982a). The growth responses observed were interpreted as being compensatory in nature, responding to the reduced photosynthetic efficiency. However, it has not been sufficiently characterised whether plants will show a similar type of growth response to NO 2 as to 0 3 and SO 2. Whitmore & Mansfield (1983) reported that the root/shoot ratio of several grasses was unaffected even when the whole plant growth was significantly reduced by exposure to NO2. The present experiment was designed to quantify the growth responses
Growth response of plants to nitrogen dioxide
363
of sunflower and maize plants in the vegetative phase to various concentrations o f N O 2 by means of growth analysis techniques and to determine whether chronic levels of N O 2 might stimulate the growth of plants by acting as a nitrogen fertilizer from the atmosphere.
MATERIALS A N D M E T H O D S Plant materials Seeds of sunflower H e l i a n t h u s annuus L. cv. Russian M a m m o t h and maize Z e a m a y s L. cv. Dento were sown in pots containing 1.8 litres of artificial soil (a mixture of vermiculite, peat, perlite and fine gravel, 2:2:1:1, v/v), 5g of Magamp K ( N : P 2 O s : K 2 0 = 6 : 4 0 : 5 , W. R. Grace Co., Tennessee, USA) and 15 g of magnesia lime. The seeds were sterilised with 0.1% solution of Benlate (Dupon, Delaware, USA) for 1 h and then washed with tap water for 24 h. Seven days after the sowing, the seedlings were thinned to one plant per pot to increase the uniformity among plants. Nutrients were supplied twice a week as 100 to 200ml of I g per litre of Hyponex ( N : P z O s : K 2 0 = 6.5:5: 19) solution. The plants were grown for 2 weeks in a naturally lit growth room with a constant temperature of 25 °C and a relative humidity of 70 %. Fourteen days after sowing, 0.72 g of potassium nitrate was added to each pot, and then the plants were transferred to four artificially lit growth cabinets with exactly the same environmental conditions (420 #E s - 1 m - 2; light period, 14 h; 25 °C, relative humidity, 70 ~ ) except for the NO 2 concentration. The light source consisted of twenty-four 400 W stannous halide lamps. Exposure to
NO 2
Exposure of plants to various concentrations of N O 2 w a s started on the fourteenth day and ended on the twenty-eighth day after sowing. The plants were watered daily, and no additional fertilizer was applied during the exposure period. Nitrogen dioxide was supplied from a pressurised cylinder of predetermined concentration. Controlled levels of N O 2 w e r e released through a thermal mass flow controller into the air stream entering the growth cabinet. The treatments were as follows: (1) control: charcoal filtered air; (2) 0.2 p p m NO2; (3) 0.5 ppm N O 2 and (4) 1.0 ppm N O 2. The concentration of N O 2 in the cabinet was continuously
364
K. Okano, T. Totsuka, T. Fukuzawa,
T. Tazaki
monitored and regulated at a required level by a controlling system based on a chemiluminescent NO-NO,-NO, analyser (Model 14, Therm0 Electron). The concentrations used in these experiments are not unduly high compared with levels actually found in Japan. In urban areas, such as Tokyo, the monthly average concentration of NO, in 1983 was about 0.05 ppm, but hourly average values were in the range from 0.1 to 0.2 ppm. Moreover, the monthly average concentration of NO, (NO, + NO) is about 0.2 ppm and hourly average values often exceed 0.5 ppm in winter. Harvest and growth analysis
Each ten plants were harvested just before the start of NO, exposure and after the end of 2 weeks’ continuous exposure. The sunflower plants were separated into leaves, stems (including petioles) and roots. The maize plants were divided into leaf blades, leaf sheaths, culms and roots. The roots were washed carefully to eliminate the potting medium. Leaf area was measured photometrically with an area meter (Model 3100, LICOR). The separated plant parts were dried in an oven at 80 “C for 3 days and then weighed. The growth analysis variables, relative growth rate (RGR), net assimilation rate (NAR), leaf area ratio (LAR), leaf weight ratio (LWR) and specific leaf area (SLA) were calculated from the formula discussed by Saeki (1965). Relative growth rate is the product of the NAR and the LAR, and the LAR is the product of the LWR and the SLA.
RESULTS No chlorosis or necrosis was observed on the surface of leaves, even when the plants were continuously exposed to NO, at l.Oppm for 2 weeks. There was a slight crinkling, or puckering, and a darkening of the green colour of the newly expanding leaves of the sunflower plants exposed to NO, at 005ppm or more. The dry weights of the whole plant, and each component part of sunflower plants exposed to various concentrations of NO, for 2 weeks are shown in Table 1. The whole dry weight of the plants exposed to NO, at 0.2 ppm was 7 % greater than that of the control plants grown under
365
Growth response o f plants to nitrogen dioxide
TABLE 1 Growth of Sunflower Plants Exposed to Various Concentrations of NO2 for 2 Weeks Plant part
Initial Control
Dry weight (g) Whole plant Leaves Stems Roots Leaf area (cm 2) Shoot/root ratio
0.41 0.26 0.10 0.05 107 7.20
11.05 3.98 4.89 2.18 1 322 4.07
NO 2 concentration (ppm) 0.2 0.5
11.83 (107) 4.04 (102) 5.28 (108) 2.53 (116) 1 264 (96)* 3.68 (90)
9.73 (88)* 3.98 (100) 3.80 (78)** 1.95 (89) 1 242 (94) 3-99 (98)
1.0
8.67 (78)*** 4.07 (102) 3-22 (66)*** 1.38 (63)*** 1 158 (88)** 5.28 (130)**
Numerals in parentheses are relative values to control. Significantly different from the control at the 5% (*), 1% (**) and 0.1 ~ (***) levels.
charcoal filtered air. Stimulation of dry weight growth induced by N O 2 at 0 . 2 p p m was marked in the roots and the stems, and 16~o and 8~o, respectively, greater than the control values. On the other hand, dry weight growth of the leaves was less affected and leaf area was slightly~ but significantly, decreased when exposed to 0.2 ppm N O 2. Exposure t o N O 2 at 0.5 ppm or more caused significant reduction in the dry weights of roots, stems and whole plants. In contrast, there was no significant difference between the leaf dry weight of NO2-exposed plants and that of control plants, although the leaf area was slightly reduced by N O 2 exposure. Consequently, the shoot/root ratio was decreased by exposure t o N O 2 at 0.2 ppm, being significantly elevated as a result of exposure to NO 2 at 1.0 ppm, indicating that the root dry weight was reduced more than the shoot dry weight. In the case of maize plants, the dry weight of whole plants tended to decrease with increasing N O 2 concentration (Table 2). However, no differential effects of N O 2 o n various plant parts were observed and no significant changes in the shoot/root ratio could be detected. The data obtained from the sunflower plants were further examined by the techniques of growth analysis and the results are shown in Figs l, 2 and 3. The relative growth rate of the sunflower plants not subjected to NO 2 exposure (control) was 0.24 mg m g - 1 day- 1 during the experimental period, which ranged from 14 to 28 days after sowing (Fig. 1). Exposure to NO 2 at 0.2 ppm resulted in a significantly greater R G R than that of the control. A slight reduction in R G R was detected on plants exposed to
366
K. Okano, T. Totsuka, T. Fukuzawa, T. Tazaki
TABLE 2 Growth of Maize Plants Exposed to Various Concentrations of NO 2 for 2 Weeks Plant part
lnitial Control
Dry weight (g) Whole plant Leaf blades Leaf sheaths + culms Roots Leaf area (cm 2) Shoot/root ratio
0.74 0.34
9.02 3.45
0-17 0.23 151 2.22
3.38 2.19 1 018 3.12
NO 2 concentration (ppm) 0.2 0.5
8-66 (96) 3.12 (90) 3.29 (97) 2.26 (103) 913 (90) 2.84 (91)
1.0
8-68 (96) 3.49 (101)
8.41 (93) 3.36 (97)
3.04 (90)* 2.16 (99) 1 074 (106) 3"02 (97)
2-93 (87) 2.13 (97) 1 042 (102) 2.95 (95)
Numerals in parentheses are relative values to control. * Significantly different from the control at the 5 ~ level. N O 2 at 0.5 ppm. Exposure t o NO 2 at 1.0 ppm significantly reduced the
RGR by 7 ~ compared with the control. The relative growth rate was divided into the NAR and the LAR (Fig. 2). The changes in the NAR caused by various concentrations of N O 2 w e r e very similar to those in the RGR, but the impact o f N O 2 o n the NAR was more pronounced than that on the RGR. A l0 ~o increase in the NAR was detected in the plants exposed t o NO 2 at 0"2 ppm. Exposures to N O 2 at 0.5ppm and 1.0ppm reduced the NAR by 9 ~ and 15~, 0.3 m
a3
0.2
1oo
102
96
93
0.5
1.0
E E 0.1
0.0 C
0.2 NO 2
(ppm)
Fig. 1. Changes in the relative growth rate (RGR) of sunflower plants exposed to various concentrations of NO2. Numerals in the Figure show the relative values to control (100), significantly different from control at the 5 ~o (*) and 0.1 ~ (***) levels.
Growth response of plants to nitrogen dioxide
367
0.2
I m "0
11o
e
E E
O'J
0.1
v
E e~
z
0
e.e-
,°I° C
0.0 0.2
O.5
C
1.0 N0 2
0.2
0.5
1.0
(~ppm)
Fig. 2. Changes in the net assimilation rate (NAR) and leaf area ratio (LAR) of sunflower plants exposed to various concentrations of NO 2. Numerals in the Figure show the relative values to control (100), significantly different from control at the 5 ~o (*), 1 ~o (**) and 0.1 ~o (***) levels.
respectively, compared with the control. The changes in the LAR were the opposite of those in the NAR. Exposure t o N O 2 at 0"2ppm brought about a reduced LAR whilst exposures t o N O 2 at 0 . 5 p p m or more resulted in a significantly greater LAR than that of the control. The leaf area ratio was further divided into the LWR and the SLA (Fig. 3). The leaf weight ratio--the proportion of leaf weight to whole plant weight--was significantly reduced by N O 2 a t 0-02 ppm. On the other hand, exposures t o NO 2 at 0.5 p p m or more conspicuously increased the LWR compared with the control, i.e. by 1 4 ~ at 0.5 ppm and by 31 ~ at 0.6
0.6 .-..
.k
I
E E
0.4
""
0.2
131
"
0.4 "k
0.2 .,.,.J ¢.n
0.0
0.0 C
0.2
0.5
1.0 N02
C
0.2
0.5
1.0
(ppm)
Fig. 3. Changes in the leaf weight ratio (LWR) and specific leaf area (SLA) of sunflower plants exposed to various concentrations of N O 2. Symbols are the same as in Fig. I.
K. Okano, T. Totsuka, T. Fukuzawa, T. Tazaki
368
TABLE 3 Growth Analysis on Maize Plants Exposed to Various Concentrations of NO 2
Treatment
Control 0-2 ppm 0'5 ppm 1.0ppm
R GR NA R LA R L WR SLA (mgmg-lday -1) (mgcm-2day -l) (cm2mg -l) (mgmg -l) (cm2mg -1) 0' 178 0' 175 0" 176 0.173
1-30 1.34 1.21 1.19
0.113 0.105 0.124 0.124
0.39 0.36 0.40 0.40
0.296 0.295 0.307 0.310
1.0 ppm. The specific leaf area significantly decreased with the increase in NO 2 concentration. The reduced SLA seemed to be the result of a crinkling of new leaves exposed to NO 2, and might not mean that thicker leaves would have been produced on the plants exposed to N O 2. Table 3 shows the results of growth analysis on the maize plants exposed to various concentrations of NO 2. The general trends of changes in the growth parameters of maize plants were similar to those of sunflower plants, although they were not so clear. The exposure to NO 2 at 0.2 ppm resulted in a greater NAR and a smaller LAR than the control. The maize plants exposed to NO 2 at 0-5 ppm or more had a reduced NAR and an increased LAR compared with the control. Therefore, the RGR, which was the product of the NAR and the LAR, was less affected by exposure to various concentrations of NO 2. Thus, in both plant species, the remarkable reduction in the NAR induced by relatively higher concentrations of NO 2 could, in part, be compensated for by the increase in the LAR, resulting in a smaller reduction of the RGR. The marked rise in the LWR caused by NO 2 could account for the increase in the LAR. DISCUSSION
Growth stimulation of plants by lower concentration of NO2 Nitrogen dioxide, even at a lower concentration, has been believed to be toxic or inhibitory to plants. Taylor & Eaton (1966) reported that continuous exposure to N O 2 at less than 0.5ppm for 10 to 22 days caused significant suppression of growth in pinto bean and tomato seedlings. Ashenden & Mansfield (1978) showed that the yield of Poa pratensis was reduced by exposure to NO 2 at 0.068 ppm for 20 weeks. In the present experiment, however, the growth of sunflower plants was slightly stimulated by exposure to NO 2 at 0.2ppm for 2 weeks. Net
Growth response of plants to nitrogen dioxide
369
assimilation rate, an approximation of photosynthetic efficiency, was also significantly increased as a result of exposure to 0.2 ppm NO 2. Nitrogen dioxide absoi'bed in plant leaves through the stomata can be converted into nitrate and nitrite, and then rapidly assimilated into amino acids and proteins if nitrate and nitrite reductases are active (Rogers and Campbell, 1979; Yoneyama & Sasakawa, 1979). Ito et al. (1984) also showed an increase in amino acid synthesis when kidney bean plants were exposed to NO 2. NO2-nitrogen incorporated into amino acids and proteins was then retransferred from the leaves to rapidly growing parts, probably through phloem, along with the photosynthates (Yoneyama et al., 1980b; Okano et al., 1983). Therefore, a beneficial effect of lower concentrations of NO 2 on plant growth might be expected--and has been actually confirmed--with sunflower (Failer, 1972), rice (Fujiwara, 1973), tomato (Troiano & Leone, 1977; Marie & Ormrod, 1984)and cucumber (Yoneyama et al., 1980a). The fixation of carbon dioxide and the translocation of assimilate were also found to be enhanced by exposure t o N O 2 (Okano et al., 1984b). If lower concentrations of N O 2 a c t as a nitrogen fertilizer [from the atmosphere, the stimulation of plant growth by N O 2 would be expected when the plants are nitrogen deficient. In the present experiment, since no additional fertilizer was applied to the plants during the 2-week exposure period, they might have become nitrogen deficient. It is necessary to quantify the contribution of NO2-nitrogen to the total nitrogen increase for a better understanding of the nutritional effect of N O 2 o n plant growth. In our next study, absorption of N O 2 by the plants was simultaneously measured by means of the 15N dilution method (details to be published). The amount of NO2-nitrogen absorbed by sunflower plants exposed to 0.2 ppm NO 2 during a 2-wEek period was about 12.2 mg per plant, accounting for 5.8 ~o of the total nitrogen increase during this period. This would be sufficient to account for the increase in dry weight growth observed in this species. ~n contrast, maize plants absorbed only 3.7 mg per plant, contributing to the total nitrogen increase by 2.3 ~. Less absorption of N O 2 by maize might be one of the reasons why the growth of maize was less affected by exposure t o N O 2 than that of sunflowers. Adaptive response of plants to higher concentrations of N O 2
Although the growth of sunflower plants was slightly enhanced by 0.2 ppm NO2, exposure to NO 2 at 0.5 ppm or more significantly reduced
370
K. Okano, T. Totsuka, T. Fukuzawa, T. Tazaki
the dry weight of the plant. Of the plant parts, the roots and stems were severely affected by NO 2, while the leaves were less affected. Consequently, the shoot/root ratio was significantly elevated by 1-0 p p m N O 2. Whitmore & Mansfield (1983) reported that the shoot/root ratio of several grasses was unaffected even when the whole plant growth was significantly reduced by exposure to N O 2. The present result, obtained with sunflowers, however, indicated that N O 2 can also exert differential effects on the growth of various plant parts. This was apparently so, also in the case of O a or SO 2. The different responses of various plant parts to these two pollutants have been extensively documented in a wide range of species such as radish (Tingey et al., 1971), crimson clover and annual ryegrass (Bennett & Runeckles, 1977), tobacco and cucumber (Mejstfik, 1980), sunflower (Shimizu et al., 1980, 1981) and green ash (Jensen, 1981). Growth analysis techniques revealed the following. The net assimilation rate was decreased by higher concentrations (above 0-5 ppm) of N O 2, whilst, in contrast, the LAR increased with the decrease in the NAR. As a result, the RGR, which is the product of the NAR and the LAR, was less affected by NO 2. This means that the inhibitory effect of N O 2 on the R G R apparently acted through the reduced NAR, and that the reduction of photosynthetic efficiency caused by NO 2 was, in part, compensated for by the increase in the LAR. The leaf area ratio is the product of the LWR and the SLA. The results shown in Fig. 3 indicated that the increase in the LAR was overwhelmingly the result of an increase in the LWR. The SLA decreased somewhat with increasing NO 2 concentration. The increase in the LWR, along with the reduced stem and root weight ratio (data not shown), implies that the pattern of dry matter partitioning among plant parts was changed in favour of the leaves as a result of exposure to NO 2. Recently, Jones & Mansfield (1982b) and Okano et al. (1984a) have directly demonstrated, by tracer experiments, t h a t the pattern of assimilate distribution in plants is altered by SO 2 or 0 3 so that a greater proportion of assimilate is partitioned.to growing leaves and a smaller proportion to the root and stem. The sunflower plants used in the present experiment might also respond to N O 2 by diverting relatively more assimilates into the photosynthetic organs (leaves) in response to the fall in photosynthetic efficiency. The increase in assimilatory area induced by NO2, however, was not enough to compensate for the decrease in photosynthetic efficiency. Hence, the growth rate declined as a result of NO2, and the dry weight of the sunflower plant was 1 2 ~ and 2 2 ~ , respectively, lower than the control after 14 days' exposure to 0"5 ppm and 1.0 ppm NO2.
Growth response of plants to nitrogen dioxide
371
Thus, the adaptive response of plants to N O 2 we identified in the present study is exactly the same as that to O 3 or S O 2 - - a fact demonstrated by a number of investigators (Oshima et al., 1978, 1979; H o r s m a n et al., 1980; Walmsley et al., 1980; Shimizu et al., 1981 ; Jones & Mansfield, 1982a). Furthermore, this kind of adaptive response seems to be brought about not only by exposure to air pollutants but also by other environmental factors which cause a reduction in the a m o u n t of available assimilate. For example, a deficiency in photosynthate has been found to have a greater effect on root growth than on shoot growth (Curtis & Clark, 1950). Blackman & Black (1959) reported, with several herbaceous species, that the N A R w a s dependent on the amount of light received, whilst, in contrast, the LAR rose as light intensity decreased. These facts clearly indicate that the proportion of leaf area to plant weight (LAR) varies easily according to environmental conditions and that changes in the LAR may assist the plant to adapt to various environmental stresses.
REFERENCES Ashenden, T. W. & Mansfield, T. A. (1978). Extreme pollution sensitivity of grasses when SO 2 and N O 2 a r e present in the atmosphere together. Nature, Lond., 273, 142-3. Bennett, J. P. & Runeckles, V. C. (1977). Effects of low levels of ozone on growth of crimson clover and annual ryegrass. Crop Sci., 17, 443-5. Blackman, G. E. & Black, J. N. (1959). Physiological and ecological studies in the analysis of plant environment. XI. A further assessment of the influence of shading on the growth of different species in the vegetative phase. Ann. Bot., 23, 51-63. Curtis, O. F. & Clark, D. G. (1950). Factors influencing root growth and the ratio between roots and tops. In An introduction to plant physiology, 666-92. New York, McGraw-Hill. Faller, V. N. (1972). Schwefeldioxid, Schwefelwasserstoff, Nitrosegase und Ammoniak als ausschliel~liche S-bzw. N-Quellen der h6heren Pflanze. Z. Pflanzenern. Bodenk., 131, 120-30. Fujiwara, T. (1973). Effects of nitrogen oxides in the atmosphere on vegetation. J. Pollut. Control, 9, 253-7. Horsman, D. C., Nicholls, A. O. & Calder, D. M, (1980). Growth responses of Dactylis glomerata, Lolium perenne and Phalaris aquatica to chronic ozone exposure. Aust. J. Plant Physiol., 7, 511-17. Ito, O., Okano, K. & Totsuka, T. (1984). Effects of NO/ and 0 3 alone or in combination on kidney bean plants. II. Amino acid pool size and composition. Res. Rep. Natl Inst. Environ. Stud., Jpn, 66, 15-25. Jensen, K. F. ( 1981). Ozone fumigation decreased the root carbohydrate content and dry weight of green ash seedlings. Environ. Pollut. (Ser. A), 26, 147-52.
372
K. Okano, T. Totsuka, T. Fukuzawa, T. Tazaki
Jones, T. & Mansfield, T. A. (1982a). The effects of SO 2 on growth and development of seedlings of Phleurn pratense under different light and temperature environments. Environ. Pollut. (Set. A), 27, 57-71. Jones, T. & Mansfield, T. A. (1982b). Studies on dry matter partitioning and distribution of 14C-labelled assimilates in plants of Phleum pratense exposed to SO 2 pollution. Environ. Pollut. (Ser. A), 28, 199-207. Kato, T., Tachibana, S. & Inden, T. (1974). Studies on the injury of crops by toxic gases in covering structure. II. Mechanisms involved in the sensitivity of plants to NO 2. Environ. Control in Biol., 12, 103-7. Marie, B. A. & Ormrod, D. P. (1984). Tomato plant growth with continuous exposure to sulphur dioxide and nitrogen dioxide. Eneiron. Pollut. (Ser. A), 33, 257-65. Mejstfik, V. (1980). The influence of low SO 2 concentrations on growth reduction of Nicotiana tabacum L. cv. Samsun and Cucumis sativus L. cv. Unikfit. Environ. Pollut. (Set. A), 21, 73-6. Okano, K., Tatsumi, J., Yoneyama, T., Kono, Y. & Totsuka, T. (1983). Investigation on the carbon and nitrogen transfer from a terminal leaf to the root system of rice plant by a double tracer method with 13C and 15N. Japan. J. Crop. Sci., 52, 33141. Okano, K., Ito, O., Takeba, G., Shimizu, A. & Totsuka, T. (1984a). Alteration of 13C_assimilate partitioning in plants of Phaseolus vulgaris exposed to ozone. New Phytol., 97, 153-63. Okano, K., Ito, O., Takeba, G., Shimizu, A. & Totsuka, T. (1984b). Effects of N O 2 and 0 3 alone or in combination on kidney bean plants. V. 3C-assimilate partitioning as affected by NO 2 and/or O a. Res. Rep. Natl Inst. Environ. Stud., Jpn., 66, 49-58. 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. Rogers, H. H. & Campbell, J. C, (1979). Nitrogen-15 dioxide uptake and incorporation by Phaseolus vulgaris (L.). Science, N.Y., 206, 333-5. Saeki, T. (1965). Growth analysis of plants. Bot. Mag. Tokyo, 78, 111-19. Shimizu, H., Furukawa, A. & Totsuka, T. (1980). Effects of low concentrations of SO 2 on the growth of sunflower plants. Environ. Control in Biol., 18, 39-47. Shimizu, H., Motohashi, S., Iwaki, H., Furukawa, A. & Totsuka, T. (1981). Effects of chronic exposures to ozone on the growth of sunflower plants. Environ. Control in Biol., 19, 13747. Taylor, O. C. & Eaton, F. M. (1966). Suppression of plant growth by nitrogen dioxide. Plant Physiol., Lancaster, 41, 132-5. Taylor, O. C., Thompson, C. R., Tingey, D.T. & Reinert, R. A. (1975). Oxides of nitrogen. In Responses ofplants to air pollution, ed. by J. B. Mudd and T. T. Kozlowski, 121-39. London, Academic Press.
Growth response of plants to nitrogen dioxide
313
Thompson, C. R., Hensel, E. G., Kats, G. & Taylor, 0. C. (1970). Effects of continuous exposure of navel oranges to nitrogen dioxide. Atmos. Environ., 4, 349-5s.
Tingey, D. T., Heck, W. W. & Reinert, R. A. (1971). Effects of low concentrations of ozone and sulfur dioxide on foliage, growth, and yield of radish. J. Am. Sot. hort. Sci., 96, 369-71. Totsuka, T., Sato, S., Yoneyama, T. & Ushijima, T. (1978). Response of plants to atmospheric NO, fumigation. 2. Effects of NO, fumigation on dry matter growth of sunflower and kidney bean plants. Res. Rep. Nat1 Inst. Environ. Stud., Jpn, 2, 77-87.
Troiano, J. J. & Leone, I. A. (1977). Changes in growth rate and nitrogen content of tomato plants after exposure to NO,. Phytopathology, 67, 1130-3. Walmsley, L., Ashmore, M. R. & Bell, J. N. B. (1980). Adaptation of radish Raphanus sativus L. in response to continuous exposure to ozone. Environ. Pollut. (Ser. A), 23, 165-77.
Whitmore, M. E. & Mansfield, T. A. (1983). Effects of long-term exposures to SO, and NO, on Poapratensis and other grasses. Environ. Pollut. (Ser. A), 31, 217-35.
Yoneyama, T. & Sasakawa. H. (1979). Transformation of atmospheric NO, absorbed in spinach leaves. Plant & Cell Physiol., 20, 263-6. Yoneyama, T., Sasakawa, H., Ishizuka, S. & Totsuka, T. (1979). Absorption of atmospheric NO, by plants and soils. II. Nitrite accumulation, nitrite reductase activity and diurnal change of NO, absorption in leaves. Soil Sci. Plant Nutr., 25, 267-75.
Yoneyama, T., Totsuka, T., Hayakawa, H. & Yazaki, J. (1980a). Absorption of atmospheric NO, by plants and soils. V. Day and night NO,-fumigation effect on the plant growth and estimation of the amount of NO,-nitrogen absorbed by plants. Res. Rep. Nat1 Inst. Environ. Stud., Jpn, 11, 31-50. Yoneyama, T., Arai, K. & Totsuka, T. (1980b). Transfer of nitrogen and carbon from a mature sunflower leaf--“NO, and 13C0, feeding studies. Plant & Cell Physiol., 21, 1367-81.
Growth Responses of Plants to Various Concentrations of Nitrogen Dioxide K. Okano & T. Totsuka Division of Environmental Biology, National Institute for Environmental Studies, Yatabe, Tsukuba, Ibaraki 305, Japan
& T. Fukuzawa & T. Tazaki Department of Biology, Faculty of Science, Toho University, Funabashi, Chiba 274, Japan
ABSTRACT Sunflower Helianthus annuus L. and maize Zea mays L. plants in the vegetative phase were exposed to nitrogen dioxide ( NO z) at 0.0 (control), 0.2, 0.5 and l'O ppm (IA litre- 1)Jbr 2 weeks. The growth responses of the plants to NO 2 were examined by the techniques of growth analysis. The sunflower plant was more susceptible to NO z than the maize plant. Exposure to NO 2 at 0.2ppm slightly stimulated the growth of the sunflower plants. The net assimilation rate ( N A R ) was also significantly increased when the plants were exposed to 0.2ppm NO z. Exposures to NO 2 at 0.5ppm or more significantly reduced the dry weight of the sunflower plant. O f the component parts, the roots and stems were severely affected, while the leaves were less affected. This resulted in an elevated shoot~root ratio. The net assimilation rate of both species was reduced by the exposures to NO 2 at 0.5 ppm or more, while, in contrast, the leaf area ratio ( L A R ) was increased. The relative growth rate (RGR), the product of the N A R and the L A R , was therefore less affected by NO 2. The increase in the L A R was overwhelmingly the result of an increase in the leaf weight ratio ( L W R ) . These results imply that a reduction in photosynthetic efficiency induced by NO 2 could be, in part, compensated for by an increase in assimilatory area, suggesting an adaptive growth response of the plants to air pollutant stresses. 361 Environ. Pollut. Ser. A. 0143-1471/85/$03.30 (0 Elsevier Applied Science Publishers Ltd, England, 1985. Printed in Great Britain
362
K. Okano, T. Totsuka, T. Fukuzawa, T. Tazaki
INTRODUCTION Nitrogen dioxide (NO2)--an important air pollutant--has been demonstrated to be phytotoxic in a wide range of species (Taylor et al., 1975). Higher concentrations (above 3 to 4 ppm, #1 litre-1) of NO 2 can cause acute injurious effects on plant leaves, probably through the accumulation of nitrite (Kato et al., 1974; Yoneyama et al., 1979). Chronic exposure to lower concentrations of NO 2 usually reduces the growth of plants (Taylor & Eaton, 1966; Thompson et al., 1970; Ashenden & Mansfield, 1978; Totsuka et al., 1978), although no nitrite can be detected in leaf tissues. The mechanism of this type of damage by NO 2 is not known. On the other hand, several investigators (Failer, 1972; Fujiwara, 1973; Troiano & Leone, 1977; Yoneyama et al., 1980a; Marie & Ormrod, 1984) found that, in some cases, lower concentrations of NO 2 were not detrimental to plants but resulted in an increased plant weight as compared with nonexposed plants. These findings were supported by the fact that NO 2 absorbed in plant leaves was rapidly metabolized and incorporated into organic nitrogen compounds (Yoneyama & Sasakawa, 1979; Rogers & Campbell, 1979). The growth responses of plants to both ozone (03) and sulphur dioxide (SO2) have been extensively documented. A consistent feature of the results obtained is that the impact of these two pollutants on the root is more drastic than that on the shoot. This results in a lowered root/shoot ratio (Tingey et al., 1971; Bennett & Runeckles, 1977; Mejst~ik, 1980; Shimizu et al., 1980; Jensen, 1981). Techniques of growth analysis revealed that the relative growth rate (RGR) was initially lowered by exposure to 0 3 or SO 2 because of a reduction in the net assimilation rate (NAR), but that, thereafter, RGR in the fumigated plants recovered above that of the control plants due to a larger leaf area ratio (LAR) (Oshima et al., 1978, 1979; Horsman et al., 1980; Walmsley et al., 1980; Shimizu et al., 1981; Jones & Mansfield, 1982a). The growth responses observed were interpreted as being compensatory in nature, responding to the reduced photosynthetic efficiency. However, it has not been sufficiently characterised whether plants will show a similar type of growth response to NO 2 as to 0 3 and SO 2. Whitmore & Mansfield (1983) reported that the root/shoot ratio of several grasses was unaffected even when the whole plant growth was significantly reduced by exposure to NO2. The present experiment was designed to quantify the growth responses
Growth response of plants to nitrogen dioxide
363
of sunflower and maize plants in the vegetative phase to various concentrations o f N O 2 by means of growth analysis techniques and to determine whether chronic levels of N O 2 might stimulate the growth of plants by acting as a nitrogen fertilizer from the atmosphere.
MATERIALS A N D M E T H O D S Plant materials Seeds of sunflower H e l i a n t h u s annuus L. cv. Russian M a m m o t h and maize Z e a m a y s L. cv. Dento were sown in pots containing 1.8 litres of artificial soil (a mixture of vermiculite, peat, perlite and fine gravel, 2:2:1:1, v/v), 5g of Magamp K ( N : P 2 O s : K 2 0 = 6 : 4 0 : 5 , W. R. Grace Co., Tennessee, USA) and 15 g of magnesia lime. The seeds were sterilised with 0.1% solution of Benlate (Dupon, Delaware, USA) for 1 h and then washed with tap water for 24 h. Seven days after the sowing, the seedlings were thinned to one plant per pot to increase the uniformity among plants. Nutrients were supplied twice a week as 100 to 200ml of I g per litre of Hyponex ( N : P z O s : K 2 0 = 6.5:5: 19) solution. The plants were grown for 2 weeks in a naturally lit growth room with a constant temperature of 25 °C and a relative humidity of 70 %. Fourteen days after sowing, 0.72 g of potassium nitrate was added to each pot, and then the plants were transferred to four artificially lit growth cabinets with exactly the same environmental conditions (420 #E s - 1 m - 2; light period, 14 h; 25 °C, relative humidity, 70 ~ ) except for the NO 2 concentration. The light source consisted of twenty-four 400 W stannous halide lamps. Exposure to
NO 2
Exposure of plants to various concentrations of N O 2 w a s started on the fourteenth day and ended on the twenty-eighth day after sowing. The plants were watered daily, and no additional fertilizer was applied during the exposure period. Nitrogen dioxide was supplied from a pressurised cylinder of predetermined concentration. Controlled levels of N O 2 w e r e released through a thermal mass flow controller into the air stream entering the growth cabinet. The treatments were as follows: (1) control: charcoal filtered air; (2) 0.2 p p m NO2; (3) 0.5 ppm N O 2 and (4) 1.0 ppm N O 2. The concentration of N O 2 in the cabinet was continuously
364
K. Okano, T. Totsuka, T. Fukuzawa,
T. Tazaki
monitored and regulated at a required level by a controlling system based on a chemiluminescent NO-NO,-NO, analyser (Model 14, Therm0 Electron). The concentrations used in these experiments are not unduly high compared with levels actually found in Japan. In urban areas, such as Tokyo, the monthly average concentration of NO, in 1983 was about 0.05 ppm, but hourly average values were in the range from 0.1 to 0.2 ppm. Moreover, the monthly average concentration of NO, (NO, + NO) is about 0.2 ppm and hourly average values often exceed 0.5 ppm in winter. Harvest and growth analysis
Each ten plants were harvested just before the start of NO, exposure and after the end of 2 weeks’ continuous exposure. The sunflower plants were separated into leaves, stems (including petioles) and roots. The maize plants were divided into leaf blades, leaf sheaths, culms and roots. The roots were washed carefully to eliminate the potting medium. Leaf area was measured photometrically with an area meter (Model 3100, LICOR). The separated plant parts were dried in an oven at 80 “C for 3 days and then weighed. The growth analysis variables, relative growth rate (RGR), net assimilation rate (NAR), leaf area ratio (LAR), leaf weight ratio (LWR) and specific leaf area (SLA) were calculated from the formula discussed by Saeki (1965). Relative growth rate is the product of the NAR and the LAR, and the LAR is the product of the LWR and the SLA.
RESULTS No chlorosis or necrosis was observed on the surface of leaves, even when the plants were continuously exposed to NO, at l.Oppm for 2 weeks. There was a slight crinkling, or puckering, and a darkening of the green colour of the newly expanding leaves of the sunflower plants exposed to NO, at 005ppm or more. The dry weights of the whole plant, and each component part of sunflower plants exposed to various concentrations of NO, for 2 weeks are shown in Table 1. The whole dry weight of the plants exposed to NO, at 0.2 ppm was 7 % greater than that of the control plants grown under
365
Growth response o f plants to nitrogen dioxide
TABLE 1 Growth of Sunflower Plants Exposed to Various Concentrations of NO2 for 2 Weeks Plant part
Initial Control
Dry weight (g) Whole plant Leaves Stems Roots Leaf area (cm 2) Shoot/root ratio
0.41 0.26 0.10 0.05 107 7.20
11.05 3.98 4.89 2.18 1 322 4.07
NO 2 concentration (ppm) 0.2 0.5
11.83 (107) 4.04 (102) 5.28 (108) 2.53 (116) 1 264 (96)* 3.68 (90)
9.73 (88)* 3.98 (100) 3.80 (78)** 1.95 (89) 1 242 (94) 3-99 (98)
1.0
8.67 (78)*** 4.07 (102) 3-22 (66)*** 1.38 (63)*** 1 158 (88)** 5.28 (130)**
Numerals in parentheses are relative values to control. Significantly different from the control at the 5% (*), 1% (**) and 0.1 ~ (***) levels.
charcoal filtered air. Stimulation of dry weight growth induced by N O 2 at 0 . 2 p p m was marked in the roots and the stems, and 16~o and 8~o, respectively, greater than the control values. On the other hand, dry weight growth of the leaves was less affected and leaf area was slightly~ but significantly, decreased when exposed to 0.2 ppm N O 2. Exposure t o N O 2 at 0.5 ppm or more caused significant reduction in the dry weights of roots, stems and whole plants. In contrast, there was no significant difference between the leaf dry weight of NO2-exposed plants and that of control plants, although the leaf area was slightly reduced by N O 2 exposure. Consequently, the shoot/root ratio was decreased by exposure t o N O 2 at 0.2 ppm, being significantly elevated as a result of exposure to NO 2 at 1.0 ppm, indicating that the root dry weight was reduced more than the shoot dry weight. In the case of maize plants, the dry weight of whole plants tended to decrease with increasing N O 2 concentration (Table 2). However, no differential effects of N O 2 o n various plant parts were observed and no significant changes in the shoot/root ratio could be detected. The data obtained from the sunflower plants were further examined by the techniques of growth analysis and the results are shown in Figs l, 2 and 3. The relative growth rate of the sunflower plants not subjected to NO 2 exposure (control) was 0.24 mg m g - 1 day- 1 during the experimental period, which ranged from 14 to 28 days after sowing (Fig. 1). Exposure to NO 2 at 0.2 ppm resulted in a significantly greater R G R than that of the control. A slight reduction in R G R was detected on plants exposed to
366
K. Okano, T. Totsuka, T. Fukuzawa, T. Tazaki
TABLE 2 Growth of Maize Plants Exposed to Various Concentrations of NO 2 for 2 Weeks Plant part
lnitial Control
Dry weight (g) Whole plant Leaf blades Leaf sheaths + culms Roots Leaf area (cm 2) Shoot/root ratio
0.74 0.34
9.02 3.45
0-17 0.23 151 2.22
3.38 2.19 1 018 3.12
NO 2 concentration (ppm) 0.2 0.5
8-66 (96) 3.12 (90) 3.29 (97) 2.26 (103) 913 (90) 2.84 (91)
1.0
8-68 (96) 3.49 (101)
8.41 (93) 3.36 (97)
3.04 (90)* 2.16 (99) 1 074 (106) 3"02 (97)
2-93 (87) 2.13 (97) 1 042 (102) 2.95 (95)
Numerals in parentheses are relative values to control. * Significantly different from the control at the 5 ~ level. N O 2 at 0.5 ppm. Exposure t o NO 2 at 1.0 ppm significantly reduced the
RGR by 7 ~ compared with the control. The relative growth rate was divided into the NAR and the LAR (Fig. 2). The changes in the NAR caused by various concentrations of N O 2 w e r e very similar to those in the RGR, but the impact o f N O 2 o n the NAR was more pronounced than that on the RGR. A l0 ~o increase in the NAR was detected in the plants exposed t o NO 2 at 0"2 ppm. Exposures to N O 2 at 0.5ppm and 1.0ppm reduced the NAR by 9 ~ and 15~, 0.3 m
a3
0.2
1oo
102
96
93
0.5
1.0
E E 0.1
0.0 C
0.2 NO 2
(ppm)
Fig. 1. Changes in the relative growth rate (RGR) of sunflower plants exposed to various concentrations of NO2. Numerals in the Figure show the relative values to control (100), significantly different from control at the 5 ~o (*) and 0.1 ~ (***) levels.
Growth response of plants to nitrogen dioxide
367
0.2
I m "0
11o
e
E E
O'J
0.1
v
E e~
z
0
e.e-
,°I° C
0.0 0.2
O.5
C
1.0 N0 2
0.2
0.5
1.0
(~ppm)
Fig. 2. Changes in the net assimilation rate (NAR) and leaf area ratio (LAR) of sunflower plants exposed to various concentrations of NO 2. Numerals in the Figure show the relative values to control (100), significantly different from control at the 5 ~o (*), 1 ~o (**) and 0.1 ~o (***) levels.
respectively, compared with the control. The changes in the LAR were the opposite of those in the NAR. Exposure t o N O 2 at 0"2ppm brought about a reduced LAR whilst exposures t o N O 2 at 0 . 5 p p m or more resulted in a significantly greater LAR than that of the control. The leaf area ratio was further divided into the LWR and the SLA (Fig. 3). The leaf weight ratio--the proportion of leaf weight to whole plant weight--was significantly reduced by N O 2 a t 0-02 ppm. On the other hand, exposures t o NO 2 at 0.5 p p m or more conspicuously increased the LWR compared with the control, i.e. by 1 4 ~ at 0.5 ppm and by 31 ~ at 0.6
0.6 .-..
.k
I
E E
0.4
""
0.2
131
"
0.4 "k
0.2 .,.,.J ¢.n
0.0
0.0 C
0.2
0.5
1.0 N02
C
0.2
0.5
1.0
(ppm)
Fig. 3. Changes in the leaf weight ratio (LWR) and specific leaf area (SLA) of sunflower plants exposed to various concentrations of N O 2. Symbols are the same as in Fig. I.
K. Okano, T. Totsuka, T. Fukuzawa, T. Tazaki
368
TABLE 3 Growth Analysis on Maize Plants Exposed to Various Concentrations of NO 2
Treatment
Control 0-2 ppm 0'5 ppm 1.0ppm
R GR NA R LA R L WR SLA (mgmg-lday -1) (mgcm-2day -l) (cm2mg -l) (mgmg -l) (cm2mg -1) 0' 178 0' 175 0" 176 0.173
1-30 1.34 1.21 1.19
0.113 0.105 0.124 0.124
0.39 0.36 0.40 0.40
0.296 0.295 0.307 0.310
1.0 ppm. The specific leaf area significantly decreased with the increase in NO 2 concentration. The reduced SLA seemed to be the result of a crinkling of new leaves exposed to NO 2, and might not mean that thicker leaves would have been produced on the plants exposed to N O 2. Table 3 shows the results of growth analysis on the maize plants exposed to various concentrations of NO 2. The general trends of changes in the growth parameters of maize plants were similar to those of sunflower plants, although they were not so clear. The exposure to NO 2 at 0.2 ppm resulted in a greater NAR and a smaller LAR than the control. The maize plants exposed to NO 2 at 0-5 ppm or more had a reduced NAR and an increased LAR compared with the control. Therefore, the RGR, which was the product of the NAR and the LAR, was less affected by exposure to various concentrations of NO 2. Thus, in both plant species, the remarkable reduction in the NAR induced by relatively higher concentrations of NO 2 could, in part, be compensated for by the increase in the LAR, resulting in a smaller reduction of the RGR. The marked rise in the LWR caused by NO 2 could account for the increase in the LAR. DISCUSSION
Growth stimulation of plants by lower concentration of NO2 Nitrogen dioxide, even at a lower concentration, has been believed to be toxic or inhibitory to plants. Taylor & Eaton (1966) reported that continuous exposure to N O 2 at less than 0.5ppm for 10 to 22 days caused significant suppression of growth in pinto bean and tomato seedlings. Ashenden & Mansfield (1978) showed that the yield of Poa pratensis was reduced by exposure to NO 2 at 0.068 ppm for 20 weeks. In the present experiment, however, the growth of sunflower plants was slightly stimulated by exposure to NO 2 at 0.2ppm for 2 weeks. Net
Growth response of plants to nitrogen dioxide
369
assimilation rate, an approximation of photosynthetic efficiency, was also significantly increased as a result of exposure to 0.2 ppm NO 2. Nitrogen dioxide absoi'bed in plant leaves through the stomata can be converted into nitrate and nitrite, and then rapidly assimilated into amino acids and proteins if nitrate and nitrite reductases are active (Rogers and Campbell, 1979; Yoneyama & Sasakawa, 1979). Ito et al. (1984) also showed an increase in amino acid synthesis when kidney bean plants were exposed to NO 2. NO2-nitrogen incorporated into amino acids and proteins was then retransferred from the leaves to rapidly growing parts, probably through phloem, along with the photosynthates (Yoneyama et al., 1980b; Okano et al., 1983). Therefore, a beneficial effect of lower concentrations of NO 2 on plant growth might be expected--and has been actually confirmed--with sunflower (Failer, 1972), rice (Fujiwara, 1973), tomato (Troiano & Leone, 1977; Marie & Ormrod, 1984)and cucumber (Yoneyama et al., 1980a). The fixation of carbon dioxide and the translocation of assimilate were also found to be enhanced by exposure t o N O 2 (Okano et al., 1984b). If lower concentrations of N O 2 a c t as a nitrogen fertilizer [from the atmosphere, the stimulation of plant growth by N O 2 would be expected when the plants are nitrogen deficient. In the present experiment, since no additional fertilizer was applied to the plants during the 2-week exposure period, they might have become nitrogen deficient. It is necessary to quantify the contribution of NO2-nitrogen to the total nitrogen increase for a better understanding of the nutritional effect of N O 2 o n plant growth. In our next study, absorption of N O 2 by the plants was simultaneously measured by means of the 15N dilution method (details to be published). The amount of NO2-nitrogen absorbed by sunflower plants exposed to 0.2 ppm NO 2 during a 2-wEek period was about 12.2 mg per plant, accounting for 5.8 ~o of the total nitrogen increase during this period. This would be sufficient to account for the increase in dry weight growth observed in this species. ~n contrast, maize plants absorbed only 3.7 mg per plant, contributing to the total nitrogen increase by 2.3 ~. Less absorption of N O 2 by maize might be one of the reasons why the growth of maize was less affected by exposure t o N O 2 than that of sunflowers. Adaptive response of plants to higher concentrations of N O 2
Although the growth of sunflower plants was slightly enhanced by 0.2 ppm NO2, exposure to NO 2 at 0.5 ppm or more significantly reduced
370
K. Okano, T. Totsuka, T. Fukuzawa, T. Tazaki
the dry weight of the plant. Of the plant parts, the roots and stems were severely affected by NO 2, while the leaves were less affected. Consequently, the shoot/root ratio was significantly elevated by 1-0 p p m N O 2. Whitmore & Mansfield (1983) reported that the shoot/root ratio of several grasses was unaffected even when the whole plant growth was significantly reduced by exposure to N O 2. The present result, obtained with sunflowers, however, indicated that N O 2 can also exert differential effects on the growth of various plant parts. This was apparently so, also in the case of O a or SO 2. The different responses of various plant parts to these two pollutants have been extensively documented in a wide range of species such as radish (Tingey et al., 1971), crimson clover and annual ryegrass (Bennett & Runeckles, 1977), tobacco and cucumber (Mejstfik, 1980), sunflower (Shimizu et al., 1980, 1981) and green ash (Jensen, 1981). Growth analysis techniques revealed the following. The net assimilation rate was decreased by higher concentrations (above 0-5 ppm) of N O 2, whilst, in contrast, the LAR increased with the decrease in the NAR. As a result, the RGR, which is the product of the NAR and the LAR, was less affected by NO 2. This means that the inhibitory effect of N O 2 on the R G R apparently acted through the reduced NAR, and that the reduction of photosynthetic efficiency caused by NO 2 was, in part, compensated for by the increase in the LAR. The leaf area ratio is the product of the LWR and the SLA. The results shown in Fig. 3 indicated that the increase in the LAR was overwhelmingly the result of an increase in the LWR. The SLA decreased somewhat with increasing NO 2 concentration. The increase in the LWR, along with the reduced stem and root weight ratio (data not shown), implies that the pattern of dry matter partitioning among plant parts was changed in favour of the leaves as a result of exposure to NO 2. Recently, Jones & Mansfield (1982b) and Okano et al. (1984a) have directly demonstrated, by tracer experiments, t h a t the pattern of assimilate distribution in plants is altered by SO 2 or 0 3 so that a greater proportion of assimilate is partitioned.to growing leaves and a smaller proportion to the root and stem. The sunflower plants used in the present experiment might also respond to N O 2 by diverting relatively more assimilates into the photosynthetic organs (leaves) in response to the fall in photosynthetic efficiency. The increase in assimilatory area induced by NO2, however, was not enough to compensate for the decrease in photosynthetic efficiency. Hence, the growth rate declined as a result of NO2, and the dry weight of the sunflower plant was 1 2 ~ and 2 2 ~ , respectively, lower than the control after 14 days' exposure to 0"5 ppm and 1.0 ppm NO2.
Growth response of plants to nitrogen dioxide
371
Thus, the adaptive response of plants to N O 2 we identified in the present study is exactly the same as that to O 3 or S O 2 - - a fact demonstrated by a number of investigators (Oshima et al., 1978, 1979; H o r s m a n et al., 1980; Walmsley et al., 1980; Shimizu et al., 1981 ; Jones & Mansfield, 1982a). Furthermore, this kind of adaptive response seems to be brought about not only by exposure to air pollutants but also by other environmental factors which cause a reduction in the a m o u n t of available assimilate. For example, a deficiency in photosynthate has been found to have a greater effect on root growth than on shoot growth (Curtis & Clark, 1950). Blackman & Black (1959) reported, with several herbaceous species, that the N A R w a s dependent on the amount of light received, whilst, in contrast, the LAR rose as light intensity decreased. These facts clearly indicate that the proportion of leaf area to plant weight (LAR) varies easily according to environmental conditions and that changes in the LAR may assist the plant to adapt to various environmental stresses.
REFERENCES Ashenden, T. W. & Mansfield, T. A. (1978). Extreme pollution sensitivity of grasses when SO 2 and N O 2 a r e present in the atmosphere together. Nature, Lond., 273, 142-3. Bennett, J. P. & Runeckles, V. C. (1977). Effects of low levels of ozone on growth of crimson clover and annual ryegrass. Crop Sci., 17, 443-5. Blackman, G. E. & Black, J. N. (1959). Physiological and ecological studies in the analysis of plant environment. XI. A further assessment of the influence of shading on the growth of different species in the vegetative phase. Ann. Bot., 23, 51-63. Curtis, O. F. & Clark, D. G. (1950). Factors influencing root growth and the ratio between roots and tops. In An introduction to plant physiology, 666-92. New York, McGraw-Hill. Faller, V. N. (1972). Schwefeldioxid, Schwefelwasserstoff, Nitrosegase und Ammoniak als ausschliel~liche S-bzw. N-Quellen der h6heren Pflanze. Z. Pflanzenern. Bodenk., 131, 120-30. Fujiwara, T. (1973). Effects of nitrogen oxides in the atmosphere on vegetation. J. Pollut. Control, 9, 253-7. Horsman, D. C., Nicholls, A. O. & Calder, D. M, (1980). Growth responses of Dactylis glomerata, Lolium perenne and Phalaris aquatica to chronic ozone exposure. Aust. J. Plant Physiol., 7, 511-17. Ito, O., Okano, K. & Totsuka, T. (1984). Effects of NO/ and 0 3 alone or in combination on kidney bean plants. II. Amino acid pool size and composition. Res. Rep. Natl Inst. Environ. Stud., Jpn, 66, 15-25. Jensen, K. F. ( 1981). Ozone fumigation decreased the root carbohydrate content and dry weight of green ash seedlings. Environ. Pollut. (Ser. A), 26, 147-52.
372
K. Okano, T. Totsuka, T. Fukuzawa, T. Tazaki
Jones, T. & Mansfield, T. A. (1982a). The effects of SO 2 on growth and development of seedlings of Phleurn pratense under different light and temperature environments. Environ. Pollut. (Set. A), 27, 57-71. Jones, T. & Mansfield, T. A. (1982b). Studies on dry matter partitioning and distribution of 14C-labelled assimilates in plants of Phleum pratense exposed to SO 2 pollution. Environ. Pollut. (Ser. A), 28, 199-207. Kato, T., Tachibana, S. & Inden, T. (1974). Studies on the injury of crops by toxic gases in covering structure. II. Mechanisms involved in the sensitivity of plants to NO 2. Environ. Control in Biol., 12, 103-7. Marie, B. A. & Ormrod, D. P. (1984). Tomato plant growth with continuous exposure to sulphur dioxide and nitrogen dioxide. Eneiron. Pollut. (Ser. A), 33, 257-65. Mejstfik, V. (1980). The influence of low SO 2 concentrations on growth reduction of Nicotiana tabacum L. cv. Samsun and Cucumis sativus L. cv. Unikfit. Environ. Pollut. (Set. A), 21, 73-6. Okano, K., Tatsumi, J., Yoneyama, T., Kono, Y. & Totsuka, T. (1983). Investigation on the carbon and nitrogen transfer from a terminal leaf to the root system of rice plant by a double tracer method with 13C and 15N. Japan. J. Crop. Sci., 52, 33141. Okano, K., Ito, O., Takeba, G., Shimizu, A. & Totsuka, T. (1984a). Alteration of 13C_assimilate partitioning in plants of Phaseolus vulgaris exposed to ozone. New Phytol., 97, 153-63. Okano, K., Ito, O., Takeba, G., Shimizu, A. & Totsuka, T. (1984b). Effects of N O 2 and 0 3 alone or in combination on kidney bean plants. V. 3C-assimilate partitioning as affected by NO 2 and/or O a. Res. Rep. Natl Inst. Environ. Stud., Jpn., 66, 49-58. 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. Rogers, H. H. & Campbell, J. C, (1979). Nitrogen-15 dioxide uptake and incorporation by Phaseolus vulgaris (L.). Science, N.Y., 206, 333-5. Saeki, T. (1965). Growth analysis of plants. Bot. Mag. Tokyo, 78, 111-19. Shimizu, H., Furukawa, A. & Totsuka, T. (1980). Effects of low concentrations of SO 2 on the growth of sunflower plants. Environ. Control in Biol., 18, 39-47. Shimizu, H., Motohashi, S., Iwaki, H., Furukawa, A. & Totsuka, T. (1981). Effects of chronic exposures to ozone on the growth of sunflower plants. Environ. Control in Biol., 19, 13747. Taylor, O. C. & Eaton, F. M. (1966). Suppression of plant growth by nitrogen dioxide. Plant Physiol., Lancaster, 41, 132-5. Taylor, O. C., Thompson, C. R., Tingey, D.T. & Reinert, R. A. (1975). Oxides of nitrogen. In Responses ofplants to air pollution, ed. by J. B. Mudd and T. T. Kozlowski, 121-39. London, Academic Press.
Growth response of plants to nitrogen dioxide
313
Thompson, C. R., Hensel, E. G., Kats, G. & Taylor, 0. C. (1970). Effects of continuous exposure of navel oranges to nitrogen dioxide. Atmos. Environ., 4, 349-5s.
Tingey, D. T., Heck, W. W. & Reinert, R. A. (1971). Effects of low concentrations of ozone and sulfur dioxide on foliage, growth, and yield of radish. J. Am. Sot. hort. Sci., 96, 369-71. Totsuka, T., Sato, S., Yoneyama, T. & Ushijima, T. (1978). Response of plants to atmospheric NO, fumigation. 2. Effects of NO, fumigation on dry matter growth of sunflower and kidney bean plants. Res. Rep. Nat1 Inst. Environ. Stud., Jpn, 2, 77-87.
Troiano, J. J. & Leone, I. A. (1977). Changes in growth rate and nitrogen content of tomato plants after exposure to NO,. Phytopathology, 67, 1130-3. Walmsley, L., Ashmore, M. R. & Bell, J. N. B. (1980). Adaptation of radish Raphanus sativus L. in response to continuous exposure to ozone. Environ. Pollut. (Ser. A), 23, 165-77.
Whitmore, M. E. & Mansfield, T. A. (1983). Effects of long-term exposures to SO, and NO, on Poapratensis and other grasses. Environ. Pollut. (Ser. A), 31, 217-35.
Yoneyama, T. & Sasakawa. H. (1979). Transformation of atmospheric NO, absorbed in spinach leaves. Plant & Cell Physiol., 20, 263-6. Yoneyama, T., Sasakawa, H., Ishizuka, S. & Totsuka, T. (1979). Absorption of atmospheric NO, by plants and soils. II. Nitrite accumulation, nitrite reductase activity and diurnal change of NO, absorption in leaves. Soil Sci. Plant Nutr., 25, 267-75.
Yoneyama, T., Totsuka, T., Hayakawa, H. & Yazaki, J. (1980a). Absorption of atmospheric NO, by plants and soils. V. Day and night NO,-fumigation effect on the plant growth and estimation of the amount of NO,-nitrogen absorbed by plants. Res. Rep. Nat1 Inst. Environ. Stud., Jpn, 11, 31-50. Yoneyama, T., Arai, K. & Totsuka, T. (1980b). Transfer of nitrogen and carbon from a mature sunflower leaf--“NO, and 13C0, feeding studies. Plant & Cell Physiol., 21, 1367-81.