Regulation of nitrate reductase activity by ammonium in wheat

Plant Science Letters, 20 (1981) 305--313

305

© Elsevier/North-Holland Sclent15c Pubhshers Ltd

R E G U L A T I O N O F N I T R A T E R E D U C T A S E ACTIVITY BY AMMONIUM IN WHEAT NEERAJDATTA, L.V M RAO, SIPRAGUHA-MUKHERJEE*andSUDHIRK SOPORY Plant Research Laboratory, School of L~fe Sciences, Jawaharlal Nehru Unwerslty, New Delhz-110067 (Indm)

(Received April 25th, 1980) (Revmlon received September 3rd, 1980) (Accepted September 25th, 1980) SUMMARY

A m m o n i u m mtrate-mduced nitrate reductase activity was more than potassium nitrate induced activity m excised s h o o t tips of wheat. This effect was more p r o n o u n c e d m the dark than in light. There was no effect of NH4H2PO4 alone, however, m the presence of nitrate, it enhanced nitrate reductase activity. When shoot tips were transferred to NH4H2PO4 alone after 18 h of KNO3 treatment, the e n z y m e actlwty increased shghtly and then stabilized. This effect was n o t noticed with KH2PO4. The mtrate reductase stabilization b y a m m o n i u m was s t o p p e d b y the mhlbltors of R N A and protein synthesis and tungstate, except a c t m o m y c m D which did n o t show any inhibitory effect. The enzyme actlwty was stablhzed by a m m o n i u m m vlvo b u t n o t m vitro.

INTRODUCTION Synthems of nitrate reductase, a substrate inducible enzyme is effected b y m a n y factors like temperature, hght, hormones, carbon dioxide, a m m o n i u m nitrate and the position of the tissue m the plant [1]. A m m o m u m , the end product, represses nitrate reductase actlwty m some systems [2,3], whereas m some others it stimulates [4--6]. Stewart [3] and Stewart and Rhodes [7] reported that a m m o n m m represses nitrate reductase actwlty m physiologically simple plants, hke algae and fungi and also m L e m n a . Smith and T h o m p s o n [8] have shown that normally a m m o n i u m increased nitrate reductase actlwty m barley roots, b u t when barley roots were pretreated with a m m o n i u m and then exposed to nitrate and amm o m u m , it repressed the e n z y m e actlwty. Boutard [9] showed that amm o n m m inhibited nitrate reductase adtlvity m barley roots and increased it *To whom correspondence should be sent.

306

m barley shoots, when etmlated seedlings were transferred from H 2 0 to KNO3. He suggested that the mtrate reductase was different m roots and shoots. In soybean cells,Oaks [10] observed that a m m o n m m cltratemhlblted mtrate-induced nitrate reductase actwlty. Recently, it has been reported from thin laboratory that a m m o n m m enhances nltrate reductase actiwty m pea buds [11] and m wheat leaves [12]. In the former system a m m o n m m was found to enhance the enzyme actlwty by stabihzmg the enzyme [13]. In the present paper a more detailed work LS presented to show that m wheat also the mtrate reductase actlwty m enhanced by stabillzmg the enzyme. METHODS Wheat seeds (Trztzcum aestwum vat. HD-2204) were o b t a m e d from the Indian Agricultural Research Institute, New DelhL E x p e n m e n t s were performed w]th excised etlolated shoots from 7-day-old seedlings of wheat. For e x p e n m e n t a l treatments, shoot tips (4--5 cm in length from the apex) were floated m different test solutions and m c u b a t e d at 25°C under white hght (1200 ~W/cm 2 ), unless otherwise stated. Sterile conditmns were mamtamed t h r o u g h o u t the m c u b a t m n period. For enzyme extractmn, shoot tips (250 mg) were removed from the incubation m e d m m , washed with dlstiUed water, dried and then homogemzed m extractmn buffer comprmlng of 50 mM phosphate buffer (pH 7.4) 1 mM EDTA and 1 mM cysteme, at 4°C. The homogenate was centrifuged at 15 000 rev./min for 15 mm. The supernatant was assayed for the enzyme activity according to the modified procedure of Hageman and Hucklesby [ 14 ] as reported earher [ 11 ]. Protein was assayed following the m e t h o d of L o w r y et al. [15]. A c c u m u l a t m n of nitrate in the tissue was estimated by the m e t h o d of Wooley et al. [16]. RESULTS Shoot tips of wheat were treated with KNO3 and NH4NO3 for 18 h in hght and dark. It was observed (Table I) that nitrate reductase activity was more m light than m dark. A m m o m u m enhanced the enzyme actlwty by 90% in hght and 142% m dark as compared to KNO3. However, more mtrate accumulated m shoot tips m KNO3 than in NH4NO3. The klnetms of reduction of nitrate reductase in KNO3 and NH4NO3, both at a concentration of 100 mM, was studied, in dark and in hght. As seen in Figs. l a and l b , in b o t h the cases a lag phase of 2 h was observed In dark, the m a x i m u m nitrate reductase activity was obtained around 18 h in KNO3 followed by a decline whereas m NH4NO3 the enzyme activity mcreased steadily till 24 h. In hght, b o t h m KNO3 and NH4NO3 the activity started dechnlng after 18 h, the fall m actwlty in KNO3 was faster than that in NH4NO3. To check, if a m m o n m m was responsible for the stimulation of nitrate

307 TABLE I E F F E C T OF LIGHT AND AMMONIUM ON N I T R A T E R E D U C T A S E ACTIVITY AND N I T R A T E UPTAKE BY THE TISSUE

Shoot tips of 7 - d a y ~ l d etiolated seedlings were incubated m 100 m_M KNO 3 and 100 mM NH4NO r The nitrate reductase activity and the nitrate uptake was assayed after 18 h The specific activity is expressed as nmol NO 2 15 rmn "~ • mg protein -j The nitrate uptake was expressed as #tool NOs g fresh w t - ' The values are mean of 3 different experiments. Treatments

Nitrate reductase activity Dark

KNO 3 NH4NO 3

Tissue mtrate Ltght

Spec act

Rel act %

Spec act

Rel act %

25 50 61 73

100 00 242 06

116,66 221 70

100 190

Dark

Light

38 40 34 80

174 00 63 00

reductase activity, s h o o t tips were incubated m a mLxture of KNO3 and NH4H:PO4 m which the concentration of KNO3 was kept constant at 100 mM and that of NH4H2PO4 was varied from 10 mM to 100 mM. A m m o n m m enhanced mtrate reductase activity b y a b o u t 39% at 10 mM and 25 mM NH4H2PO4 concentration (Table II). To s t u d y the effect of a m m o n m m on mtrate reductase activity m the absence of an external supply of nitrate, shoot tips, incubated m KNO3 for 18 h, were transferred to 10 mM NH4H2PO4 alone. After the transfer, mtrate reductase activity increased slightly till 4--6 h and then mamtmned a higher level m NH4H2PO4 than m shoot tips whmh were transferred to fresh KNO3. For nearly 24 h there was no dechne m activity m NH4H2PO4 (Fig. 2). In ten different experiments conducted, a similar pattern was observed. To ascertain whether the increase and specmlly the stablhzatmn of mtrate reductase m NH4H2PO4 was due to a m m o n m m alone or also due to phosphate runs, after 18 h of KNO3 pretreatment, one set of shoot tips were transferred to NH4H2PO4 (10 mM) second set to KH2PO4 (10 mM) third set to dlsttlled water and fourth set to fresh KNO3. As shown (Fig. 3) mtrate reductase activity increased and stabilized only m the shoot tips transferred to NH4H2PO4 To fred o u t if the a m m o n m m m e d m t e d mcrease and stabfllzatmn revolved transcnptmnal and translational processes, shoot tips were treated with a c t m o m y c m D (20 #g/ml), cyclohexlmlde (20 ug/ml), 6-methyl purme (1 mM) and sodium tungstate (1 mM) together with NH4H2PO4, after 18 h of p r e m c u b a t m n m KNO3. A c t m o m y c m D had no effect whereas m cyclohexlmlde the activity decreased sharply, after a lag o f 4 h (Fig. 3a). In 6methyl purine and tungstate also the a c t l w t y decreased sharply after a lag of 6 h and 4 h respectively (Ftg. 3b).

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(~ , , c) and 1 0 0 mM NH4NO 3 (Q - ) and t h e activity o f nitrate reductase w a s recorded in dark (b) Kinetics o f mtrate reductase reduction in hght S h o o t tips o f 7-day-old e t l o l a t e d seedlings w e r e k e p t m 1 0 0 m M K N O 3 ( " :~) and 1 0 0 m M NH4NO 3

Fig 1 (a) Kmetlcs of nitrate reductase mductlon in dark Shoot tips of 7-day-old etloiated seedlings were kept in 100 m M

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309 TABLE II EFFECT OF D I F F E R E N T CONCENTRATIONS OF NH4H2PO 4 IN COMBINATION WITH KNO 3 ON NITRATE REDUCTASE ACTIVITY Shoot tips from 7-day-old etiolated seedlings were incubated m a mixture of 100 mM KNO 3 and varying c o n c e n t r a t m n of NH4H~PO4, the concentration varying from 10 mM to 100 mM The speclfm actlwty is expressed as nmol NO 2 15 m m -~ • mg protein -~ The values are mean of 3 experiments Treatments KNO 3 100 mM + NH4H2PO 4 + NH4H2PO 4 + NH4H2PO 4 + NH4H2PO 4

10 25 50 100

Spec act

% Rel act

139 194 193 137 160

100 139 138 99 115

mM mM mM mM

00 52 16 98 16

00 94 97 16 26

To confirm that ammonmm stabilizes the mtrate reductase actwity, the m VlVO d e c a y o f t h e e n z y m e w a s s t u d m d i n K N O 3 a n d m N H 4 H 2 P O 4 . S h o o t tips were incubated for 18 h m KNO3 and then transferred to fresh KNO3 and NH4H2PO4, a l o n g w i t h t u n g s t a t e . I n K N O 3 t h e tl/2 w a s f o u n d t o b e 8 . 5 h , w h e r e a s i n NH4H2PO4, it w a s 2 0 h ( F i g . 4).

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F]g 2 Effect of a m m o n m m ion, phosphate Ion and water on mtrate reductase activity Shoot tips from 7-day-old etlolated seedlings, incubated m 100 mM KNO 3 for 18 h, were transferred to 10 mM NH4H2PO 4 (~ ~) 10 mM KH2PO 4 ( A) dmtdled water (* - - - ~ ) and fresh 100 mM KNO~ (o o), and the kinetms of mtrate r e d u c t a ~ activity was recorded

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Fig 3 (a) Effect of a c t m o m y c m D and cyclohexlmlde on the kinetics o f nitrate reductase mductzon with a m m o n m m alone Shoot tips from 7-day-old etlolated seedlings, incubated m 100 mM KNO 3 for 18 h were transferred to 10 m_M NH4H~PO , (~ ~), NH4H2PO 4 + 20 #g/ml a c t m o m y c i n D (A - ) , 10 mM NH4H2PO , + 20 #g/ml cyclohexlmlde (• •), and fresh KNO 3 (c o ). All treatments were given m hght (b) Effect of 6-methyl purme and tungstate on the kinetics of mtrate reductase induction with a m m o n m m alone S h o o t tips from 7-day-old etlolated seedlings, incubated m 100 mM KNO3 for 18 h, were transferred to 10 mM NH4H~PO 4 (~ ~), 10 mM NH4H~PO4 + 1 mM 6-methyl purlne (A A), 10 mM NH4H2PO 4 + 1 mM Na2WO 4 (* e), and fresh 100 mM KNO 3 (o o) All treatments were given m hght

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311 DISCUSSION

The stlmulatory effect of a m m o n m m on m t r a t e reductase activity m pea and wheat was reported earher [11,12]. In pea, we f o u n d t h a t mtrate reductase a c t l w t y stabihzed m the presence o f a m m o m u m [13] but no such effect could apparently be observed m wheat [12]. The present work shows t h a t even m wheat m variety HD2204, nitrate reductase actlwty IS enhanced by a m m o m u m by mcreasmg its t~/2. The difference between the earlier work [12] and the present one could be due to the use of a different variety of wheat and also the use of NH4H2PO4 instead of NH4NO3. In NH4NO3, as was seen m the present work, tl/: was 6 h less than m NH4H2PO4. As shown m Table I, a m m o m u m increased mtrate reductase activity as compared to KNO3, b o t h m hght and in dark. In KNO3, m both hght and dark, the e n z y m e activity increased up to 18 h after which it declined. In NH4NO3, m dark the activity continued to increase up to 24 h (Fig. la), m hght, however, a gradual decrease was n o t e d after 18 h (Fig. l b ) The sharp dechne m the actlwty m KNO3 as compared to the gradual decline m NH4NO3 could be explained by assuming t h a t although there is no difference m the rate of synthesis of the e n z y m e m both the cases, there m a y be a decrease m the decay of the e n z y m e m NH4NO3 as compared to t h a t m KNO3 This concept concurs vclth an earher observation of Beevers and Hageman [17] who f o u n d mtrate reductase to be an unstable enzyme, hawng a short t1/2. In several plants, it has been shown t h a t mtrate reductase has a varying and a relatively high turn over rate m wvo [18--22]. Various mechamsms have been suggested to explain this rapid loss of actlwty. It could be due to specific degradation of the e n z y m e [23--27] or because of a group of enzymes which could reversibly reactivate the e n z y m e [ 28,29] Bechet and Wmme [30] reported the presence of an inhibitory protein which reversibly reactivated the e n z y m e by binding to the enzyme from pea shoots and roots which specifically reactivated the mtrate reduced N A D H ~ y t o c h r o m e c reductase c o m p o n e n t of mtrate reductase. Wallace [31,32] reported t h a t m maize roots the mtrate reductase is Lrreverslbly reactivated by an reactivating protein which brads to the e n z y m e and forms an undlssocmble complex. In the present study, a m m o m u m e n h a n c e m e n t was not due to increase m uptake of mtrate. On the contrary, t~ssue mtrate was more m KNO3 than m 1NH4NOs incubated shoot tips This is m c o n f o r m i t y with earher studies [33]. As reported earher m another variety of wheat [12], the effect of amm o m u m could n o t be mimicked by amino acids m the present variety of wheat also All amino acids were tested at different concentrations but none could simulate a m m o m u m effect to the same degree (data n o t given). When shoot tips were transferred after 18 h of incubation m 100 mM KNO3 to 10 mM NH4H2PO4, the actlwty increased initially for 4 h, and then ~t stab~hzed. But interestingly enough, when shoot t~ps were treated w~th NH4H:PO~ from the beginning, no mtrate reductase act~wty is reduced. The

312

t~/2 m vlvo, on the basra of rate o f loss o f activity was f o u n d to be 8.5 h m KNOs and 20 h in NH4H:PO4. A m m o n m m did n o t increase the t~/2 m vitro (data n o t given). In the present s t u d y the stability o f the e n z y m e decreased m cycl ohem mlde (Fig. 3a) and m 6- m et hyl purine (Fig. 3b). 6-m et hyl p u r m e inhibited RNA synthesm by 75% and c y c l o h e x l m i d e inhibited prot ei n synthesm by 50% (data n o t given). These e x p e n m e n t s suggest t hat t he stabilization o f m t r a t e reductase activity by a m m o n m m m p r o b a b l y n o t a direct effect b u t is m e d i a t e d via a n o t h e r protein. A n o t h e r interesting observatmn was t h a t the mltlal increase and stablhty o f the e n z y m e m NH4H2PO4 was blocked by tungstate. Smce tungstate specifically replaces m o l y b d e n u m during e n z y m e synthesm, thin e x p e r i m e n t showed t h a t even after transfer o f s h o o t tips m non-inducing conditions, m t r a t e reductase synthesis p r o c e e d e d . Thin could n o t be due to endogenous m t r ate, since m water and KH2PO4 t he nitrate reductase activity decreased sharply. This strongly suggests t h a t t he m R N A for nitrate reductase m ay be stable, as has been r e p o r t e d in Neurospora crassa [ 3 4 ] , which can translate in a m m o n m m m e d m m b u t c a n n o t do so in its absence. It IS posmble t hat the e ff ect o f a m m o n i u m - i n d u c e d protein, m e n t m n e d earher is stablhzatlon of m R N A f o r nitrate reductase. ACKNOWLEDGEMENTS

Neeraj Datta and L.V.M. Rao are t hankf u l to t he Council of Scientific and Industrial Research f or t he grant o f Sem or Research Fellowship. REFERENCES 1 2 3 4 5

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