Planta (Berl.) 95, 36--44 (1970) 9 by Springer-Verlag 1970
Nitrate Reduction in the Roots and Shoots of Wheat Seedlings* P. L. M r s o r r i and W. A. J x c x s o ~ Agricultural Experiment Station, North Carolina State University, Raleigh Received May 26, 1970
Summary. Intact wheat seedlings cultured in high nitrate solutions (high-NO~ cultures) reduced NO~ when placed for 24 hr in dilute CaSOa solutions although they leaked N0~ back to solution during this period. Most of the reduction seemed to occur in shoots since in parallel experiments detached shoots reduced nearly as much of the previously absorbed NO~ as intact cultures. Detached roots leaked greater quantities of N0~ back to solution than did intact cultures, and failed to reduce any of their previously absorbed N0~ during the experimental period. Seedlings of identical age cultured without a nitrogen source and rich in carbohydrate reserves (low-N cultures) rapidly absorbed NOa from dilute Ca(NOn)2 solutions and reduced 80% of that absorbed. Detached low-N roots also absorbed N0~ and reduced 40% of that absorbed. Total NO~ reduction by intact low-N cultures over the 24-hr period was comparable to that of high-NO~ cultures in spite of the fact that in vitro nitrate reductase activity of the former did not reach the levels found initially or at the end of the period in the latter, and that the total NO~ absorbed by low-N cultures was less than that initially present in high-NO~ cultures. Introduction The extent to which nitrate is reduced in roots of plants relative to that which is transported as NO~ to shoots must be subject to a number of controls (c[. Wallace and Pate, 1967). Particularly, a suffieient amount of oxidizable carbohydrate must be present in the root to supply the required carbon skeletons and reducing equivalents. I n addition, retention of NO~ ions within the tissue requires expenditure of respiratory energy. If the utilization of respiratory substrates in roots active in NO~ reduction exceeds the rate of carbohydrate supply from the shoots, the resulting carbohydrate deficiency would restrict further NO~ reduction This deficiency would also tend to curtail the NO~ uptake process and permit accumulated NO~ to diffuse back to the external solution. * Paper No. 3155 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina. These investigations were supported by the U.S. Atomic Energy CommissionContract No. At-(40-1)-2410.
Nitrate Reduction in Wheat Seedlings
37
D a t a p r e s e n t e d p r e v i o u s l y (Minotti et al., 1969a, b) showed t h a t 2-week-old w h e a t seedlings grown in high-NO~ solutions, a n d c o n t a i n i n g high a m o u n t s of NO~ in t h e i r tissues, lost significant a m o u n t s of t h a t NO~ to m o r e d i l u t e NO~ solutions d u r i n g a 24-hr period. I n t h e s e e x p e r i m e n t s t h e r a t i o of NO~ in t h e r o o t tissue to t h a t in t h e m e d i u m was a b o u t 180:1. T h e seedlings were c a p a b l e of r e d u c i n g NO~ d u r i n g t h e p e r i o d w h e n NO~ was being lost f r o m r o o t tissue, b u t i t could n o t be said u n e q u i v o c a l l y t h a t r e d u c t i o n was occurring in roots. W e r e p o r t now d a t a f r o m p a r a l l e l e x p e r i m e n t s c o m p a r i n g NO3 r e d u c t i o n , NO~ l e a k a g e a n d / o r NO3 u p t a k e in i n t a c t seedlings w i t h t h a t in d e t o p p e d r o o t s a n d d e t a c h e d shoots. The e x p e r i m e n t s f u r t h e r d e l i n e a t e t h e NO~-leakage p h e n o m e n o n , a n d i l l u s t r a t e t h a t t h e c a p a c i t y of r o o t s to reduce, a n d t o r e t a i n , NO3 w i t h i n t h e i r tissue is r e l a t e d to t h e i r c a r b o h y d r a t e c o n t e n t .
Methods Wheat (Triticum aestivum L., cv. Atlas 66) was germinated in the dark for 3 days in 10-aM CaSO4, then transplanted in groups of six seedlings into 13-1 tanks containing 0.1 meq KC1/1, 2.0 meq MgSOJ1, and 0.5 millimoles Ca(H2POa)a/1. (Each group of six seedlings hereafter will be referred to as a culture). The tanks also contained trace elements at one-fifth the concentration recommended for goagland's complete solution (Hoagland and Arnon, 1950). Cultures produced in this basic solution will be referred to as low-N cultures. Cultures grown in basic solution modified with 15 meq NaNOs/1 or 7.5 meq NH4N03/1 are referred to as high-NO~ cultures or NH~NOa cultures. The solutions were vigorously aerated, acidity was adjusted daily to pH 5.5, and complete changes of solutions were made every 2nd or 3rd day. The cultures, grown in a controlled-environment chamber, received 16 hr of light daily from cool-white fluorescent lamps supplemented with incandescent light at an intensity of approximately 1800 ft.-e, at the surface of the tanks. Temperature was 25 ~=2 ~ during the light period and 19 =~ 2~ during the dark period. 14 days after germination, the cultures were removed from the tanks and thoroughly rinsed. Cultures of detopped roots were prepared by excising shoots 0.5 em above the seed, and cultures of detached shoots were prepared by excising roots about 1 em below the seed. (Leaving a slight amount of root in the latter case allowed contact with solution and permitted the detached shoots to remain turgid throughout the 24-hr experimental period.) Groups of four cultures (24 seedlings) were placed for 24 hr in 1-1 beakers containing 0.4 meq/1 solution of either Ca(NO3)2 or CaSO4. Treatment solutions that provided supplemental carbohydrate also contained glucose at 1%. Duplicate beakers were used for each treatment. Uniform, gentle aeration was maintained by use of capillaries. Trace elements at the concentration specified above were always present in all treatment solutions. Solutions were replaced every 6 hr, at which time water loss and pH were recorded. The solutions were then analyzed for NO~- and rates of NO~ uptake or release were determined by comparison with the initial concentration in the solution. Light was continuous at 1800 ft.-c, throughout the 24-hr experimental period and, unless otherwise indicated, temperature was 25 ~ 2~ When the cultures were removed from the tanks and placed into the treatment beakers, similar cultures (four samples of two cultures each for a total of 48 seedlings) were simultaneously harvested for NO~ analysis according to the method of Johnson
38
P.L. Minotti and W. A. Jackson:
and Ulrich (1950). The values obtained represented NOa content at the beginning of the experimental period. At the end of the experiment additional cultures were harvested and analyzed in the same manner. ]~or low-N seedlings the difference in tissue NOa at the end and the beginning of the uptake period is designated accumulated N0~ and for these seedlings the ~q0~ reduced was calculated by subtracting the amounts accumulated from the total NO~ absorbed as determined by analysis of the culture solution. For high-NO~- cultures the difference between tissue NO~ at the beginning and end of experimental periods represents both NO~ leaked to the media and :NO~-reduced in the tissue. In this case, NO~ reduced was calculated by subtracting from this difference the NO~ leaked. Nitrate reductase in roots and shoots was determined by the method of Hageman and Flesher (1960) on duplicate extracts comprising two cultures each. Carbohydrates were determined by the anthrone method (u and Willis, 1954) on 70 %-ethanol extracts and on the ethanol-insoluble residues after hydrolysis with 0.5 N HC1.
Results and Discussion When placed in dilute CaSO 4 solutions both high-NO~ seedlings and NI-IaNOa seedlings lost NO~ to solution (Table 1). The leakage resembled t h a t which occurs when high-NO~ seedlings are placed in dilute Ca(NO3)~ solutions (Minotti et al., 1969a, b) and therefore is not caused b y the initial total lack of NO~ in the CaS04 solution. Leakage from detopped roots (without shoots) was at least two fold t h a t from intact cultures. Leakage from the detopped high-NO~ seedlings accounted for 29% of their initial NO~ content; a larger proportion (42 %) was noted with the detopped NtIdNO a seedlings. I n each case NO~ leakage from detopped cultures accounted for the decrease in NO~ content of their roots, thereby indicating a failure to reduce NOa. By contrast, a marked NO~ reduction occurred in detached shoots (without roots). I n fact, the decrease in NO~ content of detached shoots exceeded the decrease in NO~ content of shoots of intact seedlings (Table 1). I f it is assumed that shoots of intact cultures have the same capacity for NO~ reduction as detached shoots, then the difference in the accumulated NO~ must be accounted for b y the transport of N 0 ~ from roots to shoots of the intact cultures during the 24 hr experimental period. If the amount of transported NO~ is estimated b y the above method ,and the extent of leakage is known, an estimate can be made of the amount of NO~ reduced b y the roots of intact seedlings. For the high-NO~ cultures, the amount of NO~ transported equals the difference in accumulated NO~ in the detached shoots and shoots from intact cultures (1696~1136~560~g). I f the amount transported (560~g) and the amount leaked into the medium (101~g) are both subtracted from the total decrease of NO~ in the roots of the intact cultures (708~g), the difference (47~g) is assumed to be reduced b y the roots of these cultures. This value is only about 3 % of the total amount reduced b y the seedlings (1743~g). Similar calcluations for
Nitrate Reduction in Wheat Seedlings
39
Table i. Nitrate leakage and reduction by high-NO~ and NH~NO~ cultures during 2d hr exposure to CaSO4 solutions at 0.4 meq/1 Previous nitrogen nutrition (meq/1)
Type of culture a
Initial Net Decrease in tissue NO~ NO a NO~ content content leakage of tissue Roots Shoots Total
Reduced
Percent reduced b
(y.g NO~-N/culture) NaNO a (15.0)
Intact Detopped Detached shoots
6 764 1082 5 952
101 309 17
708 294 --
1136 -1696
1844 294 1696
1743 -1679
26 -28
2ffI-I4NOa (7.5)
Intact Detopped Detached shoots
1942 269 1659
52 114 10
189 105 --
298 -439
487 105 439
435 -429
22 22 26
a Average fresh weight per culture: NaNO~ seedlings, roots 1.08 g shoots 3.33g; NH4NO 8 seedlings, roots 0.87 g, shoots 2.01 g. b Percent of NO~ present in tissues at beginning of 24-hr period.
t h e N H 4 N O a c u l t u r e s g i v e s 1 8 9 - - [ 5 2 + ( 4 3 9 - - 2 9 8 ) ] - - - - - - 4 or n o N O ~ r e d u c e d . T h u s , w i t h i n t a c t s e e d l i n g s t h e b u l k of t h e N O ~ r e d u c t i o n d u r i n g t h e e x p e r i m e n t a l p e r i o d a p p e a r e d t o o c c u r in shoots. I t i s p o s s i b l e t h a t t h e f a i l u r e of d e t o p p e d r o o t s to r e d u c e N O ~ w a s c a u s e d b y a l a c k of r e s p i r a t o r y s u b s t r a t e . A s s h o w n in T a b l e 2, b o t h r o o t s a n d s h o o t s of N O ~ g r o w n m a t e r i a l w e r e l o w in c a r b o h y d r a t e
Table 2. Carbohydrate, nitrate, and nitrate reductase levels o/seedlings at the time they were exposed to treatment solutions Carbohydrates (rag glucose equivalents/g) a
Low-N seedlings Shoots Roots High-NO~ seedlings Shoots Roots
NOa-N (rag/g) a
Nitrate reductase (m~ moles NO~ hr -1 nag-z protein)
Ethanolsoluble
Ethanolinsoluble
45.6 tl.4
14.8 9.3
0.01 n.d. b
n.d. n.d.
2.7 2.2
3.4 4.3
1.79 0.98
200 36
a based on fresh weight. b n. d. ~ not detectable.
40
P.L. Minotti and W. A. Jackson: ]
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Nitrate Reduction in the Roots and Shoots of Wheat Seedlings* P. L. M r s o r r i and W. A. J x c x s o ~ Agricultural Experiment Station, North Carolina State University, Raleigh Received May 26, 1970
Summary. Intact wheat seedlings cultured in high nitrate solutions (high-NO~ cultures) reduced NO~ when placed for 24 hr in dilute CaSOa solutions although they leaked N0~ back to solution during this period. Most of the reduction seemed to occur in shoots since in parallel experiments detached shoots reduced nearly as much of the previously absorbed NO~ as intact cultures. Detached roots leaked greater quantities of N0~ back to solution than did intact cultures, and failed to reduce any of their previously absorbed N0~ during the experimental period. Seedlings of identical age cultured without a nitrogen source and rich in carbohydrate reserves (low-N cultures) rapidly absorbed NOa from dilute Ca(NOn)2 solutions and reduced 80% of that absorbed. Detached low-N roots also absorbed N0~ and reduced 40% of that absorbed. Total NO~ reduction by intact low-N cultures over the 24-hr period was comparable to that of high-NO~ cultures in spite of the fact that in vitro nitrate reductase activity of the former did not reach the levels found initially or at the end of the period in the latter, and that the total NO~ absorbed by low-N cultures was less than that initially present in high-NO~ cultures. Introduction The extent to which nitrate is reduced in roots of plants relative to that which is transported as NO~ to shoots must be subject to a number of controls (c[. Wallace and Pate, 1967). Particularly, a suffieient amount of oxidizable carbohydrate must be present in the root to supply the required carbon skeletons and reducing equivalents. I n addition, retention of NO~ ions within the tissue requires expenditure of respiratory energy. If the utilization of respiratory substrates in roots active in NO~ reduction exceeds the rate of carbohydrate supply from the shoots, the resulting carbohydrate deficiency would restrict further NO~ reduction This deficiency would also tend to curtail the NO~ uptake process and permit accumulated NO~ to diffuse back to the external solution. * Paper No. 3155 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina. These investigations were supported by the U.S. Atomic Energy CommissionContract No. At-(40-1)-2410.
Nitrate Reduction in Wheat Seedlings
37
D a t a p r e s e n t e d p r e v i o u s l y (Minotti et al., 1969a, b) showed t h a t 2-week-old w h e a t seedlings grown in high-NO~ solutions, a n d c o n t a i n i n g high a m o u n t s of NO~ in t h e i r tissues, lost significant a m o u n t s of t h a t NO~ to m o r e d i l u t e NO~ solutions d u r i n g a 24-hr period. I n t h e s e e x p e r i m e n t s t h e r a t i o of NO~ in t h e r o o t tissue to t h a t in t h e m e d i u m was a b o u t 180:1. T h e seedlings were c a p a b l e of r e d u c i n g NO~ d u r i n g t h e p e r i o d w h e n NO~ was being lost f r o m r o o t tissue, b u t i t could n o t be said u n e q u i v o c a l l y t h a t r e d u c t i o n was occurring in roots. W e r e p o r t now d a t a f r o m p a r a l l e l e x p e r i m e n t s c o m p a r i n g NO3 r e d u c t i o n , NO~ l e a k a g e a n d / o r NO3 u p t a k e in i n t a c t seedlings w i t h t h a t in d e t o p p e d r o o t s a n d d e t a c h e d shoots. The e x p e r i m e n t s f u r t h e r d e l i n e a t e t h e NO~-leakage p h e n o m e n o n , a n d i l l u s t r a t e t h a t t h e c a p a c i t y of r o o t s to reduce, a n d t o r e t a i n , NO3 w i t h i n t h e i r tissue is r e l a t e d to t h e i r c a r b o h y d r a t e c o n t e n t .
Methods Wheat (Triticum aestivum L., cv. Atlas 66) was germinated in the dark for 3 days in 10-aM CaSO4, then transplanted in groups of six seedlings into 13-1 tanks containing 0.1 meq KC1/1, 2.0 meq MgSOJ1, and 0.5 millimoles Ca(H2POa)a/1. (Each group of six seedlings hereafter will be referred to as a culture). The tanks also contained trace elements at one-fifth the concentration recommended for goagland's complete solution (Hoagland and Arnon, 1950). Cultures produced in this basic solution will be referred to as low-N cultures. Cultures grown in basic solution modified with 15 meq NaNOs/1 or 7.5 meq NH4N03/1 are referred to as high-NO~ cultures or NH~NOa cultures. The solutions were vigorously aerated, acidity was adjusted daily to pH 5.5, and complete changes of solutions were made every 2nd or 3rd day. The cultures, grown in a controlled-environment chamber, received 16 hr of light daily from cool-white fluorescent lamps supplemented with incandescent light at an intensity of approximately 1800 ft.-e, at the surface of the tanks. Temperature was 25 ~=2 ~ during the light period and 19 =~ 2~ during the dark period. 14 days after germination, the cultures were removed from the tanks and thoroughly rinsed. Cultures of detopped roots were prepared by excising shoots 0.5 em above the seed, and cultures of detached shoots were prepared by excising roots about 1 em below the seed. (Leaving a slight amount of root in the latter case allowed contact with solution and permitted the detached shoots to remain turgid throughout the 24-hr experimental period.) Groups of four cultures (24 seedlings) were placed for 24 hr in 1-1 beakers containing 0.4 meq/1 solution of either Ca(NO3)2 or CaSO4. Treatment solutions that provided supplemental carbohydrate also contained glucose at 1%. Duplicate beakers were used for each treatment. Uniform, gentle aeration was maintained by use of capillaries. Trace elements at the concentration specified above were always present in all treatment solutions. Solutions were replaced every 6 hr, at which time water loss and pH were recorded. The solutions were then analyzed for NO~- and rates of NO~ uptake or release were determined by comparison with the initial concentration in the solution. Light was continuous at 1800 ft.-c, throughout the 24-hr experimental period and, unless otherwise indicated, temperature was 25 ~ 2~ When the cultures were removed from the tanks and placed into the treatment beakers, similar cultures (four samples of two cultures each for a total of 48 seedlings) were simultaneously harvested for NO~ analysis according to the method of Johnson
38
P.L. Minotti and W. A. Jackson:
and Ulrich (1950). The values obtained represented NOa content at the beginning of the experimental period. At the end of the experiment additional cultures were harvested and analyzed in the same manner. ]~or low-N seedlings the difference in tissue NOa at the end and the beginning of the uptake period is designated accumulated N0~ and for these seedlings the ~q0~ reduced was calculated by subtracting the amounts accumulated from the total NO~ absorbed as determined by analysis of the culture solution. For high-NO~- cultures the difference between tissue NO~ at the beginning and end of experimental periods represents both NO~ leaked to the media and :NO~-reduced in the tissue. In this case, NO~ reduced was calculated by subtracting from this difference the NO~ leaked. Nitrate reductase in roots and shoots was determined by the method of Hageman and Flesher (1960) on duplicate extracts comprising two cultures each. Carbohydrates were determined by the anthrone method (u and Willis, 1954) on 70 %-ethanol extracts and on the ethanol-insoluble residues after hydrolysis with 0.5 N HC1.
Results and Discussion When placed in dilute CaSO 4 solutions both high-NO~ seedlings and NI-IaNOa seedlings lost NO~ to solution (Table 1). The leakage resembled t h a t which occurs when high-NO~ seedlings are placed in dilute Ca(NO3)~ solutions (Minotti et al., 1969a, b) and therefore is not caused b y the initial total lack of NO~ in the CaS04 solution. Leakage from detopped roots (without shoots) was at least two fold t h a t from intact cultures. Leakage from the detopped high-NO~ seedlings accounted for 29% of their initial NO~ content; a larger proportion (42 %) was noted with the detopped NtIdNO a seedlings. I n each case NO~ leakage from detopped cultures accounted for the decrease in NO~ content of their roots, thereby indicating a failure to reduce NOa. By contrast, a marked NO~ reduction occurred in detached shoots (without roots). I n fact, the decrease in NO~ content of detached shoots exceeded the decrease in NO~ content of shoots of intact seedlings (Table 1). I f it is assumed that shoots of intact cultures have the same capacity for NO~ reduction as detached shoots, then the difference in the accumulated NO~ must be accounted for b y the transport of N 0 ~ from roots to shoots of the intact cultures during the 24 hr experimental period. If the amount of transported NO~ is estimated b y the above method ,and the extent of leakage is known, an estimate can be made of the amount of NO~ reduced b y the roots of intact seedlings. For the high-NO~ cultures, the amount of NO~ transported equals the difference in accumulated NO~ in the detached shoots and shoots from intact cultures (1696~1136~560~g). I f the amount transported (560~g) and the amount leaked into the medium (101~g) are both subtracted from the total decrease of NO~ in the roots of the intact cultures (708~g), the difference (47~g) is assumed to be reduced b y the roots of these cultures. This value is only about 3 % of the total amount reduced b y the seedlings (1743~g). Similar calcluations for
Nitrate Reduction in Wheat Seedlings
39
Table i. Nitrate leakage and reduction by high-NO~ and NH~NO~ cultures during 2d hr exposure to CaSO4 solutions at 0.4 meq/1 Previous nitrogen nutrition (meq/1)
Type of culture a
Initial Net Decrease in tissue NO~ NO a NO~ content content leakage of tissue Roots Shoots Total
Reduced
Percent reduced b
(y.g NO~-N/culture) NaNO a (15.0)
Intact Detopped Detached shoots
6 764 1082 5 952
101 309 17
708 294 --
1136 -1696
1844 294 1696
1743 -1679
26 -28
2ffI-I4NOa (7.5)
Intact Detopped Detached shoots
1942 269 1659
52 114 10
189 105 --
298 -439
487 105 439
435 -429
22 22 26
a Average fresh weight per culture: NaNO~ seedlings, roots 1.08 g shoots 3.33g; NH4NO 8 seedlings, roots 0.87 g, shoots 2.01 g. b Percent of NO~ present in tissues at beginning of 24-hr period.
t h e N H 4 N O a c u l t u r e s g i v e s 1 8 9 - - [ 5 2 + ( 4 3 9 - - 2 9 8 ) ] - - - - - - 4 or n o N O ~ r e d u c e d . T h u s , w i t h i n t a c t s e e d l i n g s t h e b u l k of t h e N O ~ r e d u c t i o n d u r i n g t h e e x p e r i m e n t a l p e r i o d a p p e a r e d t o o c c u r in shoots. I t i s p o s s i b l e t h a t t h e f a i l u r e of d e t o p p e d r o o t s to r e d u c e N O ~ w a s c a u s e d b y a l a c k of r e s p i r a t o r y s u b s t r a t e . A s s h o w n in T a b l e 2, b o t h r o o t s a n d s h o o t s of N O ~ g r o w n m a t e r i a l w e r e l o w in c a r b o h y d r a t e
Table 2. Carbohydrate, nitrate, and nitrate reductase levels o/seedlings at the time they were exposed to treatment solutions Carbohydrates (rag glucose equivalents/g) a
Low-N seedlings Shoots Roots High-NO~ seedlings Shoots Roots
NOa-N (rag/g) a
Nitrate reductase (m~ moles NO~ hr -1 nag-z protein)
Ethanolsoluble
Ethanolinsoluble
45.6 tl.4
14.8 9.3
0.01 n.d. b
n.d. n.d.
2.7 2.2
3.4 4.3
1.79 0.98
200 36
a based on fresh weight. b n. d. ~ not detectable.
40
P.L. Minotti and W. A. Jackson: ]
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