Planta (Berl.) 123, 41--51 (1975) 9 by Springer-Verlag 1975

Nitrate, Nitrite and Ammonia Assimilation by Leaves: Effects of Inhibitors * C. A. A t k i n s * * a n d D. T. C a n v i n * * * International Atomic Energy Agency, Seibersdorf Laboratory, A-1011 Vienna, Austria Received 26 September; accepted 17 December, 1974 Summary. The assimilation of H14C03-, 15N03-, 15NO2- and 15NH4+ by barley (Hordeum vulgate L.) leaf segments in the presence of a number of metabolic inhibitors was studied in

experiments where the substrates and inhibitors were vacuum infiltrated into the tissue. 3-(3',4'-Dichloropheriyl)-l,l-dimethylurea (DCMU), carbonyl-cyanide-m-chlorophenylhydrazone (CCCP), and iodoacetate (IOA) inhibited14C02 fixation andlSNOa- andlSN02 assimilation in the light. 15•I-I4+ e~ssimilation in the light was only inhibited 70% by 10 4M DCMU. I~NH4+ assimilation was stimulated by 10-~M CCCP but was inhibited by concentrations of CCCP above 5 • 10-5M. In double-label experiments 15N0~- assimilation was less sensitive than 14C02 fixation to both DCMU and CCCP. CCCP but not DCMU stimulated NO2- accumulation in dark and IOA was inhibitory. The rate of NO~- accumulation in the light in the presence of DCMU or atrazine was similar to that in the dark and in all cases :NO2- accumulation was inhibited about 90 % by oxygenation of the medium. The results indicate that the assimilation of all nitrogen species is closely linked to photosynthetic electron transport, that rate of assimilation of nitrogen species is independent of rate of assimilation of C02, and that the dark in-vivo nitrate reduction is a useful analogue of the mechanism operating in the light only if electron flow to oxygen is impaired.

Introduction I n a p r e v i o u s p a p e r (Canvin a n d A t k i n s , 1974) we h a v e shown t h a t n i t r a t e a n d n i t r i t e a s s i m i l a t i o n in green leaves is s t r i c t l y d e p e n d e n t on l i g h t a n d t h a t a m m o n i a a s s i m i l a t i o n is g r e a t l y s t i m u l a t e d b y light. R e c e n t evidence (Miflin, 1974; Magalhaes et al., 1974) has shown t h a t isolated s p i n a c h chloroplasts will s u p p o r t a high r a t e of l i g h t - d e p e n d e n t n i t r i t e r e d u c t i o n a n d de-novo a m i n o - N f o r m a t i o n w i t h o u t t h e a d d i t i o n of cofactors, showing t h a t p h o t o s y n t h e t i c electron t r a n s p o r t p r o v i d e s t h e reducing power for n i t r i t e r e d u c t i o n a n d t h e a s s i m i l a t i o n of a m m o n i a . The f u r t h e r effects of light on n i t r a t e a s s i m i l a t i o n a n d a m i n o - a c i d f o r m a t i o n are suggested to be m e d i a t e d v i a t h e p r o d u c t i o n of r e d u c e d c a r b o n c o m p o u n d s (Klel0per et al., 1971; Magalhaes et al., 1974) h u t we h a v e a l r e a d y shown t h a t t h e a s s i m i l a t i o n of n i t r a t e is n o t d i r e c t l y d e p e n d e n t on c o n c o m i t a n t p h o t o s y n t h e t i c CO S f i x a t i o n (Canvin a n d A t k i n s , 1974). I n t h e p r e s e n t p a p e r t h e effects of light on t h e a s s i m i l a t i o n of NOn-, NO2-, a n d NH4+ b y b a r l e y leaves h a v e * Abbreviations: atrazine, 2-chloro-4-ethylamino-6-isopropylamino-s-triazine;CCCP, carbonyl-

cyanide-m-chlorophenylhydrazone; DCMU, 3-(3',4'-diehlorophenyl)-l,l-dimethylurea; IOA, iodoacetate. ** Present address: Botany Department, University of Western Australia, Nedlands, W. A. 6009, Australia. *** Present address: Biology Department, Queen's University, Kingston, Ontario K7L 3N6, Canada.

42

C.A. Atkins and D. T. Canvin I

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Fig. 1 A--C. The effect of DCMU concentration on the fixation of 14C0 s into water-soluble compounds (A), the 14C content of basic compounds (B), and the 15N content of the basic compounds (C), in vacuum-infiltrated barley leaf segments in the light. The reaction mixture contained b o t h H14COs (25 ~zmol; 33.2 • l0 s dpm ~zmo1-1) and :Na15NOs (250 ~cmol; 96.7 at% 15N). Light intensity (8 • 104 erg em -2 s-l), temperature 25 ~ and 0.3 g fr.wt, leaf tissue per sample. 9 Control, 9 10-5~ DCMU, A 3 • 10-~M DCMU, 9 7 • 10-5 M DCMU

b e e n f u r t h e r e x a m i n e d u s i n g 15N-labeled s u b s t r a t e s a n d s e v e r a l m e t a b o l i c i n h i b i tors in an attempt to further elaborate the relationships between nitrogen assimi l a t i o n a n d p h o t o s y n t h e s i s . N i t r o g e n - 1 5 c o n t e n t of all s a m p l e s w a s d e t e r m i n e d u s i n g o p t i c a l e m i s s i o n s p e c t r o m e t r y ( F e r r a r i s a n d P r o k s c h , 1972).

Materials and Methods The culture of barley plants (Hordeum vulgare L. cv. Union), the experimental procedure for leaf-segment infiltration, the incubation conditions, extraction of the tissue after incubation, collection of the soluble basic fraction, determination of NO 2- and 15N as well as the assay for photosynthesis with H14COa- have been described previously (Canvin and Atkins, 1974). The vacuum-infiltration procedure was varied however so t h a t the tissue was preincubated with inhibitor before the 15N-labelled substrate was added. The inhibitors used (DCMU, CCCP, IOA a n d atrazine) were dissolved in 50% (v/v) ethanol/water and 0.5 ml added to the reaction medium. The same volume of solvent was added to controls. Following vacuum infiltration the vessels were placed in the light (8 • 104 erg cm -2 s-1) a t 25 ~ for 20 rain. The labeled substrate was t h e n added, the tissue again vacuum infiltrated, and the zero-time samples killed and extracted. I n experiments where NO 2- accumulation was measured the preineubation with inhibitors and addition of unlabeled substrates was performed in the same way,

Generally 1 ~ incorporation was measured after 0, 30 and 60 rain incubation, a n d NO 2accumulation after 0,60 a n d 90 rain. I n all cases the 15/~ content or the NO2- present at zero time was substracted from the determined values of subsequent samples. The rate of labeling in the presence of inhibitor in each 30 rain period was expressed as a percentage of the control rates and the m e a n value of the two periods used to construct Figs. 2 and 4. I n some experiments 02 or N 2 was bubbled through the medium in the reaction vessel during incubation using a fine plastic tube which extended to the b o t t o m of the vessel. I n some experiments (Figs. 1, 3) the concomitant assimilation of H14CO3- (25 ~tmol, 33.2 • 10 s dpm ~mo1-1) and zsN08- (250 ~mol, 96.7 at% 15N) by the tissue was measured b y

NO~-, NO2- and NH,+ Assimilation: Inhibitors

Nitrate, nitrite and ammonia assimilation by leaves: Effects of inhibitors.

The assimilation of H(14)CO3 (-), (15)NO3 (-), (15)NO2 (-) and (15)NH4 (+) by barley (Hordeum vulgare L.) leaf segments in the presence of a number of...
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