Planta (Berl.) 122, 37---44 (1975) 9 by Springer-Verlag 1975
Ammonium Inactivation of Nitrate Reductase in Lemna minor L. T. O. Orebamjo* and G. R. Stewart** Department of Botany, The University, Manchester M13 9PL, U. K. Received 16 October; accepted 14 November 1974 Summary. The addition of ammonium to nitrate induced plants of Lemna minor L. brings about a rapid loss in extractable nitrate rcductase activity. This inactivation is reversible both in vivo and in vitro. Inhibitors of RNA and protein synthesis do not protect nitrate reductase against ammonium inactivation. It is suggested that factors, in addition to ammonium ions, are components of the inactivating system. Inactivation may involve some form of protein-protein interaction. The physiological significance of rapid ammonium inactivation of nitrate reductase is discussed. Introduction
Previous studies of the regulation of nitrate reductase in L e m n a minor have established that its formation is regulated through the interaction of positive and negative control elements (Orebamjo and Stewart, 1974a, b). I n this dual control system, nitrate acts as a positive modulator, while ammonium (indirectly) acts as a negative modulator, of enzyme synthesis. The kinetics of induction and repression are consistent with the transcriptional control of enzyme synthesis (Orebamjo and Stewart, 1974b). In addition to inhibiting the induced formation of nitrate reductase ammonium brings about a very rapid loss of nitrate reductase activity when added to fully induced plants (Orebamjo and Stewart, 1974b). The rate of enzyme loss under these conditions is faster than can be accounted for by an immediate cessation of enzyme synthesis and the subsequent reduction in activity through growth. A similar response to the addition of ammonium has been reported in several species of an algae (Losado et al., 1970, 1973; l~igano et al., 1974) and some 9 species of fungi (Lewis and Fincham, 1970; Subramanian and Sorger, 1972). I n both Chlorella and Chlamydomonas ammonium inactivation is reversible and dependent upon photosynthesis (Losado etal., 1973; Herrera etal., 1972). I n contrast ammonium inactivation in C y a n i d i u m although reversible, is not dependent upon photosynthesis (Rigano et al., 1974). The mechanism of inactivation in Chlorella appears to involve chemical modification of the enzyme, since in vitro the active form can be reversibly converted into its inactive form in the presence of NADH and ADP. In the fungus Ustilago inhibitors oI RNA and protein synthesis block the ammonium inactivation of the enzyme (Lewis and Fineham, 1970), suggesting' that here the rapid loss of nitrate reductase may be dependent upon continued protein synthesis. * Present address: School of Biological Sciences, The University of Lagos, Lagos, Nigeria. ** Address for reprint requests.
38
T.O. Orebamjo and G. R. Stewart
I n this paper we report the characteristics of the ammonium mediated inactivation of nitrate reductase in the higher plant L e m n a minor. The inactivation is reversible both in vivo and i n vitro. Inhibitors of I~NA and protein synthesis do not prevent the ammonium inactivation. Possible mechanisms of inactivation are discussed. Materials and Methods The clone of Lemna minor used in the present experiments was maintained and grown as described previously (Stewart, 1972a, b). Nitrate reductase was extracted and its activity determined by the procedures used previously (Stewart, 1972a). Enzyme extracts were assayed within 1-2 minutes following extraction unless subjected to further treatment (see Results). Tissue nitrate and ammonium were extracted and determined as before (Stewart, 1972b; Orebamjo and Stewart, 1974a). In all of the experiments reported here a concentration of 5 mM nitrate was used to induce the formation of nitrate reductase. Enzyme activity is expressed as ~zmolNO2 produced/hour/gfw. Results
The addition of ammonium to fully induced plants brings about a rapid loss of extractable nitrate reductase activity (Fig. 1). T h e rate at which enzyme activity is lost in ammonium treated plants is considerably faster than that measured when either 6-methyl purine or cycloheximide are used to halt further synthesis of the enzyme. The results also show that when asparagine is added to fully induced plants the rate of enzyme loss is less than that measured in cycloheximide treated plants. This suggests that there m a y be some continued synthesis of the enzyme in the presence of asparagine. The halflife of nitrate rednctase in ammonium treated plants is 3.6 h (as determined from semi-log plots of enzyme decay), compared with those of 9 and l l h in cycloheximide and 6-methyl purine treated plants. I t is evident however, that there are two phases in the decay curve for ammonium treated plants, over the first 4 h there is a phase of rapid enzyme loss (which gives the half-life of 3.6 h), subsequently there is a marked decrease in the rate at which enzyme activity is lost. The presence of protein synthesis inhibitors such as chloramphenical and cycloheximide have no measurable effect on the initial phase of the ammonium inactivation (Fig. 2). Furthermore pre-treating plants with cycloheximide tot 1 hour before the addition of ammonium has no effect on the rapid inactivation. These results suggest that the inactivating system is dependent upon neither cytoplasmic nor chloroplast protein synthesis. These results contrast with those obtained for Ustilago where both cycloheximide and actinomycin D completely prevent the ammonium inactivation of nitrate reductase (Lewis and Fincham, 1970). The loss of nitrate reductase following the addition of ammonium is not simply the result of ammonium inhibition since the addition of 20 mM ammonium chloride has no effect on the activity of the enzyme extracted from nitrate induced plants. Surprisingly though ammonium actually stabilizes enzyme extracted from such plants (Fig. 3). This protection of the extracted enzyme by
Ammonium Inactivation of Nitrate Reductase
39
0.8
~,,0.G ~
i
._~ ~
$
. 0.2
0 T 0
1
i 4
I
I 8
Time(h)
I
I 12
Fig. 1. A m m o n i u m inactivation of nitrate reduction in vivo. Asparagine-adapted plants were induced :for 15 hours before the addition of: 9 2 mM ammonium, 9 150 iJ.g/ml 6-methyl purine; 9 1 mM asparagine; 9 2 ~zg-ml eyeloheximide
.=
U.i2
D
Time(h)
:Fig. 2. Effect of inhibitors of protein synthesis on the a m m o n i u m inactivation of nitrate reductase. Asparagine-adapted plants were induced for 15 h before the addition of: 9 $ mM a m m o n i u m ; 9 2 mM a m m o n i u m + 2 ~g-mt eyeloheximide; 9 2 mM a m m o n i u m + 500 ~zg-ml D-threoehloramphenicol; [] 2 ~zg-ml cyeloheximide added a t 14 h induction, followed by 2 mM ammonium at 15 h
40
T.O. Orebamjo and G. R. Stewart
,,~4
E ~2
0
4
8
Time (minutes)
120
Fig. 3. I n vivo stability of nitrate reductase. Enzyme extracts prepared from 15 h induced plants kept at 1-2 ~ in the presence ( 9 or absence (o) of 20 mN ammonium chloride
/ .~4
.>
uJ 2
t 0
i
I 4
i
I 8
Time(h)
I
i 12
Fig. 4. I n vivo re-activation of nitrate reductase. Asparagine-adapted plants were induced for 15 h, then treated with 2 mM ammonium for 3 h prior to being transferred to: 9 5 mlV[NOa; 1 mM asparagine ; A 5 mM NO 3 -t- 2 fxg/ml cycloheximide
a m m o n i u m is in m a r k e d c o n t r a s t to its effect i n vivo a n d suggests t h a t t h e inactiv a t i n g s y s t e m is d e p e n d e n t u p o n o t h e r factors in a d d i t i o n to a m m o n i u m ions. On t r a n s f e r r i n g n i t r a t e i n d u c e d p l a n t s t r e a t e d for 3 h with a m m o n i u m b a c k to n i t r a t e m e d i u m t h e r e is a r a p i d increase in n i t r a t e r e d u c t a s e a c t i v i t y (Fig. 4). T h e increase is o n l y p a r t l y d e p e n d e n t u p o n t h e presence of n i t r a t e since t r a n s f e r to a n a s p a r a g i n e m e d i u m results in some increase in e n z y m e a c t i v i t y . I n a d d i t i o n t h e increase in a c t i v i t y is o n l y p a r t i a l l y blocked b y cycloheximide. These results suggest t h a t t h e a m m o n i u m m e d i a t e d i n a c t i v a t i o n of n i t r a t e r e d u c t a s e is a t least p a r t i a l l y reversible i n vivo.
Ammonium Inactivation of Nitrate l~eduetase
0
20 Time
40
60
8
41
1 0
(minutes)
Fig. 5. In vitro re-activation of nitrate reductase. Asparagine-adapted plants were induced for 15 h, treated with 2 mM ammonium for a further 3 h then extracted. Incubated in 0.1 M phosphate buffer pH 7.5 at 1~ ([~), 5~ (o), and l0 ~ (m). Incubated in 0.1 M phosphate buffer and 5 mM NO 3 at 1~ (e)
Various t r e a t m e n t s were carried out on e n z y m e e x t r a c t e d from n i t r a t e i n d u c e d p l a n t s t r e a t e d with a m m o n i u m , in an a t t e m p t to r e - a c t i v a t e t h e e n z y m e in vitro. Simple i n c u b a t i o n of e x t r a c t s from such p l a n t s a t 1-2 ~ results in a m a r k e d increase in n i t r a t e r e d u c t a s e a c t i v i t y o v e r a p e r i o d of 60-80 rain (Fig. 5). A t t e m p e r a t u r e s a b o v e 5 ~ t h e r e is a r e d u c t i o n in t h e e x t e n t to which t h e e n z y m e can be r e - a c t i v a t e d . T h e a d d i t i o n of n i t r a t e to e x t r a c t s from t h e a m m o n i u m t r e a t e d p l a n t s a p p e a r s to give some e n h a n c e m e n t of t h e r e - a c t i v a t i o n . The e x t e n t to which t h e e n z y m e can be r e - a c t i v a t e d is variable, being l a r g e l y d e p e n d e n t u p o n t h e p e r i o d of prior i n a c t i v a t i o n . I n general however, low t e m p e r a t u r e i n c u b a t i o n in t h e presence of n i t r a t e gives a 15-25% increase in r e - a c t i v a t i o n o v e r t h a t o b t a i n e d on low t e m p e r a t u r e i n c u b a t i o n alone. This in vivo r e - a c t i v a t i o n of t h e e n z y m e in Lemna c o n t r a s t s with t h e s i t u a t i o n in Neurospora where it was n o t possible to r e - a c t i v a t e t h e e n z y m e from a m m o n i u m t r e a t e d m y c e l i u m ( S u b r a m a n i a n a n d Sorger, 1972). The results in Fig. 6, show t h e t i m e course of in rive i n a c t i v a t i o n a n d t h e level of e n z y m e r e c o v e r e d a f t e r in vitro r e - a c t i v a t i o n . W h e n t h e level of r e - a c t i v a t e d e n z y m e is c o m p a r e d with t h a t in p l a n t s t r e a t e d w i t h cycloheximide it can be seen t h a t t h e r e - a c t i v a t i o n p r o c e d u r e leads to over 70-85 % r e c o v e r y p r o v i d e d t h a t t h e p e r i o d of i n a c t i v a t i o n does n o t exceed 5 - 6 h. The i n a c t i v a t e d e n z y m e can o n l y be p a r t i a l l y r e - a c t i v a t e d a f t e r a p e r i o d of 10 h i n a c t i v a t i o n a n d a f t e r this t i m e no r e - a c t i v a t i o n was possible. This suggests t h a t t h e r e m a y be two c o m p o n e n t s in t h e i n a c t i v a t i o n process, i n i t i a l l y t h e r e is a p e r i o d of reversible i n a c t i v a t i o n followed a n irreversible loss of e n z y m e a c t i v i t y . This t w o - s t e p process of i n a c t i v a t i o n would seem to be similar to t h a t for t h e glucose m e d i a t e d i n a c t i v a t i o n of i s o c i t r a t e lyase r e p o r t e d in Chlorella ( J o h n et al., 1970). T h a t t h e i n a c t i v a t i o n can be physiologically d e m o n s t r a t e d , r a t h e r t h a n being an a r t i f a c t of t h e e x t r a c t i o n a n d a s s a y procedures, is shown b y t h e results in Fig. 7. On t r a n s f e r r i n g n i t r a t e i n d u c e d p l a n t s to a n a m m o n i u m or a n a s p a r a g i n e containing m e d i u m t h e r e is a r a p i d fall in tissue n i t r a t e levels. T h e r a t e a t which n i t r a t e is lost on t r a n s f e r to a m m o n i u m is less t h a n t h a t which occurs on t r a n s f e r
42
T . O . Orebamjo a n d G. 1~. Stewart
\ 2
l
I
I
]
T i m e -- hours after induction
Fig. 6. Time-course of in vivo inactivation a n d in vitro re-activation of nitrate reductase, Asparagine-adapted plants were induced for 15 h t h e n 2 m ~ a m m o n i u m was added. 9 Ammonium inactivated level; 9 in vitro re-activated level ( 6 0 m i n incubation with 0.1 IV[ phosphate buffer p H 7.5 ~- 5 mM NO 3 a t 1a C.). 9 Control on 5 mM nitrate; 9 5 mM nitrate 2~ g-ml cycloheximide
8
8
o
o /
j~
/ i/+,/
/
i/
l i /
2
~~ . o~~ 9 --.Xl ~ _ 1 %
l,
i
~
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~
~
I
I
~o
0
l
2
,
l
4
,
i
6
l
1
8
,
i
10
Time(h)
Fig. 7 a a n d b. Changes in tissue nitrate levels on t r e a t m e n t with a m m o n i u m or asparagine. (a) 15 h induced plants transferred to 2 mM a m m o n i u m (~), 1 mM asparagine (tt). (b) 2 mM a m m o n i u m (D), 1 mM asparagine (B) added after 15 h induction
Ammonium Inactivation of Nitrate Reductase
43
to asparagine, this is consistent with the rapid inactivation of nitrate reductase in the present of ammonium. This can be seen more clearly when ammonium or asparagine are added to plants on a nitrate medium (Fig. 7b). The rapid increase in tissue nitrate concentration on the addition of ammonium can be accounted for by the rapid ammonium inactivation of the enzyme in such plants. Discussion Various mechanisms have been described which can account for the rapid loss of enzyme activity which occurs in m a n y organisms in response to changes in environmental conditions. The simplest of these is where the normal in vivo rate of turnover is rapid so that cessation of enzyme synthesis leads to a rapid loss of activity (Lascelles, 1968). Other inactivation systems appears to involve more specific mechanisms although proteolytic degredation m a y be the basis of some of these also (Katsunuma et al., 1973). Among the other mechanisms of rapid inactivation the chemical modification of glutamine synthetase in E. coli is perhaps the best understood (see Holzer and Duntze, 1971). Several other enzymes are controlled through chemical modification, including nitrate reductase of Chlorella (Maldonado et al., 1973). I n L e m n a however the results presented here suggest that the ADP-dependent N A D H reduction of Chlorella nitrate reductase is not the basis of the ammonium inactivation. Incubation of extracts from nitrate induced plants with ADP, N A D H (and ammonium) has no effect on the stability of the L e m n a enzyme. Similarly the re-activation of the L e m n a enzyme at low temperature argues against chemical modification of the type described by Maldonado et al., being responsible for the ammonium inactivation. An alternative to chemical modification which has been reported is a mechanism of inactivation involving protein-protein interactions. For example the rapid loss of ornithine transcarbamylase on the addition of arginine to cells of Saeeharomyee, is the result of a reversible interaction between arginase and ornithine transcarbamylase (Wiame, 1971). This type of inactivation could well explain the reversible loss of nitrate reductase which occurs on the addition of ammonium to nitrate induced plants of Lemna. Recently we have reported that ammoniumadapted plants contain a protein which inhibits nitrate reductase (Stewart et al., 1974). I t is possible that this inhibitor protein could bind to nitrate reductase in the presence of high tissue ammonium levels and in this way bring about a rapid loss of activity. The precise role of ammonium in this process is uncertain although it could be that ammonium ions favour the formation of such a complex or alternatively activate the inhibitor protein and permit its interaction with nitrate reductase. I t is evident that if this protein is involved in the inactivation process it must be present in the cells prior to the addition of ammonium since neither 6-methyl purine nor cycloheximide prevent the ammonium inactivation. I t is possible however that some form of sequential induction of nitrate reduetase and the inhibitor protein m a y occur since the addition of ammonium to plants induced for three hours does not lead to an immediate loss of nitrate reductase activity. Attempts to re-activate enzyme from such plants have been unsuccessful, suggesting that at this stage of induction ammonium is exerting its effeet at the level of enzyme synthesis and also that the inactivating system is either nonfunctional or is absent.
44
T. 0. Orebamjo and G. R. Stewart
The physiological significance of the rapid, reversible i n a c t i v a t i o n of n i t r a t e reductase b r o u g h t a b o u t i n the presence of a m m o n i u m m a y be t h a t this is a sensitive a n d flexible m e a n s for regulating the i n p u t of reduced n i t r o g e n which p r e v e n t s the u n n e c e s s a r y r e d u c t i o n of n i t r a t e w h e n a m m o n i u m is present. C e r t a i n l y the rapid increase i n tissue n i t r a t e observed on the a d d i t i o n of a m m o n i u m shows t h a t this is at least a consequence if n o t the f u n c t i o n of the i n a c t i v a t i o n . A n a l t e r n a t i v e f u n c t i o n for t h e i n a c t i v a t i o n m a y be related to c o m p e t i t i o n for available r e d u c t a n t w h e n b o t h a m m o n i u m a n d n i t r a t e are present. The rapid i n a c t i v a t i o n of n i t r a t e reductase would have a sparing effect on N A D H cons u m p t i o n , m a k i n g more available for reductive a m i n a t i o n . This could p r e v e n t the excessive a c c u m u l a t i o n of p o t e n t i a l l y toxic a m m o n i u m levels i n the tissue. The difference i n response of n i t r a t e reductase to the a d d i t i o n of a m m o n i u m a n d asparagine suggests t h a t this, r a t h e r t h a n the r e g u l a t i o n of n i t r a t e assimilation per se, m a y be the f u n c t i o n of the rapid a m m o n i u m i n a c t i v a t i o n of n i t r a t e reduetase i n Lemna minor. T. O. Orebamjo was a Commonwealth Scholarship holder during the course of this work.
References Herrera, J., Paneque, A., Maldonado, J. ~., Barea, J. L., Losado, M. : Regulation by ammonia of nitrate reductase synthesis and activity in Chlamydomonas reinhardi. Biochem. biophys. Res. Commun. 48, 996-1003 (1972) Holzer, H., Duntze, W. : Metabolic regulation by chemical modification of enzymes. Ann. Rev. Bioehem. 40, 354-374 (1971) John, P. C. L., Thurston, C. F., Syrett, P. J. : Disappearance of isocitrate lyase enzyme from cells of Chlorella pyrenoidosa. Biochem. J. 119, 913-919 (1970) Katunuma, N., Katsunuma, T., Kominami, E., Suzuki, K., Hamaguchi, Y., Chiehibu, K., Kobayashi, K. : Regulation of intraeellular enzyme levels by group specific proteases in various organs. Adv. in Enzyme Regulation 11, 37 51 (1973) Laseelles, J.: The bacterial photosynthetic apparatus. Adv. Microbiol Physiology 2, 1-42 (1968) Lewis, C.M., Fincham, J. 1~. S.: Regulation of nitrate reductase in the basidiomycete Ustilago maydis. J. Bact. 103, 55-61 (1970) Losado, M., Herrera, J., Maldonado, J.M., Paneque, A.: Mechanism of nitrate reductase reversible inactivation by ammonium in Chlamydomonas. Plant Sci. Letters 1, 31-37 (1973) Losado, M., Paneque, A., Aparicio, P. J., Vega, J. M., Cardenas, J., Herrera, J. : Inactivation and repression by ammonium of the nitrate reducing system in Chlorella. Biochem. biophys. Res. Commun. 88, 1009-1016 (1970) Orebamjo, T.O., Stewart, G.R.: Some characteristics of nitrate reductase induction in Lemna minor L. Planta (Berl.) 117, 1-10 (1974a) Orebamjo, T. 0., Stewart, G. R. : Ammonium repression of nitrate reductase in Lemna minor L. Planta (Berl.), in press Rigano, C., Aliotaa, G., Violante, U. : Reversible inactivation by ammonia of assimilatory nitrate reductase in Cyanidium caldarium. Arch. Mikrobiol. 99, 81-90 (1974) Stewart, G. R. : Regulation of nitrite reductase level in Lemna minor L. J. exp. Bot. 23, 171-183 (1972a) Stewart, G. R. : End product repression of nitrate reductase in Lemna minor L. Symp. Biol. Hung. 13, 127-135 (1972b) Stewart, G.R., Lee, J.A., Orebamjo, T. O., Havill, D.C.: Ecological aspects of nitrogen metabolism. In Mechanisms of regulation of plant growth. Eds. Bieleski, R. L., Ferguson, A. R., Creswell, M. M. Bull. roy. Soc. New Zealand 12, Wellington, ~. Z., p. 41-47 (1974) Waime, J. M. : The regulation of arginine metabolism in Saccharomyces cerevisiae, exclusion mechanisms. Curr. Top. in Cellular Regulation 4, 1-38 (1971)
Ammonium Inactivation of Nitrate Reductase in Lemna minor L. T. O. Orebamjo* and G. R. Stewart** Department of Botany, The University, Manchester M13 9PL, U. K. Received 16 October; accepted 14 November 1974 Summary. The addition of ammonium to nitrate induced plants of Lemna minor L. brings about a rapid loss in extractable nitrate rcductase activity. This inactivation is reversible both in vivo and in vitro. Inhibitors of RNA and protein synthesis do not protect nitrate reductase against ammonium inactivation. It is suggested that factors, in addition to ammonium ions, are components of the inactivating system. Inactivation may involve some form of protein-protein interaction. The physiological significance of rapid ammonium inactivation of nitrate reductase is discussed. Introduction
Previous studies of the regulation of nitrate reductase in L e m n a minor have established that its formation is regulated through the interaction of positive and negative control elements (Orebamjo and Stewart, 1974a, b). I n this dual control system, nitrate acts as a positive modulator, while ammonium (indirectly) acts as a negative modulator, of enzyme synthesis. The kinetics of induction and repression are consistent with the transcriptional control of enzyme synthesis (Orebamjo and Stewart, 1974b). In addition to inhibiting the induced formation of nitrate reductase ammonium brings about a very rapid loss of nitrate reductase activity when added to fully induced plants (Orebamjo and Stewart, 1974b). The rate of enzyme loss under these conditions is faster than can be accounted for by an immediate cessation of enzyme synthesis and the subsequent reduction in activity through growth. A similar response to the addition of ammonium has been reported in several species of an algae (Losado et al., 1970, 1973; l~igano et al., 1974) and some 9 species of fungi (Lewis and Fincham, 1970; Subramanian and Sorger, 1972). I n both Chlorella and Chlamydomonas ammonium inactivation is reversible and dependent upon photosynthesis (Losado etal., 1973; Herrera etal., 1972). I n contrast ammonium inactivation in C y a n i d i u m although reversible, is not dependent upon photosynthesis (Rigano et al., 1974). The mechanism of inactivation in Chlorella appears to involve chemical modification of the enzyme, since in vitro the active form can be reversibly converted into its inactive form in the presence of NADH and ADP. In the fungus Ustilago inhibitors oI RNA and protein synthesis block the ammonium inactivation of the enzyme (Lewis and Fineham, 1970), suggesting' that here the rapid loss of nitrate reductase may be dependent upon continued protein synthesis. * Present address: School of Biological Sciences, The University of Lagos, Lagos, Nigeria. ** Address for reprint requests.
38
T.O. Orebamjo and G. R. Stewart
I n this paper we report the characteristics of the ammonium mediated inactivation of nitrate reductase in the higher plant L e m n a minor. The inactivation is reversible both in vivo and i n vitro. Inhibitors of I~NA and protein synthesis do not prevent the ammonium inactivation. Possible mechanisms of inactivation are discussed. Materials and Methods The clone of Lemna minor used in the present experiments was maintained and grown as described previously (Stewart, 1972a, b). Nitrate reductase was extracted and its activity determined by the procedures used previously (Stewart, 1972a). Enzyme extracts were assayed within 1-2 minutes following extraction unless subjected to further treatment (see Results). Tissue nitrate and ammonium were extracted and determined as before (Stewart, 1972b; Orebamjo and Stewart, 1974a). In all of the experiments reported here a concentration of 5 mM nitrate was used to induce the formation of nitrate reductase. Enzyme activity is expressed as ~zmolNO2 produced/hour/gfw. Results
The addition of ammonium to fully induced plants brings about a rapid loss of extractable nitrate reductase activity (Fig. 1). T h e rate at which enzyme activity is lost in ammonium treated plants is considerably faster than that measured when either 6-methyl purine or cycloheximide are used to halt further synthesis of the enzyme. The results also show that when asparagine is added to fully induced plants the rate of enzyme loss is less than that measured in cycloheximide treated plants. This suggests that there m a y be some continued synthesis of the enzyme in the presence of asparagine. The halflife of nitrate rednctase in ammonium treated plants is 3.6 h (as determined from semi-log plots of enzyme decay), compared with those of 9 and l l h in cycloheximide and 6-methyl purine treated plants. I t is evident however, that there are two phases in the decay curve for ammonium treated plants, over the first 4 h there is a phase of rapid enzyme loss (which gives the half-life of 3.6 h), subsequently there is a marked decrease in the rate at which enzyme activity is lost. The presence of protein synthesis inhibitors such as chloramphenical and cycloheximide have no measurable effect on the initial phase of the ammonium inactivation (Fig. 2). Furthermore pre-treating plants with cycloheximide tot 1 hour before the addition of ammonium has no effect on the rapid inactivation. These results suggest that the inactivating system is dependent upon neither cytoplasmic nor chloroplast protein synthesis. These results contrast with those obtained for Ustilago where both cycloheximide and actinomycin D completely prevent the ammonium inactivation of nitrate reductase (Lewis and Fincham, 1970). The loss of nitrate reductase following the addition of ammonium is not simply the result of ammonium inhibition since the addition of 20 mM ammonium chloride has no effect on the activity of the enzyme extracted from nitrate induced plants. Surprisingly though ammonium actually stabilizes enzyme extracted from such plants (Fig. 3). This protection of the extracted enzyme by
Ammonium Inactivation of Nitrate Reductase
39
0.8
~,,0.G ~
i
._~ ~
$
. 0.2
0 T 0
1
i 4
I
I 8
Time(h)
I
I 12
Fig. 1. A m m o n i u m inactivation of nitrate reduction in vivo. Asparagine-adapted plants were induced :for 15 hours before the addition of: 9 2 mM ammonium, 9 150 iJ.g/ml 6-methyl purine; 9 1 mM asparagine; 9 2 ~zg-ml eyeloheximide
.=
U.i2
D
Time(h)
:Fig. 2. Effect of inhibitors of protein synthesis on the a m m o n i u m inactivation of nitrate reductase. Asparagine-adapted plants were induced for 15 h before the addition of: 9 $ mM a m m o n i u m ; 9 2 mM a m m o n i u m + 2 ~g-mt eyeloheximide; 9 2 mM a m m o n i u m + 500 ~zg-ml D-threoehloramphenicol; [] 2 ~zg-ml cyeloheximide added a t 14 h induction, followed by 2 mM ammonium at 15 h
40
T.O. Orebamjo and G. R. Stewart
,,~4
E ~2
0
4
8
Time (minutes)
120
Fig. 3. I n vivo stability of nitrate reductase. Enzyme extracts prepared from 15 h induced plants kept at 1-2 ~ in the presence ( 9 or absence (o) of 20 mN ammonium chloride
/ .~4
.>
uJ 2
t 0
i
I 4
i
I 8
Time(h)
I
i 12
Fig. 4. I n vivo re-activation of nitrate reductase. Asparagine-adapted plants were induced for 15 h, then treated with 2 mM ammonium for 3 h prior to being transferred to: 9 5 mlV[NOa; 1 mM asparagine ; A 5 mM NO 3 -t- 2 fxg/ml cycloheximide
a m m o n i u m is in m a r k e d c o n t r a s t to its effect i n vivo a n d suggests t h a t t h e inactiv a t i n g s y s t e m is d e p e n d e n t u p o n o t h e r factors in a d d i t i o n to a m m o n i u m ions. On t r a n s f e r r i n g n i t r a t e i n d u c e d p l a n t s t r e a t e d for 3 h with a m m o n i u m b a c k to n i t r a t e m e d i u m t h e r e is a r a p i d increase in n i t r a t e r e d u c t a s e a c t i v i t y (Fig. 4). T h e increase is o n l y p a r t l y d e p e n d e n t u p o n t h e presence of n i t r a t e since t r a n s f e r to a n a s p a r a g i n e m e d i u m results in some increase in e n z y m e a c t i v i t y . I n a d d i t i o n t h e increase in a c t i v i t y is o n l y p a r t i a l l y blocked b y cycloheximide. These results suggest t h a t t h e a m m o n i u m m e d i a t e d i n a c t i v a t i o n of n i t r a t e r e d u c t a s e is a t least p a r t i a l l y reversible i n vivo.
Ammonium Inactivation of Nitrate l~eduetase
0
20 Time
40
60
8
41
1 0
(minutes)
Fig. 5. In vitro re-activation of nitrate reductase. Asparagine-adapted plants were induced for 15 h, treated with 2 mM ammonium for a further 3 h then extracted. Incubated in 0.1 M phosphate buffer pH 7.5 at 1~ ([~), 5~ (o), and l0 ~ (m). Incubated in 0.1 M phosphate buffer and 5 mM NO 3 at 1~ (e)
Various t r e a t m e n t s were carried out on e n z y m e e x t r a c t e d from n i t r a t e i n d u c e d p l a n t s t r e a t e d with a m m o n i u m , in an a t t e m p t to r e - a c t i v a t e t h e e n z y m e in vitro. Simple i n c u b a t i o n of e x t r a c t s from such p l a n t s a t 1-2 ~ results in a m a r k e d increase in n i t r a t e r e d u c t a s e a c t i v i t y o v e r a p e r i o d of 60-80 rain (Fig. 5). A t t e m p e r a t u r e s a b o v e 5 ~ t h e r e is a r e d u c t i o n in t h e e x t e n t to which t h e e n z y m e can be r e - a c t i v a t e d . T h e a d d i t i o n of n i t r a t e to e x t r a c t s from t h e a m m o n i u m t r e a t e d p l a n t s a p p e a r s to give some e n h a n c e m e n t of t h e r e - a c t i v a t i o n . The e x t e n t to which t h e e n z y m e can be r e - a c t i v a t e d is variable, being l a r g e l y d e p e n d e n t u p o n t h e p e r i o d of prior i n a c t i v a t i o n . I n general however, low t e m p e r a t u r e i n c u b a t i o n in t h e presence of n i t r a t e gives a 15-25% increase in r e - a c t i v a t i o n o v e r t h a t o b t a i n e d on low t e m p e r a t u r e i n c u b a t i o n alone. This in vivo r e - a c t i v a t i o n of t h e e n z y m e in Lemna c o n t r a s t s with t h e s i t u a t i o n in Neurospora where it was n o t possible to r e - a c t i v a t e t h e e n z y m e from a m m o n i u m t r e a t e d m y c e l i u m ( S u b r a m a n i a n a n d Sorger, 1972). The results in Fig. 6, show t h e t i m e course of in rive i n a c t i v a t i o n a n d t h e level of e n z y m e r e c o v e r e d a f t e r in vitro r e - a c t i v a t i o n . W h e n t h e level of r e - a c t i v a t e d e n z y m e is c o m p a r e d with t h a t in p l a n t s t r e a t e d w i t h cycloheximide it can be seen t h a t t h e r e - a c t i v a t i o n p r o c e d u r e leads to over 70-85 % r e c o v e r y p r o v i d e d t h a t t h e p e r i o d of i n a c t i v a t i o n does n o t exceed 5 - 6 h. The i n a c t i v a t e d e n z y m e can o n l y be p a r t i a l l y r e - a c t i v a t e d a f t e r a p e r i o d of 10 h i n a c t i v a t i o n a n d a f t e r this t i m e no r e - a c t i v a t i o n was possible. This suggests t h a t t h e r e m a y be two c o m p o n e n t s in t h e i n a c t i v a t i o n process, i n i t i a l l y t h e r e is a p e r i o d of reversible i n a c t i v a t i o n followed a n irreversible loss of e n z y m e a c t i v i t y . This t w o - s t e p process of i n a c t i v a t i o n would seem to be similar to t h a t for t h e glucose m e d i a t e d i n a c t i v a t i o n of i s o c i t r a t e lyase r e p o r t e d in Chlorella ( J o h n et al., 1970). T h a t t h e i n a c t i v a t i o n can be physiologically d e m o n s t r a t e d , r a t h e r t h a n being an a r t i f a c t of t h e e x t r a c t i o n a n d a s s a y procedures, is shown b y t h e results in Fig. 7. On t r a n s f e r r i n g n i t r a t e i n d u c e d p l a n t s to a n a m m o n i u m or a n a s p a r a g i n e containing m e d i u m t h e r e is a r a p i d fall in tissue n i t r a t e levels. T h e r a t e a t which n i t r a t e is lost on t r a n s f e r to a m m o n i u m is less t h a n t h a t which occurs on t r a n s f e r
42
T . O . Orebamjo a n d G. 1~. Stewart
\ 2
l
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I
]
T i m e -- hours after induction
Fig. 6. Time-course of in vivo inactivation a n d in vitro re-activation of nitrate reductase, Asparagine-adapted plants were induced for 15 h t h e n 2 m ~ a m m o n i u m was added. 9 Ammonium inactivated level; 9 in vitro re-activated level ( 6 0 m i n incubation with 0.1 IV[ phosphate buffer p H 7.5 ~- 5 mM NO 3 a t 1a C.). 9 Control on 5 mM nitrate; 9 5 mM nitrate 2~ g-ml cycloheximide
8
8
o
o /
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/
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l,
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~o
0
l
2
,
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4
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8
,
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Time(h)
Fig. 7 a a n d b. Changes in tissue nitrate levels on t r e a t m e n t with a m m o n i u m or asparagine. (a) 15 h induced plants transferred to 2 mM a m m o n i u m (~), 1 mM asparagine (tt). (b) 2 mM a m m o n i u m (D), 1 mM asparagine (B) added after 15 h induction
Ammonium Inactivation of Nitrate Reductase
43
to asparagine, this is consistent with the rapid inactivation of nitrate reductase in the present of ammonium. This can be seen more clearly when ammonium or asparagine are added to plants on a nitrate medium (Fig. 7b). The rapid increase in tissue nitrate concentration on the addition of ammonium can be accounted for by the rapid ammonium inactivation of the enzyme in such plants. Discussion Various mechanisms have been described which can account for the rapid loss of enzyme activity which occurs in m a n y organisms in response to changes in environmental conditions. The simplest of these is where the normal in vivo rate of turnover is rapid so that cessation of enzyme synthesis leads to a rapid loss of activity (Lascelles, 1968). Other inactivation systems appears to involve more specific mechanisms although proteolytic degredation m a y be the basis of some of these also (Katsunuma et al., 1973). Among the other mechanisms of rapid inactivation the chemical modification of glutamine synthetase in E. coli is perhaps the best understood (see Holzer and Duntze, 1971). Several other enzymes are controlled through chemical modification, including nitrate reductase of Chlorella (Maldonado et al., 1973). I n L e m n a however the results presented here suggest that the ADP-dependent N A D H reduction of Chlorella nitrate reductase is not the basis of the ammonium inactivation. Incubation of extracts from nitrate induced plants with ADP, N A D H (and ammonium) has no effect on the stability of the L e m n a enzyme. Similarly the re-activation of the L e m n a enzyme at low temperature argues against chemical modification of the type described by Maldonado et al., being responsible for the ammonium inactivation. An alternative to chemical modification which has been reported is a mechanism of inactivation involving protein-protein interactions. For example the rapid loss of ornithine transcarbamylase on the addition of arginine to cells of Saeeharomyee, is the result of a reversible interaction between arginase and ornithine transcarbamylase (Wiame, 1971). This type of inactivation could well explain the reversible loss of nitrate reductase which occurs on the addition of ammonium to nitrate induced plants of Lemna. Recently we have reported that ammoniumadapted plants contain a protein which inhibits nitrate reductase (Stewart et al., 1974). I t is possible that this inhibitor protein could bind to nitrate reductase in the presence of high tissue ammonium levels and in this way bring about a rapid loss of activity. The precise role of ammonium in this process is uncertain although it could be that ammonium ions favour the formation of such a complex or alternatively activate the inhibitor protein and permit its interaction with nitrate reductase. I t is evident that if this protein is involved in the inactivation process it must be present in the cells prior to the addition of ammonium since neither 6-methyl purine nor cycloheximide prevent the ammonium inactivation. I t is possible however that some form of sequential induction of nitrate reduetase and the inhibitor protein m a y occur since the addition of ammonium to plants induced for three hours does not lead to an immediate loss of nitrate reductase activity. Attempts to re-activate enzyme from such plants have been unsuccessful, suggesting that at this stage of induction ammonium is exerting its effeet at the level of enzyme synthesis and also that the inactivating system is either nonfunctional or is absent.
44
T. 0. Orebamjo and G. R. Stewart
The physiological significance of the rapid, reversible i n a c t i v a t i o n of n i t r a t e reductase b r o u g h t a b o u t i n the presence of a m m o n i u m m a y be t h a t this is a sensitive a n d flexible m e a n s for regulating the i n p u t of reduced n i t r o g e n which p r e v e n t s the u n n e c e s s a r y r e d u c t i o n of n i t r a t e w h e n a m m o n i u m is present. C e r t a i n l y the rapid increase i n tissue n i t r a t e observed on the a d d i t i o n of a m m o n i u m shows t h a t this is at least a consequence if n o t the f u n c t i o n of the i n a c t i v a t i o n . A n a l t e r n a t i v e f u n c t i o n for t h e i n a c t i v a t i o n m a y be related to c o m p e t i t i o n for available r e d u c t a n t w h e n b o t h a m m o n i u m a n d n i t r a t e are present. The rapid i n a c t i v a t i o n of n i t r a t e reductase would have a sparing effect on N A D H cons u m p t i o n , m a k i n g more available for reductive a m i n a t i o n . This could p r e v e n t the excessive a c c u m u l a t i o n of p o t e n t i a l l y toxic a m m o n i u m levels i n the tissue. The difference i n response of n i t r a t e reductase to the a d d i t i o n of a m m o n i u m a n d asparagine suggests t h a t this, r a t h e r t h a n the r e g u l a t i o n of n i t r a t e assimilation per se, m a y be the f u n c t i o n of the rapid a m m o n i u m i n a c t i v a t i o n of n i t r a t e reduetase i n Lemna minor. T. O. Orebamjo was a Commonwealth Scholarship holder during the course of this work.
References Herrera, J., Paneque, A., Maldonado, J. ~., Barea, J. L., Losado, M. : Regulation by ammonia of nitrate reductase synthesis and activity in Chlamydomonas reinhardi. Biochem. biophys. Res. Commun. 48, 996-1003 (1972) Holzer, H., Duntze, W. : Metabolic regulation by chemical modification of enzymes. Ann. Rev. Bioehem. 40, 354-374 (1971) John, P. C. L., Thurston, C. F., Syrett, P. J. : Disappearance of isocitrate lyase enzyme from cells of Chlorella pyrenoidosa. Biochem. J. 119, 913-919 (1970) Katunuma, N., Katsunuma, T., Kominami, E., Suzuki, K., Hamaguchi, Y., Chiehibu, K., Kobayashi, K. : Regulation of intraeellular enzyme levels by group specific proteases in various organs. Adv. in Enzyme Regulation 11, 37 51 (1973) Laseelles, J.: The bacterial photosynthetic apparatus. Adv. Microbiol Physiology 2, 1-42 (1968) Lewis, C.M., Fincham, J. 1~. S.: Regulation of nitrate reductase in the basidiomycete Ustilago maydis. J. Bact. 103, 55-61 (1970) Losado, M., Herrera, J., Maldonado, J.M., Paneque, A.: Mechanism of nitrate reductase reversible inactivation by ammonium in Chlamydomonas. Plant Sci. Letters 1, 31-37 (1973) Losado, M., Paneque, A., Aparicio, P. J., Vega, J. M., Cardenas, J., Herrera, J. : Inactivation and repression by ammonium of the nitrate reducing system in Chlorella. Biochem. biophys. Res. Commun. 88, 1009-1016 (1970) Orebamjo, T.O., Stewart, G.R.: Some characteristics of nitrate reductase induction in Lemna minor L. Planta (Berl.) 117, 1-10 (1974a) Orebamjo, T. 0., Stewart, G. R. : Ammonium repression of nitrate reductase in Lemna minor L. Planta (Berl.), in press Rigano, C., Aliotaa, G., Violante, U. : Reversible inactivation by ammonia of assimilatory nitrate reductase in Cyanidium caldarium. Arch. Mikrobiol. 99, 81-90 (1974) Stewart, G. R. : Regulation of nitrite reductase level in Lemna minor L. J. exp. Bot. 23, 171-183 (1972a) Stewart, G. R. : End product repression of nitrate reductase in Lemna minor L. Symp. Biol. Hung. 13, 127-135 (1972b) Stewart, G.R., Lee, J.A., Orebamjo, T. O., Havill, D.C.: Ecological aspects of nitrogen metabolism. In Mechanisms of regulation of plant growth. Eds. Bieleski, R. L., Ferguson, A. R., Creswell, M. M. Bull. roy. Soc. New Zealand 12, Wellington, ~. Z., p. 41-47 (1974) Waime, J. M. : The regulation of arginine metabolism in Saccharomyces cerevisiae, exclusion mechanisms. Curr. Top. in Cellular Regulation 4, 1-38 (1971)
