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Photosynthetic photon flux effects on bean response to nitrogen dioxide
Environmentaland ExperimentalBotany, Vol. 30, No. 4, pp. 463467, 1990
0098 8472/90 $3.00 + 0.00 {) 1990 Pergamon Press plc
Printed in Great Britain.
P H O T O S Y N T H E T I C P H O T O N F L U X EFFECTS O N BEAN RESPONSE TO NITROGEN DIOXIDE H. S. SRIVASTAVA,* D. P. ORMROD~ and B. HALE MARIE t *Department of Plant Science, Rohilkhand University, Bareilly 243005, India and ~Department of Horticultural Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1
(Received 18 October 1989; accepted in final revisedform 23 May 1990) SRIVASTAVA H. S., ORMROD D. P. and HALE MARIE B. Photosynthetic photon flux effects on bean response to nitrogen dioxide. ENVIRONMENTALAND EXPERIMENTAL BOTANY 30, 463467, 1990. Phaseolus vulgaris L. cv. Kinghorn Wax seedlings, supplied with nutrient solution containing either 0 or 5 mM nitrate as sole N source, were exposed to 0.25 #1/1 NO2 for 6 hr each day for 10 days at continuous photosynthetic photon flux (PPF) of 100, 300, 500 or 700 #mol m -2 sec- ~. There was a significant interaction of PPF and nitrate. Shoot and root dry weights increased with increasing PPFs only when nitrate was supplied. The main effects of NO 2 on plant growth were significant; none of the interactions involving NO2 were significant. Exposure to NO2 decreased shoot and root dry weight in both the presence and absence of nutrient N and at all PPF levels. All interactions were significant for in vitro leaf nitrate reductase activity (NRA), which increased markedly at PPFs above 100 #tool m -2 sec ~ when nitrate was supplied. Treatment with N O 2 strongly inhibited enzyme activity in the presence of nitrate, particularly at the 300 #tool m ~ sec i PPF level. These experiments demonstrated that PPF level does not modify the effect of NOz on growth but does have a major effect on NRA and on NO 2 effects on NRA in the presence of nutrient nitrate.
Key words: Photosynthetic photon flux, light, nitrogen dioxide, Phaseolus vulgaris, bean, growth, nitrate reductase activity.
INTRODUCTION MODIFICATION o f p l a n t responses to gaseous air pollutants b y e n v i r o n m e n t a l factors is well k n o w n ] 3'5'6/ RUNECKLES(1°) postulated that the degree of injury c o r r e l a t e d with the a m o u n t of p o l l u t a n t a b s o r b e d b y the plant. Since s t o m a t a are the p r i n c i p a l portals of gaseous air p o l l u t a n t uptake, (4/ e n v i r o n m e n t a l factors t h a t influence s t o m a t a l opening/closing m a y influence overall p o l l u t a n t effects. T h e injurious effects of NO2 are well known; nevertheless, nitrate and nitrite generated from NO2 dissolution in the cell sap are also, to some extent, r e d u c e d a n d assimilated by plants to serve as a source of n u t r i e n t N . (7'8'12'13) T h e injurious effects of the p o l l u t a n t m a y d e p e n d 463
u p o n the a c c u m u l a t i o n of its p r i m a r y p r o d u c t , nitrite; r a p i d assimilation of this m e t a b o l i t e reduces injury. T h e reduction a n d assimilation of nitrate and nitrite generated from N O 2 require large amounts of A T P and N A D ( P ) H . A m o n g other biochemical processes in green plants, the light reaction of photosynthesis is the most i m p o r t a n t generator of these cofactors. Thus, increasing photosynthetic p h o t o n flux (PPF) is expected not only to enhance the u p t a k e a n d assimilation of NO2 a n d nutrient nitrate, b u t also to decrease the potential for injury to the p l a n t by the pollutant. A l t h o u g h the influence of P P F on p l a n t responses to NO2 has been e x a m i n e d with respect to gaseous exchange (9'll/ a n d some aspects of growth, (2/ no
464
H . S . SRIVASTAVA et al.
studies have been undertaken with respect to PPF effects on nitrate and N O 2 gas assimilation. The current investigation was undertaken in this context with the objective of discovering the influence of PPF on NO~ effects on growth and nitrate reductase activity (NRA) in bean. Continuous light was used to evaluate the influence of PPF level on NO2 effects. Any interruption with dark periods would have depleted the A T P and N A D ( P ) H and possibly dampened any influence of PPF. The hypothesis was that increasing PPF should decrease NO2 injury in terms of shoot and root growth retardation and be explainable on the basis of increased assimilation of NO2 as indicated by N R A . MATERIALS AND M E T H O D S
Seeds of snap bean (Phaseolus vulgaris L. cv. Kinghorn Wax) were sown in 15 cm diameter plastic pots filled with coarse vermiculite. Seedlings were grown in a controlled environment chamber under PPF of approximately 350 #mol m -2 sec ~ with a 10-hr photoperiod and temperature of 25°C (light) and 20°C (dark). Seedlings were irrigated daily with deionized water. Seedlings were thinned to one per pot 7 days after sowing and thereafter irrigated with modified half-strength Hoagland's nutrient solution containing no N. ~j/On day 8, the plants were divided into four lots and transferred to exposure chambers (75 x 75 x 75 cm plexiglass chambers). Thereafter they were irrigated with the same nutrient solution but containing either no N ( - N) or 5 m M KNO3 as the sole rooting medium N source ( + N), and exposed to either charcoal filtered air ( - NO2) or 0.25 #1/1 NO2 ( + NO2) for 6 hr each day for 10 days. During the exposure and growth period (days 8 18), the plants were exposed at a temperature of 25__2°C to continuous PPFs of 100, 300, 500 or 700 #mol m 2 sec-l at canopy level from high pressure sodium lamps as measured with a q u a n t u m meter (LIC O R model LI-185). The differential light levels were obtained with neutral density shade cloth. The pollutant was introduced into the chamber from a compressed gas cylinder and its level in the exposure c h a m b e r monitored with a Thermoelectron Model 14T chemiluminescent N O 2 analyzer. Leaf, root and shoot samples were col-
lected on day 19. Roots were washed in running tap water. Generally the seedlings had not developed any nodules on the roots prior to harvest. A few roots at 500 and 700 #mol m - 2 sec 1 PPF developed juvenile nodules. Such plants were not included in any analyses. Dry weights of roots and shoots were measured after drying them in a hot air oven at 70°C for 48 hr. Five plants were measured in each treatment. For measurement of in vitro leaf N R A , freshly harvested leaves from each of two additional treated plants were sliced into thin sections and 0.5 g extracted with a mortar and pestle in 2.0 ml of cold (below 4°C) extraction medium. T h e medium consisted of 0.2 M sodium phosphate buffer (pH 7.4), 2.0 m M E D T A , 2 m M cysteine, and 0.5% casein. Cysteine and casein were added to the medium just before use, The extract was centrifuged for 40 min at 17,000 x g below 4°C. T h e N R A was assayed in the clear supernatant by a colorimetric procedure./~2/The reaction mixture consisted of 0.8 ml of 0.2 M N a - K phosphate buffer (pH 7.8), 0.2 ml of 0.2 M KNO3, 0.2 ml of 2 m M N A D H and 0.3 ml of the enzyme extract. The mixture was incubated at 28°C for 30 min and the reaction terminated by the addition of 1.5 ml of 1 o~ sulfanilamide (in 1.5 N HC1) followed by 1.5 ml of 0.02% naphthylethylene diamine dihydrochloride. After 15 min, the mixture was centrifuged at 17,000 x g for 5 min and the absorbance was measured at 540 nm using a Beckman DU-65 spectrophotometer. The entire experiment was repeated for both root and shoot weights and N R A determination to provide two independent replications each consisting of five subsamples (plants) for weights and two subsamples (plants) for N R A determination. All data were subjected to an analysis of variance on the basis of two replicates in a split-plot design with PPF and NO2 levels as whole units and nitrate levels as sub-units (Table 1). Treatment effects and interactions were considered significant at P ~< 0.05. RESULTS
The dry weights of bean shoots and roots were significantly affected by PPF, nutrient nitrate (N)
PPF EFFECTS ON BEAN RESPONSE TO NO2
465
Table 1. Analyses of variancefor response of shoot dry weight, root dry weight and leaf NRA to PPF, NO2 and N Shoot dry weight Source
DF
Mean square
Pr > F
Root dry weight Mean square
Pr > F
Leaf NRA Mean square
Pr > F
Whole unit analysis Replicate PPF NO~ PPF x NO~ Error A
1 3 1 3 7
1275 1,493,202 186,050 5201 2929
0.53 0.0001 0.0001 0.24
933 59,078 45,904 1105 460
0.20 0.0001 0.0001 0.15
294 839,773 923,100 405,588 4581
0.81 0.0001 0.0001 0.0001
1 3 1 3 8
8,694,450 1,039,200 21 4117 4539
0.0001 0.0001 0.94 0.48
205,761 26,693 1326 297 398
0.0001 0.0001 0.11 0.55
6,056,070 623,371 990,176 457,999 4451
0.0001 0.0001 0.0001 0.0001
Sub-unit analysis N PPF x N NO~ x N PPF x NO2 x N Error B
supply, a n d NO2 exposure ( T a b l e 1). T h e m a i n effects of PPF, nitrate a n d NO2, a n d the interaction of P P F a n d nitrate were significant for both growth response variables. T h e r e was no significant interaction of P P F a n d N 0 2 , nitrate and N 0 2 , or PPF, nitrate a n d NO2. T h e significant effects of P P F a n d nitrate are a reflection of the overall increase in growth with increasing P P F or a d d i t i o n of nitrate, b u t must be interpreted in terms of the significant P P F a n d nitrate interaction. T h e significant i n t e r a c t i o n occurred because the shoot a n d root d r y weights, unaffected by P P F in the absence of nitrate, increased very m a r k e d l y with increasing P P F above 100 #mol m 2 sec-1 when nitrate was a d d e d (Figs 1, 2). T h e significant effect on N 0 2 , without interaction with either P P F or nitrate, indicates that the r e t a r d a t i o n of shoot a n d root g r o w t h by NO2 took place regardless of P P F level or w h e t h e r nitrate was present or not. T h e leaf N R A was significantly affected by all the t r e a t m e n t variables. T h e m a i n effects of PPF, nitrate a n d NO~ a n d all interactions were significant for the response of N R A to the treatments ( T a b l e 1). This means that the response of N R A for each factor must be i n t e r p r e t e d in terms of specific levels of each of the other factors. Exposure to NO2, in the absence of n u t r i e n t N, had no effect on leaf N R A (Fig. 3). L e a f N R A increased sharply with increased P P F from 100 to 300 # m o l m -2 sec 1 in + N plants. However,
i 2400=
2000-
'7
E ~ 1200e~
m 80@
P P F ,~mol.m-~,sec -'
Fro. 1. PPF effects on shoot dry weights of bean plants grown without or with nitrate and not exposed or exposed to NO2. Means_S.D. for 10 plants (two replicates and five plants per replicate). C), - N , - N O 2 ; D, - N , +NO2; 0 , +N, - N O 2 ; I , + N , +NO2.
in + N plants, N O 2 inhibited N R A with a very large inhibition at 300 p m o l m -2 sec 1 PPF. L e a f N R A in + N plants declined with increasing P P F a b o v e 300 #mol m -2 sec l a n d there was m u c h
466
H . S . SRIVASTAVA et al. 2400-
I
2000-
400 -
7m,1600m
In
.c
E
zO'1200_ m o
g200-
<
~800100400100
300 PPF
500
700
p m o l . m - 2 . s e c -~
FIG. 2. PPF effects on root dry weights of bean plants grown without or with nitrate and not exposed or exposed to NO 2. Means + S.D. for 10 plants (two replicates and five plants per replicate). O, - N , -NO2; D, - N , +NO2; O, +N, -NO~; II, +N, +NO2.
less N O 2 inhibition of leaf N R A at 500 and 700 ~mol m 2 sec-t than at 300 #mol m -2 sec -z. Plants grown without nutrient N did not show any visible injury due to N O 2 exposure. However, characteristic symptoms of NO2 injury were quite a p p a r e n t on the leaves of plants grown with nitrate N and exposed to NO2. T h e symptoms included t a n n e d to necrotic leaf margins and brownish rust-like lesions on the p r i m a r y leaves and yellow chlorotic lesions on the first trifoliolate leaves. T h e injury to the leaves was more severe at 300 and 500 # m o l m -2 see -I than at other PPFs. A t 100 # m o l m -2 sec -~ only occasional necrotic margins were noted.
DISCUSSION T h e NO~-induced inhibition of shoot growth was not affected by light level as indicated by the lack of a significant P P F x NO2 interaction. Light level did not therefore modify in any m a j o r w a y the growth response of bean shoots to NO~. Similarly, the light level did not have an effect on NO2-induced inhibition of root growth. T h e r e
o
lOO
360
7oo
PPF /~mol.m -2.sec -~
FIG. 3. PPF effects on NRA in leaves of bean plants grown without or with nitrate and not exposed or exposed to NO2. Means_S.D. for four plants (two replicates and two plants per replicate). O, - N , -NO2; [], - N , +NO2; 0 , +N, -NO2; IB, +N, + NO~.
was significant a n d similar r e t a r d a t i o n at all light levels. T h e nitrate level also h a d no effect on N O 2 response within the p a r t i c u l a r nutrient regimes used in these experiments. I n contrast the light level had a m a r k e d effect on N R A response to N O 2 in plants supplied with nutrient nitrate. T h e shoot to root d r y weight relationship increased with NO2 exposure as a result of differences in m a g n i t u d e of the N O 2 response between root and shoot. A n increase in shoot to root ratio with SO2 and NO2 t r e a t m e n t also has been observed in wheat./2/This suggests that t r e a t m e n t with NO2 results in inhibition of d r y m a t t e r translocation from shoot to root. It is not likely that the NO~ gas t r e a t m e n t has any direct effect on roots as the rooting m e d i u m would absorb the NO2 before it reached root surfaces. Assimilation of nitrate originating fi"om N O 2 or a b s o r b e d by roots from the nutrient solution is initiated with its reduction to nitrite and to a m m o n i a by the sequential action of nitrate a n d nitrite reductases. Exposure to NO2 is known to
PPF EFFECTS ON BEAN RESPONSE T O NO2 increase N R A in m a n y systems, including bean leaves, especially in the absence o f nutrient N. !12) I n the present research, leaf N R A in the absence of n u t r i e n t nitrate was not affected by NO2 and increased only slightly with increasing PPF. T h e l e a f N R A in plants fed n u t r i e n t nitrate increased greatly c o m p a r e d with N-starved plants, except at the lowest P P F level, b u t was m a r k e d l y inhibited by N O 2 at the 300/~mol m -2 sec 1 light level and, to a lesser extent, at the 500 a n d 700 # m o l m 2 sec 1 light levels. T h e hypothesis that increasing P P F would decrease N O 2 injury to plants was not s u p p o r t e d in this study. Increasing continuous P P F did not a m e l i o r a t e the NO2 injury to p l a n t d r y weights found at the n u t r i e n t - N level used in these experiments. I n addition, more r a t h e r t h a n less leaf injury was noted at higher PPFs on + N plants. F u r t h e r research should include a c o m p a r i s o n of continuous P P F with l i g h t - d a r k cycles to determine if the continuous P P F used in the present research was the correct a p p r o a c h to testing the increasing PPF, decreasing NO2 injury hypothesis. A very strong interaction (P < 0.0001) between PPF, N O 2 a n d n u t r i e n t - N for N R A was noted in this research. I n c r e a s i n g continuous P P F h a d a m a j o r effect in N-fed plants on leaf N R A both without a n d with N 0 2 . T h e i n h i b i t o r y effect of NO2 on leaf N R A of nitrate-fed plants was extreme at the 300 #tool P P F level suggesting a different m e c h a n i s m for P P F effects on N R A c o m p a r e d with effects on shoot a n d root growth. F u r t h e r research is required to d e t e r m i n e w h e t h e r the P P F a n d NO2 effects on N R A are directly on the enzyme system or m e d i a t e d indirectly via effects on nitrogen m e t a b o l i s m that, in turn, affect N R A , as well as to d e t e r m i n e the mechanisms of the P P F - N O 2 interaction. Such research should include consideration of the effect of PPFs on NO2 uptake. Increasing n u t r i e n t N a n d P P F could lead to increased NO2 u p t a k e with consequent greater NO2 p e r t u r b a t i o n of e n z y m e action.
Acknowledgements The authors thank Larry Pyear and Kelli Payne for technical assistance and Professor R. Austin Fletcher tbr the use of some laboratory facilities.
467
This research was supported by an Operating Grant to D.P.O. from the Natural Sciences and Engineering Research Council of Canada. H.S.S. was the recipient of an International Scientific Exchange Award from the Natural Sciences and Engineering Research Council of Canada. REFERENCES
1. ARDITTIJ. and DUNNA. (1969) Experimental plant physiology. Holt, Rinehart, and Winston, New York. 2. GOULD R. P. and MANSHELDJ. A. (1988) Effects of sulfur dioxide and nitrogen dioxide on growth and translocation in winter wheat. J. exp. Bot. 39, 389-399. 3. HECK W. W. (1968) Factors influencing expression of oxidant damage to plants. A. Rev. Phytopath. 6, 165 188. 4. MANSFIELDT. A. and MAJERNIK 0 . (1970) Can stomata play a part in protecting plants against air pollutants? Envir. PoUut. 1, 149-154. 5. MUDD B. J. and KOZLOWSKIT. T. (1975) Pages 9-51 in Responses of plants to air poUution. Academic Press, New York. 6. ORMROD D. P. (1986) Gaseous air pollution and horticultural crop production. Hortic. Rev. 8, 1-42. 7. ROGERS H. H., CAMPBELLJ. C. and VOLK R. J. (1979) Nitrogen-15 dioxide uptake and incorporation by Phaseolus vulgaris (L.). Science206, 333335. 8. ROWLAND A . J . (1986) Nitrogen uptake, assimilation and transport in barley in the presence of atmospheric nitrogen dioxide. Pl. Soil 91,353-356. 9. ROWLAND-BAMFORDA.J. and DREW M. C. (1988) The influence of plant nitrogen status on NO 2 uptake, NO 2 assimilation and on the gas exchange characteristics of barley plants exposed to atmospheric NO2. J. exp. Bot. 39, 1287 1297. 10. RUNECKLESV. C. (1974) Dosage of air pollutants and damage to vegetation. Envir. Conservation 1, 305-308. 11. SRIVASTAVAH. S., JOLLIFFE P. A . and RUNECKLES V. C. (1975) Effects of environmental conditions on the inhibition of gas exchange in bean leaves. Can. J. Bot. 53, 475-482. 12. SRIVASTAVA H. S. and ORMROD D. P. (1984) Effects of nitrogen dioxide and nitrate nutrition on growth and nitrate assimilation in bean leaves. Pl. Physiol. 76, 418-423. 13. YONEYAMAT. and SASAKAWAH. (1979) Transformation of atmospheric NO 2 absorbed in spinach leaves. Pl. Cell Physiol. 20, 263-265.
0098 8472/90 $3.00 + 0.00 {) 1990 Pergamon Press plc
Printed in Great Britain.
P H O T O S Y N T H E T I C P H O T O N F L U X EFFECTS O N BEAN RESPONSE TO NITROGEN DIOXIDE H. S. SRIVASTAVA,* D. P. ORMROD~ and B. HALE MARIE t *Department of Plant Science, Rohilkhand University, Bareilly 243005, India and ~Department of Horticultural Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1
(Received 18 October 1989; accepted in final revisedform 23 May 1990) SRIVASTAVA H. S., ORMROD D. P. and HALE MARIE B. Photosynthetic photon flux effects on bean response to nitrogen dioxide. ENVIRONMENTALAND EXPERIMENTAL BOTANY 30, 463467, 1990. Phaseolus vulgaris L. cv. Kinghorn Wax seedlings, supplied with nutrient solution containing either 0 or 5 mM nitrate as sole N source, were exposed to 0.25 #1/1 NO2 for 6 hr each day for 10 days at continuous photosynthetic photon flux (PPF) of 100, 300, 500 or 700 #mol m -2 sec- ~. There was a significant interaction of PPF and nitrate. Shoot and root dry weights increased with increasing PPFs only when nitrate was supplied. The main effects of NO 2 on plant growth were significant; none of the interactions involving NO2 were significant. Exposure to NO2 decreased shoot and root dry weight in both the presence and absence of nutrient N and at all PPF levels. All interactions were significant for in vitro leaf nitrate reductase activity (NRA), which increased markedly at PPFs above 100 #tool m -2 sec ~ when nitrate was supplied. Treatment with N O 2 strongly inhibited enzyme activity in the presence of nitrate, particularly at the 300 #tool m ~ sec i PPF level. These experiments demonstrated that PPF level does not modify the effect of NOz on growth but does have a major effect on NRA and on NO 2 effects on NRA in the presence of nutrient nitrate.
Key words: Photosynthetic photon flux, light, nitrogen dioxide, Phaseolus vulgaris, bean, growth, nitrate reductase activity.
INTRODUCTION MODIFICATION o f p l a n t responses to gaseous air pollutants b y e n v i r o n m e n t a l factors is well k n o w n ] 3'5'6/ RUNECKLES(1°) postulated that the degree of injury c o r r e l a t e d with the a m o u n t of p o l l u t a n t a b s o r b e d b y the plant. Since s t o m a t a are the p r i n c i p a l portals of gaseous air p o l l u t a n t uptake, (4/ e n v i r o n m e n t a l factors t h a t influence s t o m a t a l opening/closing m a y influence overall p o l l u t a n t effects. T h e injurious effects of NO2 are well known; nevertheless, nitrate and nitrite generated from NO2 dissolution in the cell sap are also, to some extent, r e d u c e d a n d assimilated by plants to serve as a source of n u t r i e n t N . (7'8'12'13) T h e injurious effects of the p o l l u t a n t m a y d e p e n d 463
u p o n the a c c u m u l a t i o n of its p r i m a r y p r o d u c t , nitrite; r a p i d assimilation of this m e t a b o l i t e reduces injury. T h e reduction a n d assimilation of nitrate and nitrite generated from N O 2 require large amounts of A T P and N A D ( P ) H . A m o n g other biochemical processes in green plants, the light reaction of photosynthesis is the most i m p o r t a n t generator of these cofactors. Thus, increasing photosynthetic p h o t o n flux (PPF) is expected not only to enhance the u p t a k e a n d assimilation of NO2 a n d nutrient nitrate, b u t also to decrease the potential for injury to the p l a n t by the pollutant. A l t h o u g h the influence of P P F on p l a n t responses to NO2 has been e x a m i n e d with respect to gaseous exchange (9'll/ a n d some aspects of growth, (2/ no
464
H . S . SRIVASTAVA et al.
studies have been undertaken with respect to PPF effects on nitrate and N O 2 gas assimilation. The current investigation was undertaken in this context with the objective of discovering the influence of PPF on NO~ effects on growth and nitrate reductase activity (NRA) in bean. Continuous light was used to evaluate the influence of PPF level on NO2 effects. Any interruption with dark periods would have depleted the A T P and N A D ( P ) H and possibly dampened any influence of PPF. The hypothesis was that increasing PPF should decrease NO2 injury in terms of shoot and root growth retardation and be explainable on the basis of increased assimilation of NO2 as indicated by N R A . MATERIALS AND M E T H O D S
Seeds of snap bean (Phaseolus vulgaris L. cv. Kinghorn Wax) were sown in 15 cm diameter plastic pots filled with coarse vermiculite. Seedlings were grown in a controlled environment chamber under PPF of approximately 350 #mol m -2 sec ~ with a 10-hr photoperiod and temperature of 25°C (light) and 20°C (dark). Seedlings were irrigated daily with deionized water. Seedlings were thinned to one per pot 7 days after sowing and thereafter irrigated with modified half-strength Hoagland's nutrient solution containing no N. ~j/On day 8, the plants were divided into four lots and transferred to exposure chambers (75 x 75 x 75 cm plexiglass chambers). Thereafter they were irrigated with the same nutrient solution but containing either no N ( - N) or 5 m M KNO3 as the sole rooting medium N source ( + N), and exposed to either charcoal filtered air ( - NO2) or 0.25 #1/1 NO2 ( + NO2) for 6 hr each day for 10 days. During the exposure and growth period (days 8 18), the plants were exposed at a temperature of 25__2°C to continuous PPFs of 100, 300, 500 or 700 #mol m 2 sec-l at canopy level from high pressure sodium lamps as measured with a q u a n t u m meter (LIC O R model LI-185). The differential light levels were obtained with neutral density shade cloth. The pollutant was introduced into the chamber from a compressed gas cylinder and its level in the exposure c h a m b e r monitored with a Thermoelectron Model 14T chemiluminescent N O 2 analyzer. Leaf, root and shoot samples were col-
lected on day 19. Roots were washed in running tap water. Generally the seedlings had not developed any nodules on the roots prior to harvest. A few roots at 500 and 700 #mol m - 2 sec 1 PPF developed juvenile nodules. Such plants were not included in any analyses. Dry weights of roots and shoots were measured after drying them in a hot air oven at 70°C for 48 hr. Five plants were measured in each treatment. For measurement of in vitro leaf N R A , freshly harvested leaves from each of two additional treated plants were sliced into thin sections and 0.5 g extracted with a mortar and pestle in 2.0 ml of cold (below 4°C) extraction medium. T h e medium consisted of 0.2 M sodium phosphate buffer (pH 7.4), 2.0 m M E D T A , 2 m M cysteine, and 0.5% casein. Cysteine and casein were added to the medium just before use, The extract was centrifuged for 40 min at 17,000 x g below 4°C. T h e N R A was assayed in the clear supernatant by a colorimetric procedure./~2/The reaction mixture consisted of 0.8 ml of 0.2 M N a - K phosphate buffer (pH 7.8), 0.2 ml of 0.2 M KNO3, 0.2 ml of 2 m M N A D H and 0.3 ml of the enzyme extract. The mixture was incubated at 28°C for 30 min and the reaction terminated by the addition of 1.5 ml of 1 o~ sulfanilamide (in 1.5 N HC1) followed by 1.5 ml of 0.02% naphthylethylene diamine dihydrochloride. After 15 min, the mixture was centrifuged at 17,000 x g for 5 min and the absorbance was measured at 540 nm using a Beckman DU-65 spectrophotometer. The entire experiment was repeated for both root and shoot weights and N R A determination to provide two independent replications each consisting of five subsamples (plants) for weights and two subsamples (plants) for N R A determination. All data were subjected to an analysis of variance on the basis of two replicates in a split-plot design with PPF and NO2 levels as whole units and nitrate levels as sub-units (Table 1). Treatment effects and interactions were considered significant at P ~< 0.05. RESULTS
The dry weights of bean shoots and roots were significantly affected by PPF, nutrient nitrate (N)
PPF EFFECTS ON BEAN RESPONSE TO NO2
465
Table 1. Analyses of variancefor response of shoot dry weight, root dry weight and leaf NRA to PPF, NO2 and N Shoot dry weight Source
DF
Mean square
Pr > F
Root dry weight Mean square
Pr > F
Leaf NRA Mean square
Pr > F
Whole unit analysis Replicate PPF NO~ PPF x NO~ Error A
1 3 1 3 7
1275 1,493,202 186,050 5201 2929
0.53 0.0001 0.0001 0.24
933 59,078 45,904 1105 460
0.20 0.0001 0.0001 0.15
294 839,773 923,100 405,588 4581
0.81 0.0001 0.0001 0.0001
1 3 1 3 8
8,694,450 1,039,200 21 4117 4539
0.0001 0.0001 0.94 0.48
205,761 26,693 1326 297 398
0.0001 0.0001 0.11 0.55
6,056,070 623,371 990,176 457,999 4451
0.0001 0.0001 0.0001 0.0001
Sub-unit analysis N PPF x N NO~ x N PPF x NO2 x N Error B
supply, a n d NO2 exposure ( T a b l e 1). T h e m a i n effects of PPF, nitrate a n d NO2, a n d the interaction of P P F a n d nitrate were significant for both growth response variables. T h e r e was no significant interaction of P P F a n d N 0 2 , nitrate and N 0 2 , or PPF, nitrate a n d NO2. T h e significant effects of P P F a n d nitrate are a reflection of the overall increase in growth with increasing P P F or a d d i t i o n of nitrate, b u t must be interpreted in terms of the significant P P F a n d nitrate interaction. T h e significant i n t e r a c t i o n occurred because the shoot a n d root d r y weights, unaffected by P P F in the absence of nitrate, increased very m a r k e d l y with increasing P P F above 100 #mol m 2 sec-1 when nitrate was a d d e d (Figs 1, 2). T h e significant effect on N 0 2 , without interaction with either P P F or nitrate, indicates that the r e t a r d a t i o n of shoot a n d root g r o w t h by NO2 took place regardless of P P F level or w h e t h e r nitrate was present or not. T h e leaf N R A was significantly affected by all the t r e a t m e n t variables. T h e m a i n effects of PPF, nitrate a n d NO~ a n d all interactions were significant for the response of N R A to the treatments ( T a b l e 1). This means that the response of N R A for each factor must be i n t e r p r e t e d in terms of specific levels of each of the other factors. Exposure to NO2, in the absence of n u t r i e n t N, had no effect on leaf N R A (Fig. 3). L e a f N R A increased sharply with increased P P F from 100 to 300 # m o l m -2 sec 1 in + N plants. However,
i 2400=
2000-
'7
E ~ 1200e~
m 80@
P P F ,~mol.m-~,sec -'
Fro. 1. PPF effects on shoot dry weights of bean plants grown without or with nitrate and not exposed or exposed to NO2. Means_S.D. for 10 plants (two replicates and five plants per replicate). C), - N , - N O 2 ; D, - N , +NO2; 0 , +N, - N O 2 ; I , + N , +NO2.
in + N plants, N O 2 inhibited N R A with a very large inhibition at 300 p m o l m -2 sec 1 PPF. L e a f N R A in + N plants declined with increasing P P F a b o v e 300 #mol m -2 sec l a n d there was m u c h
466
H . S . SRIVASTAVA et al. 2400-
I
2000-
400 -
7m,1600m
In
.c
E
zO'1200_ m o
g200-
<
~800100400100
300 PPF
500
700
p m o l . m - 2 . s e c -~
FIG. 2. PPF effects on root dry weights of bean plants grown without or with nitrate and not exposed or exposed to NO 2. Means + S.D. for 10 plants (two replicates and five plants per replicate). O, - N , -NO2; D, - N , +NO2; O, +N, -NO~; II, +N, +NO2.
less N O 2 inhibition of leaf N R A at 500 and 700 ~mol m 2 sec-t than at 300 #mol m -2 sec -z. Plants grown without nutrient N did not show any visible injury due to N O 2 exposure. However, characteristic symptoms of NO2 injury were quite a p p a r e n t on the leaves of plants grown with nitrate N and exposed to NO2. T h e symptoms included t a n n e d to necrotic leaf margins and brownish rust-like lesions on the p r i m a r y leaves and yellow chlorotic lesions on the first trifoliolate leaves. T h e injury to the leaves was more severe at 300 and 500 # m o l m -2 see -I than at other PPFs. A t 100 # m o l m -2 sec -~ only occasional necrotic margins were noted.
DISCUSSION T h e NO~-induced inhibition of shoot growth was not affected by light level as indicated by the lack of a significant P P F x NO2 interaction. Light level did not therefore modify in any m a j o r w a y the growth response of bean shoots to NO~. Similarly, the light level did not have an effect on NO2-induced inhibition of root growth. T h e r e
o
lOO
360
7oo
PPF /~mol.m -2.sec -~
FIG. 3. PPF effects on NRA in leaves of bean plants grown without or with nitrate and not exposed or exposed to NO2. Means_S.D. for four plants (two replicates and two plants per replicate). O, - N , -NO2; [], - N , +NO2; 0 , +N, -NO2; IB, +N, + NO~.
was significant a n d similar r e t a r d a t i o n at all light levels. T h e nitrate level also h a d no effect on N O 2 response within the p a r t i c u l a r nutrient regimes used in these experiments. I n contrast the light level had a m a r k e d effect on N R A response to N O 2 in plants supplied with nutrient nitrate. T h e shoot to root d r y weight relationship increased with NO2 exposure as a result of differences in m a g n i t u d e of the N O 2 response between root and shoot. A n increase in shoot to root ratio with SO2 and NO2 t r e a t m e n t also has been observed in wheat./2/This suggests that t r e a t m e n t with NO2 results in inhibition of d r y m a t t e r translocation from shoot to root. It is not likely that the NO~ gas t r e a t m e n t has any direct effect on roots as the rooting m e d i u m would absorb the NO2 before it reached root surfaces. Assimilation of nitrate originating fi"om N O 2 or a b s o r b e d by roots from the nutrient solution is initiated with its reduction to nitrite and to a m m o n i a by the sequential action of nitrate a n d nitrite reductases. Exposure to NO2 is known to
PPF EFFECTS ON BEAN RESPONSE T O NO2 increase N R A in m a n y systems, including bean leaves, especially in the absence o f nutrient N. !12) I n the present research, leaf N R A in the absence of n u t r i e n t nitrate was not affected by NO2 and increased only slightly with increasing PPF. T h e l e a f N R A in plants fed n u t r i e n t nitrate increased greatly c o m p a r e d with N-starved plants, except at the lowest P P F level, b u t was m a r k e d l y inhibited by N O 2 at the 300/~mol m -2 sec 1 light level and, to a lesser extent, at the 500 a n d 700 # m o l m 2 sec 1 light levels. T h e hypothesis that increasing P P F would decrease N O 2 injury to plants was not s u p p o r t e d in this study. Increasing continuous P P F did not a m e l i o r a t e the NO2 injury to p l a n t d r y weights found at the n u t r i e n t - N level used in these experiments. I n addition, more r a t h e r t h a n less leaf injury was noted at higher PPFs on + N plants. F u r t h e r research should include a c o m p a r i s o n of continuous P P F with l i g h t - d a r k cycles to determine if the continuous P P F used in the present research was the correct a p p r o a c h to testing the increasing PPF, decreasing NO2 injury hypothesis. A very strong interaction (P < 0.0001) between PPF, N O 2 a n d n u t r i e n t - N for N R A was noted in this research. I n c r e a s i n g continuous P P F h a d a m a j o r effect in N-fed plants on leaf N R A both without a n d with N 0 2 . T h e i n h i b i t o r y effect of NO2 on leaf N R A of nitrate-fed plants was extreme at the 300 #tool P P F level suggesting a different m e c h a n i s m for P P F effects on N R A c o m p a r e d with effects on shoot a n d root growth. F u r t h e r research is required to d e t e r m i n e w h e t h e r the P P F a n d NO2 effects on N R A are directly on the enzyme system or m e d i a t e d indirectly via effects on nitrogen m e t a b o l i s m that, in turn, affect N R A , as well as to d e t e r m i n e the mechanisms of the P P F - N O 2 interaction. Such research should include consideration of the effect of PPFs on NO2 uptake. Increasing n u t r i e n t N a n d P P F could lead to increased NO2 u p t a k e with consequent greater NO2 p e r t u r b a t i o n of e n z y m e action.
Acknowledgements The authors thank Larry Pyear and Kelli Payne for technical assistance and Professor R. Austin Fletcher tbr the use of some laboratory facilities.
467
This research was supported by an Operating Grant to D.P.O. from the Natural Sciences and Engineering Research Council of Canada. H.S.S. was the recipient of an International Scientific Exchange Award from the Natural Sciences and Engineering Research Council of Canada. REFERENCES
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