Planta (Berl.) 86, 267 271 (1969)

The Influence of Ammonium on Nitrate Reduction in Wheat Seedlings* P. L. MT~OT~I, Don~s CRAIG WILLIAMS, and W. A. JACKSON Department of Soil Science, North Carolina State University, Raleigh Received February 12, 1969

Summary. Ammonium markedly inhibited nitrate absorption by nitrogenstarved wheat seedlings but did not decrease the proportion of absorbed nitrate that was reduced. Seedlings high in nitrate (absorbed prior to the experimental periods) reduced similar amounts of this nitrate regardless of whether or not ammonium was present and being absorbed during the period of measurement. Ammonium or products of ammonium assimilation did not interfere with the induction, stability, or activity of nitrate reductase. Consequently, the hypothesis that ammonium depresses nitrate uptake indirectly by inhibiting nitrate reduction is rejected, and it is suggested that the ammonium effect is directly on the nitrateuptake process. Introduction Seedlings of wheat (W~IsSMA~, 1951 ; MINOTTI et al., 1969), seedlings and plants of rye grass (LYcKLAMA, 1963), and slices of potato tuber (EL-SHIsHINu 1955) have been shown to absorb less nitrate-N from media also containing a m m o n i n m - N t h a n from media containing exclusively nitrate-N. The precise manner in which N H 4 inhibits N03 uptake is not known. I t has been postulated, however, t h a t N H 4 (or assimilation products of NItd) affect NOs uptake indirectly b y inhibiting the reduction of NOa in the tissue, possibly by inhibiting nitrate reductase (Nt~) (LYcI;J,AMA, 1963). I t iS reasonable t h a t effects of NI-I4 on NO 3 reduction m a y regulate NO 3 absorption under conditions where tissues of higher plants had already accumulated sizeable amounts of NO a (LYcKLAMA, 1963). Under these conditions u p t a k e from the medium represents a replacement of NO3 assimilated b y the cells. This does not mean, however, t h a t NO 3 u p t a k e is always regulated b y effects on NO~ reduction or t h a t N I t 4 decreases 2NTO3 uptake in this manner. The postulate can be tested by experiments which examine (a) the uptake, accumulation, and reduction of NO 3 in the presence and absence of 2NTI{a under conditions where rapid * Paper No. 2800 of the Journal Series of the North Carolina State University Agricultural Experiment Station. These investigations were supported by the U. S. Atomic Energy Commission, Contract No. AT-(40-1)-2410.

268

P . L . MINOTTI, D. C. WmLIAMS and W. A. JACKSON:

i n f l u x of N O d occurs, (b) t h e effects of N H 4 on t h e r e d u c t i o n of N O d a b s o r b e d p r i o r to e x p o s u r e to NH4, a n d (e) effects of N H 4 on t h e a c t i v i t y of N R . T h e results of s u c h e x p e r i m e n t s , r e p o r t e d here, s u g g e s t t h a t N H 4 c a n d i r e c t l y i n f l u e n c e t h e N O d - u p t a k e process.

M a t e r i a l s and Methods Wheat (Triticum aestivum L., cv. Atlas 66) was germinated for 3 days in the dark in 10-4 M CaSOr aud transplanted in groups of 6 seedlings to 13--L tanks containing 0.1 meq KC1/L, 0.2 meq MgSOJL, and 0.5 millimoles Ca(H~PO~)2/L. Each group of 6 seedlings hereaiter will be referred to as a culture. The tanks also contained trace elements at one-fifth the concentration of Hoagland's A-Z solution (I-IoAGLAND and AR~oN, 1950). This comprised the basic solution which produced what is referred to as Low-N seedlings. Culture grown in the basic solution plus 15 meq NaNO~/L are referred to as NO 8 seedlings. The cultures were grown in a controlled environment chamber with 16 hr of light daily. Light intensity was approximately 1800 ft-c at the surface of the tanks, and temperature was 25 • 2 ~ during the light period and 19 4- 2 ~ during the dark period. The solutions were aerated rapidly, acidity was adjusted daily to p i t 5.5, and complete changes of solutions were made every other day. 14 days after germination, the cultures were removed from the tanks and thoroughly rinsed. After rinsing, groups of 4 cultures (24 seedlings) were placed in 1-L beakers containing nitrogen solutions, usually at 0.4 meq/L. 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 the treatment solution. In the 24-hr experiments, solutions were replaced every 6 hr; at this time, water loss and pI-I were recorded. The solutions were then analyzed for ionic composition. Light was continuous at 1800 ft-c throughout the experimental period and, unless otherwise indicated, temperature was 25 4- 1~ Rates of ion uptake or release were determined by comparison with the initial concentration in the solution. When the cultures were removed from tanks and placed into the treatment beakers, similar cultures (4 samples of 2 cultures each for a total of 48 seedlings) were simultaneously harvested for NO~ analysis according to the method of JoHnsoy and ULRIC~ (1950). The value obtained represented NO~ contents at the beginning of experimentM periods. At the end of the experiment the cultures were harvested and analyzed in the same manner. The difference between tissue NO 3 at the end of uptake periods and that measured at the outset represents accumulated NO 8. The amounts reduced were calculated by subtracting the amounts accumulated from the total absorbed. N R in shoot extracts was determined by the method of I-IAGEMANand FLEStIER (1960).

Results and Discussion A m m o n i u m s u b s t a n t i a l l y d e c r e a s e d t h e a m o u n t s of NOd a c c u m u l a t i n g in tissues as well as i n h i b i t i n g N O a u p t a k e (Table 1). I f N O d u p t a k e h a d b e e n a f f e c t e d solely b y a n i n f l u e n c e of N H 4 on NOd r e d u c t i o n we w o u l d h a v e e x p e c t e d N O d to c o n t i n u e t o a c c u m u l a t e . Since i t did n o t we r e j e c t t h e p o s t u l a t e t h a t N H a r e s t r i c t s N O a u p t a k e in t h e s e seedlings i n d i r e c t l y t h r o u g h a n effect on NOd r e d u c t i o n . T h e effect a p p e a r s to be a m o r e

The Influence of Ammonium on Nitrate Reduction in Wheat Seedlings 269 direct one on t h e u p t a k e process itself (el. i~[INOTTI et al., 1969). The p e r c e n t a g e of a b s o r b e d NOa r e d u c e d (which m i g h t be t a k e n as one measure of NOa-reduetion efficiency) was similar w h e t h e r or n o t N H 4 was present. The a d d i t i o n of CaS04 to cultures in NH4NO a increased t h e u p t a k e of NO 3 w i t h o u t a n y a p p a r e n t increase in NOa-reduction capacity, which Table 1. Nitrate uptalce, accumulation, and reduction by Low-N cultures after 6 and 2~ hr exposure to the indicated salt solutions

Values, except last column, are ~g N Q - N per culture; average fresh weight per culture for 6-hr experiment: roots 1.74 g, shoots 0.94 g, for 24-hr experiment: roots 1.58 g, shoots 1.10 g. Treatment (meq/L)

Uptake

Accumulated

Reduced

% reduced

Roots

Shoots

Total

56I 382 45I

155 148 210

54 12 26

219 160 236

342 222 215

60 58 48

510

236

62

298

212

42

2,220 561

234 49

50 17

284 66

1,936 495

87 88

6 hour8

Ca(NOa) 2 (0.4) NH4NO~ (0.4) NHaNO 3 (0.4) + CaSQ (1.0) NI/4NO 8 (0.4) ~- CaSO~ (10.0) 24 hours

Ca(NOa) 2 (0.4) NH4N03 (0.4)

of course resulted in a lower figure for p e r c e n t n i t r a t e r e d u c e d (Table 1). The m o d i f i c a t i o n of t h e NH~ effects on u p t a k e b y CaSO 4 w i t h o u t commensurate m o d i f i c a t i o n of NO a r e d u c t i o n is suggestive t h a t u p t a k e was proceeding quite i n d e p e n d e n t l y of r e d u c t i o n during the initial 6 hr after exposure to NO a. Because cultures in NI-I4NO a r e d u c e d similar p o r t i o n s of t h e i r i n c o m i n g NO 3 as did those in Ca(NOa) 2 (Table 1), a n d because we k n e w t h a t s i m u l t a n e o u s a b s o r p t i o n of N H 4 b y these cultures g r e a t l y e x c e e d e d a b s o r p t i o n of NOa (MI~OTTI et al., 1969) we questioned w h e t h e r N t t a or its a s s i m i l a t i o n p r o d u c t s was i n h i b i t i n g NOa r e d u c t i o n a t all in our e x p e r i m e n t s . Since NOa m u s t be a b s o r b e d to be reduced, it is difficult to answer this question b y working with a s y s t e m of L o w - N seedlings whose tissues are i n i t i a l l y low in NO a. W e h a d p r e v i o u s l y n o t e d t h a t when seedlings grown on high c o n c e n t r a t i o n s of NO 3 were p l a c e d in dilute solutions similar to t h e t r e a t m e n t solutions in Table 1, t h e y l e a k e d NO 3 b a c k to solution b u t r e t a i n e d the c a p a c i t y to absorb NI-I4 a n d to reduce some of

P.L. MI~OTTI,]:). C. WILLIAMSand W. A. JAcxso~:

270

Table 2. Nitrate lea]cage, accumulation, and reduction by NOa-cultures after 24 hr exposure to nitrate solutions with and without ammonium Values, except last column, are ~g NOa-N per culture; average fresh weight per culture: roots 1.52 g, shoots 2.84 g. Treatment

Leak-

Accumulated

(meq/L)

~ge

Roots

Shoots

Total

Reduced

%

Ca(NO3)2 (0.2)

107

-- 427

-- 757

-- 1,184

1,077

20

Ca(N03) ~ (0.2) 316 + (NH4)2SQ (10.0)

-- 637

-- 817

-- 1,536

1,220

25

reduceda

a Percent of NO a present in tissues at beginning of 24-hr period. t h ei r previously absorbed N O 3. This p r o v i d e d a s y s t e m in which we could assess t h e effects of N t t a on N Q r e d u c t i o n r a t h e r i n d e p e n d e n t l y of absorption. Table 2 shows t h a t in such a s y s t e m high c o n c e n t r a t i o n s of (NH4)2SO a (10 m e q / L ) did n o t inhibit r e d u c t i o n of p r e v i o u s l y absorbed n i t r a t e over a 24-hr period e v e n t h o u g h such t r e a t m e n t increased n e t leakage of NO~ to solution. Assays for NI~ in t h e shoot tissue showed no significant difference due to t h e 24-hr exposure to (NH4)2SO 4 (approxim a t e l y 7.0 ~zmoles N O 2 hr -1 g m i fresh tissue). A d d i n g N H aN O a i n st ead of K N O a to the s t a n d a r d assay m i x t u r e t e n d e d to slightly enhance act i v i t y . I t is possible, however, t h a t products of N H 4 assimilation which m i g h t i n h i b i t NOa r e d u c t i o n would n o t h a v e built up sufficiently in a 24-hr period. Seedlings were therefore grown for 2 weeks on NH4NO ~ and on N a N Q . Their a b i li ty to reduce p r e v i o u s l y absorbed NOa over a 24-hr period in C a S Q is shown in Table 3. Cultures g r o w n on NH4NO 3 reduced less NO 3 a p p a r e n t l y because t h e y c o n t a i n e d so m u c h less at t h e beginning of th e 24-hr period. T h e fact t h a t t h e y r ed u ced a similar Table 3. Reduction and leakage o/previously absorbed NOa nitrogen by cultures grown /or 2 weeks with or without NHa Cultures were placed into CaSO 4 solutions (0.4 meq/L) for a 24 hr period. Previous nutrition a

Fresh wt. per culture (g)

Net NO 3 leakage

Initial nitrate (~zg NOa-N per culture)

Final nitrate

Reduced

% redueed

NaN0 a NH~NO 3

4.41 2.88

101 52

6,764 1,942

4,920 1,455

1,743 435

26 22

a Pretreatment consisted of growth for 2 weeks in the basic nutrient solution receiving N as either NaNO 3or NH4NO 3 at 15 meq of N/L. Data are for 48 seedlings and include both roots and shoots.

The Influence of Ammonium on Nitrate Reduction in Wheat Seedlings 271 p e r c e n t a g e of the NO a t h e y d i d c o n t a i n is a t least suggestive t h a t 2 weeks on N H 4 d i d n o t seriously i m p a i r their c a p a c i t y for NO 3 reduction. Some N R a c t i v i t y was f o u n d in cultures grown for 2 weeks in t h e basic solution plus 15 meq (NH4)2SO4/L , b u t this m a y have been due to some n i t r i f i c a t i o n in t h e non-sterile media. W h e n e x p o s e d to NO a t h e N R a c t i v i t y of these cultures r a p i d l y increased; thus, our results agree with those of others in o t h e r p l a n t s (e.g. BE:EVEnS et al., 1965), n a m e l y , t h a t NO3 induces N R in the presence of N H 4. Considered as a whole t h e evidence p r e s e n t e d a b o v e leads us to r e j e c t t h e idea of NOa u p t a k e being r e g u l a t e d via NO 3 reduction. Specifically we f o u n d no evidence to s u p p o r t t h e p o s t u l a t e t h a t N H 4 or its a s s i m i l a t i o n p r o d u c t s were interfering with NO a r e d u c t i o n in our t y p e s of systems. W e therefore believe t h e p r o n o u n c e d depression of NO 3 u p t a k e b y N H 4 to be a n effect on t h e u p t a k e process itself.

References BEEVERS, L., L. E. SC~AJ)ER, DONNAFLESHER, and R. H. HAG~MA~: The role of light and nitrate in the induction of nitrate reductase in radish cotyledons and maize seedlings. Plant Physiol. 40, 691--698 (1965). EL-SttIStlINu E. D. H.: Absorption and assimilation of inorganic nitrogen from different sources of storage root tissue. J. exp. Bot. 6, 6--16 (1955). HAGEMA~, R. H., and D. FLES~ER: Nitrate reductase activity in corn seedlings as affected by light and nitrate content of nutrient media. Plant Physiol. 35, 700--708 (1960). HOAGLA~D,D. R., and D. I. AR~o~: The water-culture method for growing plants without soil California Agr. Expt. Sta. Cir. No 347 (1950). Joy,so?c, C.M., and A. ULI~IC~: Determination of nitrate in plant material. Analyt. Chem. 2,o, 1526--1529 (1950). LYCKLA?SA,J. C. : The absorption of ammonium and nitrate by perennial rye grass. Acta bot. neerl. 12, 3 1 6 4 2 3 (1963). ~/[INOTTI,t 3. L., DORIS CRAIG WILLIAMS, and W. A. JACKSON: Nitrate absorption by wheat as influenced by ammonium and other cations. Crop Sci., 9, 9--14 (1969). WEISSMA~V,G. S. : Nitrogen metabolism of wheat seedlings as influenced by the ammonium:nitrate ratio and the hydrogen-ion concentration. Amer. J. Bot. 38, 163--174 (1951). Dr. P. L. ~INOTTI Department of Vegetable Crops Plant Science Building Cornell University Ithaca, New York 14850, USA