6
ELSEVIER Environmental Polllution 86 (1994) 37~2 © 1994 Elsevier Science Limited Printed in Great Britain. All rights reserved 0269-7491/94/$07.00 RESPONSES OF GREENING BEAN SEEDLING LEAVES TO NITROGEN DIOXIDE AND NUTRIENT NITRATE SUPPLY H. S. Srivastava Department of Plant Science, Rohilkhand University, Bareilly 243005, India D. P. Ormrod* & B. A. Hale Department of Horticultural Science, University of Guelph, Guelph, Ontario, Canada, NIG 2 WI (Received 17 March 1993; accepted 16 July 1993) Abstract Phaseolus vulgaris cv. Kinghorn Wax seedlings grown in darkness at 25°C for 7 days with half strength Hoagland's nutrient solution containing no nitrogen, were transferred to lit continuous stirred tank reactors (CSTRs) in atmo- spheres containing 0 or 0.3 ppm NO2 and irrigated with a nutrient solution containing 0 or 5 mM nitrate as sole ni- trogen source and allowed to grow for a period of up to 5 days in a 14 h photoperiod. Exposure to NO2 increased total Kjeldahl nitrogen in the leaves. Further, the exposure to NO2 increased chlorophyll content from day 3 onwards and inhibited the leaf dry weight substantially on days 4 and 5. The primary leaves of the seedlings exposed to 0.3 ppm NO2 and supplied with nitrate accumulated some nitrite after 5 days of exposure. Some of the seedlings were returned from CSTRs to growth chambers and allowed to grow for a further period of 5 days in a 14 h photoperiod without NO2. The growth which developed after the NO2 exposure growth period, as measured by fresh and dry weights of the leaves, was significantly less in NO2-exposed plants than in nitrate-grown plants. The experiments demonstrate that the leaves of greening seedlings are able to assimilate NO2 and that a reduction in leaf dry weight by prolonged NO2 exposure in the presence of nutrient nitrate can be associated with nitrite accumulation, and that NO2 has a carry-over effect beyond the duration of NO2 exposure. It is apparent that NO2 induces some durable biochemical or cytological aberration in the presence of nutrient nitrate, which adversely affects subsequent leaf growth. Keywords: Nitrogen dioxide, leaf growth, nitrate nutri- tion, chlorophyll, bean seedlings. INTRODUCTION As for other gases, nitrogen dioxide (NO2) is believed to be absorbed by the plant leaves through the stomata and its effect appears to be related to its dose. In many cases, low doses of NO2 are known to increase plant * To whom correspondence should be addressed. 37 growth (Capron & Mansfield, 1977; Kress et al., 1982: Whitmore & Freer-Smith, 1982: Marie & Ormrod, 1984). Increase in chlorophyll content of the leaves in the presence of NO2 has been reported in bean (Srivas- tava & Ormrod, 1984; Sandhu & Gupta, 1989), pea (Horsman & Wellburn, 1975), soyabean (Gupta & Narayanan, 1992) and spruce (Tischner et al., 1988). The simulation of plant growth is believed to be due to the assimilation of NO2-derived nitrogen, as exposure to NO2 often increases organic nitrogen content of the plant organs (Srivastava & Ormrod 1984, 1986; Row- land, 1986). The relative contribution of NO2-derived nitrogen to the plant is greater in plants raised with low levels of nutrient nitrogen than in those with adequate nitrogen (Rowland-Bamford & Drew, 1988). The uptake and assimilation of NO,_ may also account for toxic physiological effects and reduced plant growth. The assimilation could deplete the essential co-factors ATP and NAD(P)H leading to the accumulation of toxic species of nitrogen such as nitrite and ammonium in certain situations (see Wellburn, 1992). The current investigation was undertaken to test the following hypotheses/possibilities. (a) Dark-grown etiolated seedlings do not possess developed leaves with functional stomata and therefore it is possible that the uptake and assimilation of NO2 by these leaves will be restricted and assimilation-linked phytotoxic effects, if any, will be minimal. (b) During greening of leaves, any effect of NO2 on chlorophyll content could be viewed as its effect on the synthesis of the pigment, and if due to the assimilation of NO2-nitrogen into nitroge- nous precursors of chlorophyll, the effect would be related to the increase in organic nitrogen during NO2 exposure and with increased nitrate supply. (c) The effects of NO2 may persist during post-NO2- exposure growth of the new leaves as well. It was intended that these studies provide some in- sight into the mechanism of NO 2 phytotoxicity.

Responses of greening bean seedling leaves to nitrogen dioxide and nutrient nitrate supply

Embed Size (px)

Citation preview

Page 1: Responses of greening bean seedling leaves to nitrogen dioxide and nutrient nitrate supply

ELSEVIER

Environmental Polllution 86 (1994) 37~2 © 1994 Elsevier Science Limited

Printed in Great Britain. All rights reserved 0269-7491/94/$07.00

RESPONSES OF GREENING BEAN SEEDLING LEAVES TO NITROGEN DIOXIDE A N D

NUTRIENT NITRATE SUPPLY

H. S. Srivastava Department of Plant Science, Rohilkhand University, Bareilly 243005, India

D. P. Ormrod* & B. A. Hale

Department of Horticultural Science, University of Guelph, Guelph, Ontario, Canada, NIG 2 WI

(Received 17 March 1993; accepted 16 July 1993)

Abstract Phaseolus vulgaris cv. Kinghorn Wax seedlings grown in darkness at 25°C for 7 days with half strength Hoagland's nutrient solution containing no nitrogen, were transferred to lit continuous stirred tank reactors (CSTRs) in atmo- spheres containing 0 or 0.3 ppm NO2 and irrigated with a nutrient solution containing 0 or 5 mM nitrate as sole ni- trogen source and allowed to grow for a period of up to 5 days in a 14 h photoperiod. Exposure to NO2 increased total Kjeldahl nitrogen in the leaves. Further, the exposure to NO2 increased chlorophyll content from day 3 onwards and inhibited the leaf dry weight substantially on days 4 and 5. The primary leaves of the seedlings exposed to 0.3 ppm NO2 and supplied with nitrate accumulated some nitrite after 5 days of exposure. Some of the seedlings were returned from CSTRs to growth chambers and allowed to grow for a further period of 5 days in a 14 h photoperiod without NO2. The growth which developed after the NO2 exposure growth period, as measured by fresh and dry weights of the leaves, was significantly less in NO2-exposed plants than in nitrate-grown plants. The experiments demonstrate that the leaves of greening seedlings are able to assimilate NO2 and that a reduction in leaf dry weight by prolonged NO2 exposure in the presence of nutrient nitrate can be associated with nitrite accumulation, and that NO2 has a carry-over effect beyond the duration of NO2 exposure. It is apparent that NO2 induces some durable biochemical or cytological aberration in the presence of nutrient nitrate, which adversely affects subsequent leaf growth.

Keywords: Nitrogen dioxide, leaf growth, nitrate nutri- tion, chlorophyll, bean seedlings.

INTRODUCTION

As for other gases, nitrogen dioxide (NO2) is believed to be absorbed by the plant leaves through the stomata and its effect appears to be related to its dose. In many cases, low doses of NO2 are known to increase plant

* To whom correspondence should be addressed.

37

growth (Capron & Mansfield, 1977; Kress et al., 1982: Whitmore & Freer-Smith, 1982: Marie & Ormrod, 1984). Increase in chlorophyll content of the leaves in the presence of NO2 has been reported in bean (Srivas- tava & Ormrod, 1984; Sandhu & Gupta, 1989), pea (Horsman & Wellburn, 1975), soyabean (Gupta & Narayanan, 1992) and spruce (Tischner et al., 1988). The simulation of plant growth is believed to be due to the assimilation of NO2-derived nitrogen, as exposure to NO2 often increases organic nitrogen content of the plant organs (Srivastava & Ormrod 1984, 1986; Row- land, 1986). The relative contribution of NO2-derived nitrogen to the plant is greater in plants raised with low levels of nutrient nitrogen than in those with adequate nitrogen (Rowland-Bamford & Drew, 1988). The uptake and assimilation of NO,_ may also account for toxic physiological effects and reduced plant growth. The assimilation could deplete the essential co-factors ATP and NAD(P)H leading to the accumulation of toxic species of nitrogen such as nitrite and ammonium in certain situations (see Wellburn, 1992).

The current investigation was undertaken to test the following hypotheses/possibilities.

(a) Dark-grown etiolated seedlings do not possess developed leaves with functional stomata and therefore it is possible that the uptake and assimilation of NO2 by these leaves will be restricted and assimilation-linked phytotoxic effects, if any, will be minimal.

(b) During greening of leaves, any effect of NO2 on chlorophyll content could be viewed as its effect on the synthesis of the pigment, and if due to the assimilation of NO2-nitrogen into nitroge- nous precursors of chlorophyll, the effect would be related to the increase in organic nitrogen during NO2 exposure and with increased nitrate supply.

(c) The effects of NO2 may persist during post-NO2- exposure growth of the new leaves as well. It was intended that these studies provide some in- sight into the mechanism of N O 2 phytotoxicity.

Page 2: Responses of greening bean seedling leaves to nitrogen dioxide and nutrient nitrate supply

38 H. S. Srivastava, D. P. Ormrod, B. A. Hale

MATERIALS AND M E T H O D S

Plant culture Seeds of Phaseolus vulgaris L. cv. Kinghorn Wax were washed with deionised water and sown in 15 cm dia- meter plastic pots filled with medium-sized vermiculite. The pots were placed in a controlled environment growth chamber in darkness at 25°C for 7 days. During this period, pots were irrigated daily with 1/2 strength Hoagland's solution containing no nitrogen. On day 8 (taking seeding day as day 0), just before transferring the plants to NO2-exposure chambers, seedlings were thinned to 10 per pot; at the same time the cotyledons were removed to avoid any interference from the stored nitrogen in cotyledons.

for 3 min and the supernatant brought to 25 ml with the extraction solvent. The absorption of the super- natant was recorded in a DU-64 Beckmann spectro- photometer at 663, 646 and 440 nm and total chlorophyll and carotenoid contents were calculated according to Barnes et al. (1992) and Ikan (1969), respectively.

Total nitrogen determination Total organic nitrogen in the dried leaf sample was determined by a micro-Kjeldahl method. A 50 mg aliquot of the powdered leaf sample was digested with 2.5 ml of conc . H2SO 4. The nitrogen in the digested clear liquid was measured by Nesselerisation using (NH4)2SO 4 for the standard curve.

NO2 exposure and nutrient nitrate supply The pots with 8-day-old dark-grown plants were trans- ferred to lit continuous stirred tank reactors (CSTRs) for exposure to either 0 or 0-3 ppm NO2 During expo- sure, they were irrigated daily with 1/2 strength Hoagland's solution containing either no nitrogen or 5 mM KNO3 as the sole nitrogen source; the environ- mental conditions were 23 + 1 °C, photosynthetic photon flux density approximately 300 /~mol/m2s and photo- period 14 h. The exposure to NO2, and treatment with nutrient nitrate continued for 5 days with a break in NO: exposure for 30 min each day to permit harvesting and irrigating plants. The NO: otherwise was intro- duced continuously into CSTRs from a NO2 com- pressed gas cylinder and its concentration monitored with a Thermoelectron model 12T chemiluminescent NOx analyser. Plants were harvested each 24 h for weight measurements and leaf tissue analyses.

Post-exposure growth Some of the plants exposed to 0 or 0-3 ppm NO2 and treated with nutrient solution containing either no or 5 mM nitrate, for 5 days, were returned from CSTRs to the growth chamber and allowed to grow for a further 5 days. During this period, they were irrigated with the same nutrient solution containing 0 or 5 mM nitrate as sole nutrient nitrogen source as during NO2 expo- sure. The environmental conditions were 25°C, PPFD 300 /xmol/m 2 s and photoperiod 14 h. Plants were harvested only at the end of the 5 day post-exposure growth period.

Growth measurements The first bifoliate (primary) and the first trifoliate (secondary) leaves were removed along with their petioles, and weighed for the determination of fresh weight. The dry weights were determined after drying in a forced draught oven at 70-80°C for 48 h.

Total chlorophyll and carotenoid determination About 0.5 g of finely chopped fresh leaf samples was homogenised with a Polytron in 25 ml of 80% acetone (v/v) containing 2 ml/litre of NH4OH saturated with MgCO3. The homogenate was centrifuged at 16,000 × g

Nitrite determination An aliquot of 100 mg dried leaf powder was extracted with 10 ml of hot water (70-80°C). The extract was cleared by filtration and the volume standardised to 10 ml with water. To a 1.0 ml aliquot of the clear filtrate, I-0 ml of 1% sulphanilamide (in 1.5 M HC1) and 1.0 ml of 0.2% naphthyl ethyldiamine dichloride were added. After 10 min, the absorbance was read at 540 nm, and the nitrite-nitrogen content was calculated using a standard curve prepared from KNO2.

Replicates and statistical analys~s For determination of growth effects, 50 primary leaves or 30 secondary leaflets were weighed in one lot. For pigment determination, five leaves were treated collec- tively and an aliquot of 0-5 g was taken for extraction and analysis. For Kjeldahl-nitrogen and nitrite-nitrogen determination, all 50 or 30 leaves used for dry weight determination were powdered together and an aliquot of either 50 or 100 mg was taken for further treatment and analysis. All the experiments were conducted in three independent replicates and the data presented in this paper are the average of three independent values. Least significant difference (LSD (0.05)) values were calculated for comparison of nitrogen treatment effects within harvests (Table 1).

Table 1. Least significant difference (0.05) values applicable to Figs 1-5 for comparisons of nitrogen treatments within days

Days

1 2 3 4 5

Total nitrogen 1.65 2.26 4-33 3.45 3-92 (Fig. 1)

Chlorophyll 0-126 0.198 0-211 0.33 0.179 (Fig. 2)

Carotenoid 50-0 13.0 18.9 13,0 22-5 (Fig. 3)

Fresh weight 8.9 7-9 13.7 23,5 26.1 (Fig. 4)

Dry weight 1.20 1.94 1.48 4,07 2.58 (Fig. 5)

Page 3: Responses of greening bean seedling leaves to nitrogen dioxide and nutrient nitrate supply

Bean leaf response to nitrogen dioxide 39

40-

38" .....

o~ 36 o3 E d 34 . . . . g

~ 32-

~-- 30-

"~ 2 8

aa]

II

1 2 3 4 Days

800] 7501

7°° 1

1- "~ 600-'

c 550- co "6

500- (D

450q

400

/ ,¢ , , - /

./ 5 3 4

Days

-N-NO2 • -N +NO2 ~ +N-NO2 +N +NO2

Fig. 1. Effects of N O 2 exposure and nutrient nitrate supply on total organic nitrogen content of the leaves from greening seedlings. Seedlings raised for 7 days in the dark were trans- ferred to CSTRs in the light, where they were irrigated with 1/3 strength Hoagland's solution containing either no nitro- gen (-N) or 5 mM KNO 3 (+N) as sole nutrient nitrogen and were grown in atmosphere containing 0 (-NO2) or 0.3 ppm

(+NO_~) NO2 for 1, 2, 3, 4 or 5 days.

RESULTS

Responses of greening leaves to nitrate treatment and/or to NO~ exposure

Total nitrogen During a greening period of one to 5 days, total Kjel- dahl nitrogen in the primary leaves on a dry weight basis remained almost unchanged when the seedlings were not supplied with nitrate or NO2 (Fig. 1). Supply- ing nitrate in the nutrient medium or exposure t o N O 2

both increased total nitrogen (over control) from day 1 onward. When both the nitrogen sources were supplied simultaneously, the increase in total nitrogen was rela- tively high on day 1 and thereafter almost the same as for nitrate or NO2 alone.

Pigment content Total chlorophyll increased by almost two- to three- fold in all treatments during day 1 and day 2 and there-

2.4

2.2 ~ ~ m ~

1.8 / / j

~ 1.2 ° / 0.8 ~ '

0.6 ,

Days

--e---N-NO2 - • - - N + N O 2 . x . +N-NO2 ' ~ +N +NO2

Fig. 2. Effects of N O 2 exposure and nutrient nitrate supply on total chlorophyll content of the leaves from greening

seedlings. Details as in Fig. 1.

-4=J-- -N-NO2 l - N +NO2 × +N-NO2 A ~N +NO2

Fig. 3. Effects of N O 2 exposure and nutrient nitrate supply on total carotenoid content of the leaves from greening

seedlings. Details as in Fig. 1.

after increased more slowly (Fig. 2). Nutrient nitrate supply and the NO2 exposure had little effect on chlorophyll content throughout the greening period. Exposure of seedlings without nitrate to NO2 did increase total chlorophyll on day 3 and onwards. Total carotenoid content of the leaves increased by 1.5 to 1-8 times between day 1 and day 2 of seedling greening and thereafter it remained unchanged (Fig. 3). Nitrate or NO2 treatment had some effect on carotenoid content on day 1 only. Notably, exposure to NO, alone de- creased carotenoid content on day 1 compared with other treatments.

Leaf growth During the greening period of 5 days, fresh weight of the leaves increased similarly in all treatments (Fig. 4). Nitrate supply and NO2 exposure had little effect on leaf fresh weight. There was an increase in the dry weight of leaves during the greening period with nutrient nitrate supply having little effect until day 4 (Fig. 5). NO, exposure, in either the absence or the presence of nitrate, caused a reduction in leaf dry weight by day 5, while it had no effect during the first 3 days.

250

200-

% E 150-

100-

50+------q

, x / j '

• >~ / <

O h

Days

-N -NO2 - --- -N +NO2 x.. +N -NO2 +N +NO2

Fig. 4. Effects of N O 2 exposure and nutrient nitrate supply on fresh weight of the leaves from greening seedlings. Details

as in Fig. 1.

Page 4: Responses of greening bean seedling leaves to nitrogen dioxide and nutrient nitrate supply

40 H. S. Srivastava, D. P. Ormrod, B. A. Hale

26.

24

22

~ 2o

~ 18-

E: 16 ._~

D 12

10

8-

6

*J

Days

- e - - N - N O 2 - m - . N +NO2 - -~ . +N-N02 • ~ + N + N 0 2

Fig. 5. Effects of NO 2 exposure and nutrient nitrate supply on dry weight of the leaves from greening seedlings. Details

as in Fig. I.

Post-exposure growth and nitrogen and pigment contents of the primary and secondary leaves In the primary leaves, which were also present during the NO2 exposure period, nitrate supply during the post-exposure growth period increased total nitro.~ep content compared with no nitrate in both NO2 er and unexposed seedlings (Table 2). The leaves o, ,~o2 exposed seedlings grown without nutrient nitrate also had higher (about 22%) nitrogen content than those of unexposed seedlings. Nitrate supply had little effect on chlorophyll and carotenoid contents in the primary leaves of unexposed plants. However, the leaves of

NO2-exposed nitrate-supplied seedlings had lower chlorophyll content than control leaves. The carotenoid content of the leaves from NO:-exposed nitrate grown seedlings was slightly lower than in other treatments. The fresh and dry weights of the primary leaves were substantially higher in nitrate supplied plants, although this increase was less in the leaves of the seedlings which had been previously exposed to N O 2.

The secondary leaves, which were not present during N O 2 exposure and formed only during the post-expo- sure growth period, were also affected by prior N O 2

exposure of the plant. Their nitrogen contents increased only slightly with the nutrient nitrate supply in unex- posed seedlings, but substantially in exposed ones (Table 2). On the other hand, nitrate supply increased total chlorophyll content of the secondary leaves from unexposed seedlings. No other treatment had any effect on chlorophyll content and no treatments affected carotenoid contents. Nitrate supply to unexposed seedlings increased fresh and dry weights of the leaves, but reduced growth to some extent in N O : exposed seedlings.

Nitrite content of the leaves None of the leaf samples from either NO2 or nitrate treatment experiments or from post-exposure growth experiments contained measurable nitrite. The seedlings raised with nitrate and exposed to NO: for 5 days did contain nitrite. These leaves accumulated 7 11 k~g nitrite nitrogen per g dry leaves, which disappeared after post-exposure growth of 5 days.

Table 2. Nitrogen and pigment contents and fresh and dry weights of the primary and secondary leaves after post-exposure growth of the seedlings for 5 days a

Parameters Nitrogen treatments

None +Nitrate +NO2 +Nitrate LSD +NO 2 (P -- 0-05)

A. Primary leaves Total nitrogen 21-7 + 0.35 26.9 + 0.30 26.5 + 0.55 28.5 + 0-15 0.681

(mg (g dry wt) l) (100) (124) (122) (131) Chlorophyll 1 870 + 93 1 957 + 20 1 905 + 78 1 514 + 121 162.3

(/xg (g fresh wt) ~) (100) (105) (102) (81) Carotenoids 752 + 1-5 757 + 2.3 751 + 4.6 698 + 24.6 23.7

(/xg (g fresh wt) -~) (100) (101) (100) (93) Fresh weight 326 + 7-7 512 + 14.9 326 _+ 9.8 419 + 9.4 20.3

(mg leaf ~) (100) (157) (100) (108) Dry weight 27-33 + 0.65 34.57 + 1-15 27.37 + 0.55 29-63 + 2.04 2-30

(mg leaf ~) (100) (127) (100) (108) B. Secondary leaves

Total nitrogen 24-5 + 0-91 26.1 + 0.97 24.9 + 0-85 28.5 + 0-78 1.66 (mg (g dry wt) ~) (100) (106) (102) (116)

Chlorophyll 2 060 + 155 2 329 + 183 2 047 + 10 2 043 + 201 296 (/xg (g fresh wt) q) (100) (113) (99) (99)

Carotenoids 762 + 6.2 779 + 6.4 764 + 1.1 761 + 5.0 9.8 (/zg (g fresh wt) ~) (100) (102) (100) (99)

Fresh weight 72.9 + 2.9 118.7 + 4-1 73.2 + 3-0 66.5 + 9-0 10.1 (mg leaf l) (1000) (163) (100) (91)

Dry weight 8.38 + 0.84 12.33 + 2.12 8.22 _+ 0.34 7.52 + 0.58 2.20 (mg leaf ~) (100) (147) (98) (90)

a The data are means of three independent replications _+ standard deviation. The percentages relative to no nitrogen control are given in parentheses.

Page 5: Responses of greening bean seedling leaves to nitrogen dioxide and nutrient nitrate supply

Bean leaf response to nitrogen dioxide 41

Visible symptoms of injury None of the seedlings had any visible symptoms of injury due to NO2 exposure.

DISCUSSION

Most studies of the physiological effects of NO2 on crop plants have used light-grown healthy plants which are able to absorb the pollutant largely through leaf stomata. In the present study, 7-day-old dark-grown seedlings which do not have functional stomata were exposed to NO2 during a 5-day greening period. A sub- stantial increase in total Kjeldahl nitrogen content of the leaves, even within 1 day from the start of the NO2 exposure and the greening period (Fig. 1) suggests that non-stomatal acquisition of NO2 was operational in these bean seedlings. This might have been partly through the surface deposition of NO2 on the leaves and partly through rooting medium deposition and absorption of nitrite and nitrate generated from NO2 through the roots. However, the rooting medium depo- sition route probably did not contribute substantially to tissue nitrogen content because the surface layer of rooting medium would strongly absorb the NO2 and also because the clark grown seedlings would have poorly developed conducting tissues to transport root absorbed nitrogen to the leaves. The importance of non-stomatal acquisition of NO2 and other nitrogenous pollutants also has been realized in other studies (Hanson & Lindberg, 1991). Some of the bryophytes which do not have functional stomata and cuticle also respond to NO> as indicated by increased nitrate reductase activity (Morgan et al., 1992).

The increases in total nitrogen with NO r exposure during greening were similar in the presence or absence of nutrient nitrate (Fig. 1). A moderate increase in total nitrogen during NO2 exposure in the presence of nutrient nitrate has been found in earlier studies involv- ing light-grown bean and other species (Srivastava & Ormrod, 1984; Rowland-Bamford & Drew, 1988).

At the NO~ concentration (0.3 ppm) used, the effects of NO2 on pigment contents and growth of the leaves were dependent upon duration of the exposure. The increase in total chlorophyll content on days 3 and 4 in the absence of nutrient nitrate in greening leaves indicates that NO2 is able to promote chlorophyll syn- thesis to a limited extent. This increase does not seem to be linked to NO2, nitrogen assimilation, which could perhaps provide the nitrogenous precursors of chloro- phyll because (i) there was a substantial increase in total organic nitrogen on the first day of NO 2 exposure itself, but no increase in chlorophyll was observed before day 3; and (ii) nutrient nitrate supply did not increase chlorophyll content, Obviously, nitrogen was not the limiting factor in chlorophyll synthesis during greening of decotyledonised bean seedlings. The reason for the decreased carotenoid content in the NO2- exposed leaves during the first day of exposure is not known. Our experiments do demonstrate that the responses of these two chloroplastic pigments to NO2 differ in greening seedlings (Figs 2 and 3).

The present study did not indicate that the depletion of ATP and NAD(P)H during NO2 assimilation is a cause of phytotoxic effects of NO2. While exposure to NO2 caused a substantive increase in organic nitrogen on the first day, any appreciable inhibition of leaf growth in terms of dry weight was not seen until day 4. Further, some inhibition of leaf dry weight also was observed in secondary leaves in the nitrate-supplied seedlings which developed alter the exposure to NO2 was terminated. These substantial carry-over effects suggest that it is possible that NO,, in the presence of nutrient nitrate either induces or activates certain durable and toxic physico-chemical or cytochemical aberration(s) in the seedlings. From the data presented here and also from other work, some speculation may be made about these aberrations. In the primary leaves of nitrate-grown seedlings exposed to NO2 for 5 days, nitrite accumulation might have been partly responsible for the reduced leaf growth. Accumulation of nitrite has been identified as a phytotoxic mechanism in sun- flower (Yoneyama et aL, 1978), spinach and kidney bean (Shimazaki et al., 1992). The NO2 also appears to be interfering with the water balance of the leaves. In the present study, while dry weight of the leaves was in- hibited on day 3 and afterwards, the fresh weight was either unaffected or even increased slightly (Figs 4 and 5). Apparently, the NO2 increased water content of the tissue, which may be due to the accumulation of osmo- tically active nitrogenous species. Reduced transloca- tion of photosynthates from source (primary leaves) to sink (secondary leaves) might have also contributed to the lesser growth of secondary leaves in NO2 exposed seedlings (Table 2). Altered partitioning of photosyn- thates in NO2 and SO3 exposed bean plants (Ito et aL, 1984) and NO2 and SO2 exposed wheat plants (Gould & Mansfield, 1988) has been demonstrated. Other proposed mechanisms of NO 2 phytotoxicity such as increased acidity, ammonium accumulation (Srivastava, 1992) and free-radical generation and interference with the critical enzymes (Wellburn, 1992) may be function- ing as well. These possibilities need further study, as it is likely that more than one mechanism is involved in NO2 phytotoxicity.

ACKNOWLEDGEMENTS

This research was supported by an International Scien- tific Exchange Award of NSERC Canada to H.S.S. and D.P.O. and by Research Grants to D.P.O. and B.H. The authors are thankful to Larry Pyear for his expert technical assistance.

REFERENCES

Barnes, J. D., Palaguer, L., Manrique, E., Elvira, S. & Davison, A. W. (1992). A reprisal of the use of DMSO for the extraction and determination of chlorophylls a and b in lichens and higher plants. Environ. Exp. Bot., 32, 85-100.

Capron, T. M. & Mansfield, T. A. (1977). Inhibition of net photosynthesis in tomato in air polluted with NO and NO 2. 3. Exp. Bot., 27, 1181-6.

Page 6: Responses of greening bean seedling leaves to nitrogen dioxide and nutrient nitrate supply

42 H. S. Srivastava, D. P. Ormrod, B. A. Hale

Gould, R. P. & Mansfield, T. A. (1988). Effects of sulfur dioxide and nitrogen dioxide on growth and translocation in winter wheat. J. Exp. Bot., 39, 389-99.

Gupta, G. & Narayanan, R. (1992). Nitrogen fixation in soybean treated with nitrogen dioxide and molybdenum. J. Environ. Quality, 21, 4~49.

Hanson, P. J. & Lindberg, S. E. (1991). Dry deposition of reactive nitrogen compounds. A review of leaf, canopy and non-foliar measurements. Atmos. Environ., 25A, 1615-34.

Horsman, D. C. & Wellburn, A. R. (1975). Synergistic effects of SO2 and NO2 polluted air upon enzyme activity in pea seedlings. Environ. Pollut., 8, 123-33.

Ikan, R. (1969). Natural Products." A Laboratory Guide. Academic Press, New York, USA, p. 101.

Ito, O., Okano, K., Kuroiwa, M. & Totsuka, T. (1984). Effects of NO 2 and 03 alone or in combination on kidney bean plants (Phaseolus vulgaris L.). Growth, partitioning of assimilates and root activities. J. Exp. Bot., 36, 652-62.

Kress, L. W., Skelly, J. M. & Kinkelmann, K. H. (1982). Growth impact of 03, NO2 and/or SO 2 on Platanus occi- dentalis. Agric. Environ., 7, 265 74.

Marie, B. A. & Ormrod, D. P. (1984). Tomato plants grown with continuous exposure to sulfur dioxide and nitrogen dioxide. Environ. Pollut., 33, 257-65.

Morgan, S. M., Lee, J. M. & Ashenden, T. W. (1992). Effects of nitrogen dioxide on nitrate assimilation in bryophytes. New Phytol., 120, 89-92.

Rowland, A. J. (1986). Nitrogen uptake, assimilation and transport in barley in the presence of atmospheric nitrogen dioxide. Plant Soil, 91, 3563-6.

Rowland-Bamford, A. J. & Drew, M. C. (1988). The influence of plant nitrogen status on NO2 uptake, NO 2 assimilation and the gas exchange characteristics of barley

plants exposed to atmospheric NO 2. J. Exp. Bot., 39, 1287-97.

Sandhu, R. & Gupta, G. (1989). Effects of nitrogen dioxide on growth and yield of black turtle bean (Phaseolus vulgaris L.) cv. Domino. Environ. Pollut., 59, 337~t4.

Shimazaki, K.-I., Yu, S.-W., Sakai, T. & Tanaka, K. (1992). Differences between spinach and kidney bean plants in terms of sensitivity to fumigation with NO2. Plant Cell Physiol., 33, 267-73.

Srivastava, H. S. (1992). Nitrogenous pollutants in the atmo- sphere: their assimilation and phytotoxicity. Current Sci., 63, 310-17.

Srivastava, H. S. & Ormrod, D. P. (1984). Effects of nitrogen dioxide and nitrate nutrition on growth and nitrate assimi- lation in bean leaves. Plant Physiol., 76, 418-23.

Srivastava, H. S. & Ormrod, D. P. (1986). Effects of nitrogen dioxide and nitrate nutrition on nodulation, nitrogenase activity, growth and nitrogen content of bean plants. Plant PhysioL, 81,737~,1.

Tischner, R., Peuke, A., Godbold, D. L., Peif, R., Merg, G. & Hutterman, A. (1988). The effect of NO2 fumigation on aseptically grown spruce seedlings. J. Plant Physiol., 133, 243-6.

Wellburn, A. R. (1992). Why are atmospheric oxides of nitrogen usually phytotoxic and not alternative fertilizers? New Phytol., 115, 395~,29.

Whitemore, M. E. & Freer-Smith, P. H. (1982). Growth effects of SO e and/or NO 2 on woody plants and grasses during spring and summer. Nature, 300, 55 57.

Yoneyama, T., Sasagawa, H., Totsuka, T. & Yamamoto, Y. (1978). Response of plants to atmospheric NO 2 fumigation (5). Measurements of 15NO 2 uptake, nitrite accumulation and nitrite reductase in herbaceous plants. Res. Rep. Nat. Inst. Environ. Stud., (Ibaraki, Japan), 2, 103-11.