Planta (Berl.) 113, 229--240 (1973) 9 by Springer-Verlag 1973
Effect of Chloramphenicol and Cycloheximide on the Induction of Nitrate Reductase and Nitrite Reductase in Bean Leaves * C. M. Th. Sluiters-Seholten Department of Plant Physiology, University of Amsterdam, IJdijk 26, Amsterdam, The Netherlands Received March 13/May 29, 1973 Summary. In etiolated leaves of Phaseolus vulgaris L. cv. Prelude only low levels of/qADH-nitrate oxidoreductase (E.C. 1.6.6.2; NAR) and reduced benzyl viologen-nitrite oxidoreductase (E.C. 1.6.6.4; NIR) could be detected, even in the presence of nitrate. When nitrate was available illumination of leaves of 10-day-old etiolated seedlings resulted in an induction of both NAR and ~qIR. In the absence of nitrate no induction of the enzymes took place, although greening of the leaves was normal. Chloramphenicol (CAP) and cycloheximide (CHI), applied at the beginning of the light period, inhibited the induction of both NAR and NIle. Administered after 24 h of illumination CttI still inhibited the induction of both enzymes whereas CAP was no longer inhibitory. The induction of NAR and NIR by nitrate in green leaves in light was inhibited by CHI but not by CAP. From these results it seems likely that both the enzymes NAR and ~ I R are synthesized on cytoplasmic ribosomes. Before the enzymes can be manufactured in the cytoplasm some chloroplast development is required.
Introduction I n green leaves of different plants the enzymes N A g and N I R are induced u p o n the addition of nitrate (see review b y Beevers and Hageman, 1969). Schrader et al. (1967) found in maize seedlings t h a t the induction of N I R b u t not t h a t of N A R was inhibited b y CAP, an inhibitor of protein synthesis on chloroplast ribosomes (Smillie et al., 1969) ; t h e y concluded t h a t this agreed with the localization of N I R in the chloroplast (Ramirez et al., 1966; R i t e n o u r et al., 1967) and of N A R in the cytoplasm ( g i t e n o u r et al., 1967). However, Coup6 et al. (1967) reported t h a t N A R is a chloroplast enzyme. According to Ritenour et al., (1967) and Eaglesham and H e w i t t (1971) the enzyme p r o b a b l y is b o u n d to the outside of the external chloroplast membrane. The effect of CAP on the induction of N A R b y nitrate is n o t clear in itself. Afridi and H e w i t t (1965) f o u n d t h a t the induction of N A g was inhibited b y CItI, an inhibitor of protein synthesis on cytoplasmic * Abbreviations: CAP, ehloramphenicol; CHI, eycloheximide; G-6-P(-dh), glucose6-phosphate (dehydrogenase) ; NAI~, nitrate reductase; NIR, nitrite reductase.
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ribosomes (Smillie et al., 1968), b u t n o t b y CAP. This is in agreement with the results of Schrader et al. (1967), mentioned above, and also with those of Stewart (1968) and J o y (1969). On the other hand, several other authors reported t h a t the induction of N A R was inhibited b y CAP (Beevers et al., 1965) and b y both C A P and C H I (Ingle, 1965; Oji and Izawa, 1969; Shen, 1969; Sawhney and Naik, 1972). Etiolated leaves have low levels of N A R . During light-induced greening of the leaves the activity of NAI~ increases. This rise in activity is inhibited b y CAP (Beevers et al., 1965; Shibata et al., 1969). Travis et al. (1970) and Travis and K e y (1971) suggested t h a t the role of light in the induction of N A R m a y be attributed to the production of a high a m o u n t of polyribosomes. Recently it was found t h a t N I R , like N A R , is synthesized upon illumination of etiolated rice seedlings (Sawhney and Naik, 1972). The synthesis of b o t h enzymes was inhibited b y CAP and CHI. Sawhney and Naik assumed a close link between the synthesis of N A R and N I R and the development of functional chloroplasts. I n the present paper we report t h a t in green leaves of Phaseolus vulgaris the induction of both N A R and N I R is affected b y C H I only. No inhibitory effect of CAP on the induction of the enzymes during the light-induced greening of the leaves was observed when the developm e n t of the chloroplasts was allowed to proceed for 24 h before the application of the inhibitor.
Material and Methods Plant Material. One batch of seeds of Phaseolus vulgaris L. cv. Prelude, obtained from Pannevis en West-Friesland, Enkhuizen, The Netherlands, was used for all experiments. Greening Experiments. Seedlings were grown in darkness in the presence of 0.01 M KN03 for 10 d and transferred to light without addition of extra nitrate as reported previously (Slniters-Scholten et al., 1973). CAP and CHI were given to the attached leaves 1.5 h before the onset of illumination by wetting the lower sides of each leaf pair with about 0.25 ml of a solution of 500/~g/ml CAP and 50 #g/ml CHI under dim, green safelight. When the inhibitors were given after 24 li of illumination both sides of the leaves were wetted. When plants were grown in the absence of nitrate, KN03 was omitted from the nutrient medium. Inductlon Experiments with Nitrate in Green Leaves. Plants were grown on the same nutrient medium as described previously (Slniters-Scholten et al., 1973), except that the pit was adjusted to 7.0 rather than 4.0 and that nitrate was omitted, in a controlled environment room under 16 h light (7000-8000 lux, 6 Philips fluorescent lamps 65 W/29) at 26~ and 8 h dark at 24~ at a relative humidity of 90%. To induce the enzymes by nitrate, 25 leaf discs, diameter 1 cm, from 7- to 10day-old plants were floated on 50 ml of the same nutrient medium (pH 4.0) in Petri dishes in the presence of the concentration KNO 8 indicated.
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W h e n CAP a n d C t t I were given, the leaf discs were preincubated for 1.5 h in the presence of the inhibitor before nitrate was added. After the induction period the discs were washed with t a p water a n d with deionized water, dried between filter paper, a n d homogenized. Lea/Extract. For the greening experiments, 10 leaf pairs were homogenized in a solution containing 0.025 M KH2PO ~ a n d 0.005 M EDTA, p i t 8.5; (1 : 10, w/v) during 2 rain in a Virtis 45 homogenizer a t medium speed. For the induction experiments, 25 leaf discs were homogenized in 6 ml of the same solution. The homogenate was pressed through 4 layers of cheese cloth. There was no difference in the activity of N A R a n d N I R a n d in the stability of NAI~ when the leaves were homogenized at p H 7.5 or at pI-I 8.5. No cysteine was added, for eysteine in the extraction medium inhibited the activity of N A R (at 5 X 10 -3 M b y 11%, at 10 -3 M b y 59 %). This is in agreement with the behavior of the enzyme from Perilla leaves (Kannangara a n d Woolhouse, 1967), b u t in contrast to the hehavior of the enzyme from m a n y other sources (Beevers a n d Hageman, 1969). Enzyme Assays. The activity of N A R was measured b y the estimation of the a m o u n t of nitrite formed. Because N A R is very labile, the incubation was started within 5 m i n after the onset of the homogenization. The incubation mixture consisted of 140/tmol potassium-phosphate buffer (ptI 7.0), 20/~mol KNOs, 1.5 ~mol NADH, 100/~mol acetaldehyde, 0.4 ml leaf extract in a final volume of 2.1 ml. The reaction was started b y adding N A D H immediately followed b y the leaf extract. A control without N A D H was incubated at the same time. After 30 m i n incubation a t 30 ~ the reaction was stopped b y adding 25 units of alcohol dehydrogenase (E.C. 1.1.1.1) in 0.1 ml of 0.1 M potassium phosphate buffer, p H 7.0. The reaction was stopped b y alcohol dehydrogenase because acetaldehyde was included in the incubation mixture. Acetaldehyde has no effect on the assay. After 1 min, 1 m] of 1% sulfanilamide in 1.5 N HC1 a n d 1 ml of 0.02% N-(1-naphthyl)ethylenediamine dihydroehloride were added. 5 min later the tubes were centrifuged for 10 m i n a t 3000 X g to remove turbidity. The absorbance was read at 540 rim. The reaction rate was constant for at least 40 min and proportional to the a m o u n t of leaf extract added. W h e n the reaction was stopped b y sulfanilamide instead of b y removing N A D t t , much less nitrite was found, depending on the concentration of N A D H present (see also Medina and Nicholas, 1957). The high concentration of N A D H was necessitated b y the low affinity of the enzyme to N A D H in a crude extract of bean leaves (Km = 0.4 raM). No reduction of nitrite b y N A D H was found; thus, there was no interference of N I R in the assay of NAIl. The activity of M R was measured in the same leaf extract with reduced benzyl viologen as the electron donor. The a m o u n t of nitrite t h a t disappeared was determined. The incubation mixture consisted of 120/~mol potassium phosphate buffer, p g 7.0, 1.0 gmol benzyl viologen, 0.5 gmol KN02, 0.4 ml leaf extract in a final volume of 2.4 ml. The reaction was started b y adding 15 ~mol Na-dithionite in 0.6 ml of 0.2 M potassium phosphate buffer, p H 7.0. The tubes were closed with a r u b b e r stopper, gently stirred, a n d incubated a t 30 ~ for 20 min. The reaction was stopped b y shaking the tubes vigorously on a Vortex whirlmix until a t lea@ 5 s after the blue color of the reduced benzyl viologen h a d disappeared. For the zero-time controls the tubes were shaken immediately after the addition of dithionite. To measure the a m o u n t of nitrite left, 1.6 ml H~O was added to 0.4 ml of the incubation medium, followed b y the reagents. After 20 min the absorbanee was read at 540 nm. U p to 20 rain the reaction rate was constant a n d was proportional to the a m o u n t of leaf extract added. W h e n nitrite was replaced b y 20 ~mol KNOs no production of nitrite was measured. I n m a n y plants N A R activity with reduced benzyl viologen is found (Beevers a n d Hageman, 1969). I t is possible t h a t in our case the nitrite formed is reduced immediately b y NIR.
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Table 1. Induction of nitrate reductase and nitrite reductase by nitrate in green leaves of Phaseolus vulgaris plants grown in the absence of nitratea KNOa cone. (M) 0 0.01 0.05 0.1 0.2
Enzyme activityb
Protein content (mg/25 leaf discs)
NAR
NIR
G-6-P-dh
0.03 0.88 1.39 1.54 0.21
4.6 14.9 28.4 31.4 ~ 11.3
11.4 -15.1 15.5 9.4
12.3 12.3 13.1 13.8 8.4
a Nitrate was added at the beginning of the induction period. Induction for 18 h in the light. b NAR = /~mol NO 2- produeed/h 25 leaf discs; NIt~ ~ #mol NO 2- reduced/h 25 leaf discs; G-6-P-dh = /~mol NADPH/h 25 leaf discs.
G.6-P-dh (E.C. 1.1.4.49) was measured in the 15 rain 15000 • g supernatant of the above leaf extract. The reduction of NADP was followed at 340 nm according to Fiiner and Klein (1968). Other Assays. Chlorophyll was determined in 80O/o aqueous acetone (Arnon, 1949) and protein by the Folin method (Lowry et at., 1951) after precipitation of the protein with 5O/o trichloroacetic acid and redissolving in 0.1 N NaOH. Sources o/ Chemicals. Yeast alcohol dehydrogenase, G-6-P, NADH, NADP: Boehringer, Mannheim, Germany; benzyl viologen and Folin and Ciocalteu's phenol reagent: British Drug House, Poole, U.K. ; CAP: de Watermolen, Zaandam, The Netherlands; CHI: Calbiochem, Los Angeles, Calif., USA.
Reslflts Induction o / N A R and N I R by Nitrate P l a n t s grown i n light on a n u t r i e n t m e d i u m w i t h o u t n i t r a t e have low levels of N A R a n d N I R activity. I n c r e a s i n g c o n c e n t r a t i o n s of n i t r a t e lead to a n i n d u c t i o n of b o t h enzymes with a m a x i m u m a t 0.1 M. A t higher c o n c e n t r a t i o n s of n i t r a t e the i n d u c t i o n is m u c h lower, p r o b a b l y because of the high osmotic value of the i n d u c t i o n m e d i u m (Table 1). A t the same time as the increase i n a c t i v i t y of N A R a n d N I R a slight increase of the p r o t e i n c o n t e n t as well as an increase of the a c t i v i t y of a reference enzyme, G-6-P-dh, were also observed. The time-course of i n d u c t i o n is shown i n Fig. 1. The i n d u c t i o n of N A R starts a t once, the i n d u c t i o n of N I R shows a lag phase of a b o u t 3h. As is seen i n Table 2, C H I i n h i b i t s the i n d u c t i o n of b o t h N A R a n d N I R . Also the a m o u n t of p r o t e i n a n d the a c t i v i t y of G-6-P-dh decreases i n the presence of CHI.
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Fig. 1. Time-course of the induction of NAR (x) and NIR (o) by nitrate in green leaves of Phaseolus vulgaris plants grown in the absence of nitrate. The induction was carried out in the light in the presence of 0.1 M KNOa
Table 2. Effect of cycloheximide on the induction of nitrate reductase and nitrite reduetase by nitrate in green leaves of P. vulgarls plants grown in the absence of nitratea CHI cone. (gg/ml) 0 0.2 2 20
Enzyme activityb
Protein content (rag/25 leaf discs)
NAI~
NIR
G-6-P-dh
1.37 1.13 0.61 0.11
27.0 16.5 7.0 3.4
19.0 15.7 10.2 5.6
15.0 14.2 12.3 12.4
a The leaf discs were preineubated for 1.5 h in the presence of the concentration CHI indicated, before adding 0.1 M I ~ O a. Induction in the presence of nitrate for 18 h in the light. b NAR = #tool NO~- produced/h 25 leaf discs; NII~ = #tool NO 2- reduced/h 25 leaf discs; G-6-P-dh = ~mol NADPH/h 25 leaf discs.
The effect of CAP on the i n d u c t i o n of N A R a n d N I R is v e r y different. Fig. 2 shows t h a t a t 0.1 m g / m l there is a clear s t i m u l a t i o n of the i n d u c t i o n of N A R a n d N I R . The increase of G-6-P-dh u p o n the a d d i t i o n of n i t r a t e is also s t i m u l a t e d b y CAP. The i n h i b i t i o n a t higher concent r a t i o n s is p r o b a b l y due to side effects. W i t h rising a m o u n t s of CAP there is a slow decline of t h e p r o t e i n c o n t e n t of the leaf discs. These results i n d i c a t e t h a t the i n d u c t i o n of the enzymes N A R a n d N I R b y n i t r a t e i n green leaves is i n h i b i t e d b y CHI, b u t n o t b y CAP.
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Induction o / N A R and NIR by Light During the tight-induced greening of leaves of etiolated bean seedtings an induction of N A R and N i l { takes place w h e n nitrate is present in the nutrient m e d i u m . N o increase of the a c t i v i t y of the e n z y m e s occurs in the dark (Fig. 3). I n the absence of nitrate the a c t i v i t y of the e n z y m e s does not increase although normal greening of the leaves is observed (Fig. 4). During the 10 days of darkness preceding the illumination period no distinct increase in a c t i v i t y of N A R and N i l { is found, in contrast to the e n z y m e G-6-P-dh (Fig. 5). The lack of increase of N A R and N i l { in the dark in leaves of etiolated bean seedlings is therefore not, due
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Induction of Nitrate and Nitrite Reductase
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Fig. 4. Effect of nitrate on the hght-induced increase of chlorophyll content (~), activity of NAR (~) and Nil% (o) of leaves of 10-day-old, etiolated P. vulgaris seedlings. Left, seedlings grown in the presence of 0.01 M KN03; right, seedlings grown in the absence of nitrate "C m
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Fig. 5. Activity of NAR (.), NII~ (,) and G-6-P-dh (o) and protein content (• in leaves of 0- to 10-day-old, etiolated P. vulgaris seedlings grown in the presence of 0.01 M KN03
to the absence of an active protein-synthesizing system, as was proposed b y Travis et al. (1970) and Travis and K e y (1971) for corn leaves. Both CAP, applied at 500 ~g/ml and CHI, applied at 50 ~g/ml, inhibit the increase in activity of N A R and N I R (Fig. 6B, C). The same holds true for the synthesis of chlorophyll (Fig. 6A). As reported previously, the increase of the activity of G-6-P-dh b y light is not influenced by this concentration of CAP. CHI at 50 Fg/ml inhibits the increase of the activity of G-6-P-dh b y almost 100% (Sluiters-Scholten et al., 1973). The experiments on induction of NAI% and N i l { b y nitrate in green leaves have shown that the increase in activity of both enzymes is in-
236
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Fig. 6A--C. Effect of CAP and CHI, applied at the beginning of the illumination period, on the light-induced increase of chlorophyll content (A), activity of NAR (B) and NIR (C) of leaves of 10-day-old, etiolated P. vulgarls seedlings grown in the presence of 0.01 M KNO 3. o light control; * CAP 500/*g/ml; , CHI 50/~g/ml
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Fig. 7A--C. Effect of CAP and CHI, applied after 24 h of illumination, on the lightinduced increase of chlorophyll content (A), activity of NAR (B) and NIR (C) of leaves of 10-day-old, etiolated P. vulgaris seedlings grown in the presence of 0.01 M KNO~. o light control; • CAP 500/~g/ml; ~ CHI 50 #g/ml
hibited by C H I only. W e therefore assume that during the light-induced greening of the leaves CAP affects the increase in a c t i v i t y of the e n z y m e s in an indirect way. To t e s t this assumption w e gave the inhibitors after a light period of 24 h, rather than before the exposure of the leaves to light. I n this case (Fig. 7) no inhibition of the increase in activity of N A R and N I R by CAP is observed. The lack of inhibition is n o t due to the i m p e r m e a b i l i t y of the leaves to CAP, for the further synthesis of chlorophyll is inhibited. C H I applied after 24 h of illumination stops the increase in a c t i v i t y of both e n z y m e s promptly.
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These experiments show that CHI blocks the increase in activity of NAI% and N I R during the light-induced greening of leaves of etiolated seedlings in all circumstances. The inhibitory effect of CAP is not present when the inhibitor is applied after 24 h of illumination.
Discussion In leaves of Phaseolus vulgaris the induction of NAR and N I R is influenced by nitrate and by light, like in other plants. The lag phase in the induction of NII~ by nitrate was also ibund in radish cotyledons by Ingle et al. (1966). I t was concluded that nitrate induces NAIl, which reduces nitrate to nitrite; the nitrite thus formed induces I~IR in turn. This is in agreement with the induction of N I R by nitrite (Ingle et al., 1966; Chroboczek-Kelker and Filner, 1971 ; Hucklesby et al., 1973). However, the first two papers report an induction of NAR by nitrite, too. We did not succeed in obtaining a distinct induction of I~IR by nitrite. At 5 mM a slight induction of N I R as well as of NAR was observed. At higher concentrations, nitrite interfered with the estimation of nitrite in the assay of the activity of NAI~ and NIle. Besides, the inhibition by tungstate of the induction by nitrate of NAI~ but not of that of N I R in cultured tobacco cells (Chroboczek-Kelker and Filner, 1971) suggests that I~IR is induced directly by nitrate. Thus, the origin of the lag phase in the induction of NI]~ by nitrate is not yet clear. The induction of IqAR and 5IIR by nitrate is inhibited by CHI. CAP inhibits the induction only at high concentrations. At these high levels the activity of a reference enzyme, G-6-P-dh, is also affected. From this we conclude the inhibition by CAP to be due to side effects, such as inhibition of oxidative phosphorylation (Firkin and Linnane, 1968). In maize leaves Schrader et al. (1967) found an inhibition of the induction of NIR, but not that of NAR, by a concentration of CAP 10 times higher than used in our experiments. The reason for this lack of inhibition of NAR is obscure. I t appears questionable that it can be attributed only to the difference in material. At lower concentrations CAP stimulates the increase in activity of NAR and NIl%. Ireland and Bradbeer (1971) suppose that the stimulation of the cytoplasmic protein-synthesizing system by CAP is caused by the availability of extra energy and of amino acids because another protein-synthesizing system in the cell, namely that of the chloroplasts, is blocked. The concentration of CAP used in the greening experiments causes side effects in the induction experiments by nitrate. However, this concentration of CAP has no influence on the light-induced increase of the activity of G-6-P-dh (Sluiters-Seholten et al., 1973). The etiolated leaves were treated only once with the inhibitor whereas the leaf discs were floated on a solution with the inhibitor.
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Thus, the total amount of CAP offered to the leaf discs was much higher. Besides, leaf discs m a y be more penetrable to CAP than whole leaves. From the effects of CAP and C I I I on the induction of N A R and N I R it seems likely t h a t both enzymes are synthesized on cytoplasmic ribosomes. However, it cannot be ruled out t h a t cytoplasmic protein synthesis leads to activation of a latent enzyme. There is evidence t h a t in Chlorella NAR can exist in a latent form (Moreno et al., 1972). On the other hand, the labeling experiments of Zielke and Filner (1971) show t h a t the induction of N A R by nitrate in cultured tobacco cells is a result of de novo protein synthesis. The synthesis of N I R in the cytoplasm does not rule out its localization inside the chloroplast. I n our previous paper (Sluiters-Scholten et al., 1973) we found aminolevulinate dehydratase from bean leaves to be manufactured in the cytoplasm, although we and m a n y other authors have shown t h a t this enzyme is at least partly localized in the chloroplast. Before the induction of N A R and N I R can occur, some chloroplast development must have taken place. I n green leaves the induction of the enzymes is not affected by CAP. When the light-induced chloroplast development is inhibited b y CAP (Smillie et al., 1968) from the beginning of the illumination period no induction of NAR and N I R is observed. Applied after 24 h of illumination CAP has no effect on the induction of NAR and N I R . Sawhney and Naik (1972) suppose t h a t photosynthetic reactions are necessary for the synthesis of N A R and N I R . The results of their experiments with DCMU suggest t h a t a non-cyclic electron transport is required to allow the induction. I n our case, after 24 h of illumination the chloroplast development should be advanced far enough for electron transport. Gyldenholm and Whatley (1968) found between 15 and 25 h of illumination of 14- to 16-day-old leaves of ctiolated bean seedlings a distinct increase of the ability t o photorcduce N A D P with water as electron donor. I wish to thank Miss A. KSnst and Mrs. N. Stoter-Tims for their technical assistance. This investigation was supported by the Netherlands Organization for the Advancement of Pure Scientific Research (Z.W.O.), Grant 82003.
References Afridi, M. M. R. K., Hewitt, E. J.: The inducible formation and stability of nitrate reduetase in higher plants I I Effects of environmental factors, antimetabolites and amino-acids on induction. J. exp. Bot. 16, 628-645 (1965). Arnon, D. I.: Copper enzymes in isolated chloroplasts. Plant Physiol. 24, 1-15
(1949).
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Beevers, L., tIageman, R. It.: Nitrate reduction in higher plants. Ann. Rev. Plant Physiol. 20, 495-522 (1969). Beevers, L., Sehrader, L. E., Flesher, D., Hageman, R. H.: The role of fight and nitrate in the induction of nitrate reductase in radish cotyledons and maize seedlings. Plant Physiol. 40, 691-698 (1965). Chroboczek-Kelker, ~., Filner, P.: Regulation of nitrite reductase and its relationship to the regulation of nitrate reductase in cultured tobacco cells. Biochim. biophys. Acts (Amst.) 252, 69-82 (1971). Coup6, M., Champigny, M. L., Moyse, A.: Sur la localisation intracellalaire de Ia nitrate r4ductase dans les feuilles et les racines d'Orge. Physiol. v6g. 5, 271291 (1967). Eaglesham, A. R. J., Hewitt, E. J.: The regulation of nitrate reductase activity from Spinach (Spinacea oleracea L.) leaves by thiol compounds in the presence of adenosine-5'-diphosphate. FEBS Letters 16, 315-318 (1971). Yflner, B., Klein, A. O.: Changes in enzymatic activities in etiolated bean seedling leaves after a brief illumination. Plant Physiol. 48, 1587-1596 (1968). Firkin, F. C., Linnane, A. W.: Differential effects of ch]oramphenieol on the growth ~nd respiration of m~mmalian eetIs. Biochem. biophys, l%es. Comman. 8~, 898402 (1968). Gyldenholm, A. 0., Whatley, Y. R.: The onset of photophosphorylation in chloroplasts isolated from developing bean leaves. New Phytol. 67,461-468 (1968). Hucklesby, D. P., Dalling, M. J., Hageman, R. H.: Some properties of two forms of nitrite reductase from corn (Zea mays L.) scutellum. Plants (Berl.) 194, 220-233 (1972). Ingle, J.: Nucleic acid and protein synthesis associated with the induction of nitrate reductase activity in radish cotyledons. Biocfiem. J. 108, 715-724 (1968). Ingle, J., Joy, K. W., Hageman, 1~. H.: The regulation of activity of the enzymes involved in the assimilation of nitrate by higher plants. Biochem. J. 100, 577 to 588 (1966). Ireland, H. M. M., Bradbeer, J. W.: Plastid development in primary leaves of Phaseolus vulgaris. The effects of D-threo and L-threo chloramphenieol on the fight-induced formation of enzymes of the photosynthetic carbon pathway. Plants (Berl.) 96, 254-261 (1971). Joy, K. W.: Nitrogen metabofism of Lemna minor I I Enzymes of nitrate assimilation and some aspects of their regulation. Plant Physiol. 44, 849-853 (1969). Kannangara, C. G., Woolhouse, tt. W.: The role of carbon dioxide, fight and nitrate in the synthesis and degradation of nitrate reductase in leaves of Per glla /rutescsns. New Phytol. 66, 553-561 (1967}. Lowry, O. It., Rosebrough, N. J., 1~arr, A. L., Randall, R. J.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275 (1951). Medina, A., Nicholas, D. J. D.: Interference by reduced pyridine nucleotides in the diazotization of nitrite. Biochim. biophys. Acts (Amst.) 23, 440-442 (1957). Moreno, C. G., Aparicio, P. J., Palacis E., Losada, M.: Interconversion of the active and inactive forms of Chlorella nitrate reductase. FEBS Letters 26, 11-13 (1972). Oji, Y., Izawa, G.: Effects of univalent cations on the formation of nitrate reductase and nitrite reductase in rice seedlings. Plant Cell Physiol. 10, 665-674 (1969). l~amirez, J. M., Del Campo, F. F., Paneque, A., Losada, M.: Ferredoxin-nitrite reductase from spinach. Biochim. biophys. Acts (Amst.) 118, 58-71 (1966). Ritenour, G. L., Joy, K. W., Dunning, J., Hageman, 1%.H.: Intracellular localization of nitrate reductase, nitrite rcductase and glutamic acid dehydrogenase in green leaf tissue. Plant Physiol. 42, 233-237 (1967).
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Sawhney, S. K., Naik, M. S.: Role of light in the synthesis of nitrate reductase and nitrite reductase in rice seedlings. Biochem. J. 130, 475-485 (1972). Schrader, L. E., Beevers, L., Hageman, R. H.: Differential effects of chloramphenieol on the induction of nitrate and nitrite reductase in green leaf tissues. Biochem. biophys. Res. Commun. 26, 14-17 (1967). Shen, T. C.: The induction of nitrate reductase and the preferential assimilation of ammonium in germinating rice seedlings. Plant Physiol. 44, 1650-1655 (1969). Shibata, M., Kobayashi, M., Takahashi, E.: The possibility of photoindueed induction of nitrate reduetase in rice seedlings. Plant Cell Physiol. 10, 337-348 (1969). Slniters-Seholten, C. M. Th., Berg, F. M. van den, Stegwee, D.: Aminolaevulinate dehydratase in greening leaves of Phaseolus vulgaris L. Z. Pflanzenphysiol. 69, 217-227 (1973). Smillie, R. M., Steele Scott, N., Graham, D.: Biogenesis of chloroplasts: Roles of chloroplast DNA and chloroplast ribosomes. In: Comparative biochemistry and biophysics of photosynthesis, p. 332-353, Shibata, K., Takamiya, A., Jagendoff, A. T., Fuller, R., eds. Tokyo: Univ. of Tokyo Press 1968. Stewart, G. R.: The effect of cycloheximide on the induction of nitrate and nitrite reductase in Lemna minor L. Phytoehem. 7, 1139-1142 (1968). Travis, R. L., Huffaker, R. C., Key, J. L.: Light-induced development of polyribosomes and the induction of nitrate reductase in corn leaves. Plant Physiol. 46, 800-805 (1970). Travis, R. L., Key, J. L.: Correlations between polyribosome level and the ability to induce nitrate reductase in dark-grown corn seedlings. Plant Physiol. 48, 617 to 620 (1971). Zielkc, H. R., Fflner, P.: Synthesis and turnover of nitrate reductase induced by nitrate in cultured tobacco cells. J. biol. Chem. 246, 1772-1779 (1971).
Effect of Chloramphenicol and Cycloheximide on the Induction of Nitrate Reductase and Nitrite Reductase in Bean Leaves * C. M. Th. Sluiters-Seholten Department of Plant Physiology, University of Amsterdam, IJdijk 26, Amsterdam, The Netherlands Received March 13/May 29, 1973 Summary. In etiolated leaves of Phaseolus vulgaris L. cv. Prelude only low levels of/qADH-nitrate oxidoreductase (E.C. 1.6.6.2; NAR) and reduced benzyl viologen-nitrite oxidoreductase (E.C. 1.6.6.4; NIR) could be detected, even in the presence of nitrate. When nitrate was available illumination of leaves of 10-day-old etiolated seedlings resulted in an induction of both NAR and ~qIR. In the absence of nitrate no induction of the enzymes took place, although greening of the leaves was normal. Chloramphenicol (CAP) and cycloheximide (CHI), applied at the beginning of the light period, inhibited the induction of both NAR and NIle. Administered after 24 h of illumination CttI still inhibited the induction of both enzymes whereas CAP was no longer inhibitory. The induction of NAR and NIR by nitrate in green leaves in light was inhibited by CHI but not by CAP. From these results it seems likely that both the enzymes NAR and ~ I R are synthesized on cytoplasmic ribosomes. Before the enzymes can be manufactured in the cytoplasm some chloroplast development is required.
Introduction I n green leaves of different plants the enzymes N A g and N I R are induced u p o n the addition of nitrate (see review b y Beevers and Hageman, 1969). Schrader et al. (1967) found in maize seedlings t h a t the induction of N I R b u t not t h a t of N A R was inhibited b y CAP, an inhibitor of protein synthesis on chloroplast ribosomes (Smillie et al., 1969) ; t h e y concluded t h a t this agreed with the localization of N I R in the chloroplast (Ramirez et al., 1966; R i t e n o u r et al., 1967) and of N A R in the cytoplasm ( g i t e n o u r et al., 1967). However, Coup6 et al. (1967) reported t h a t N A R is a chloroplast enzyme. According to Ritenour et al., (1967) and Eaglesham and H e w i t t (1971) the enzyme p r o b a b l y is b o u n d to the outside of the external chloroplast membrane. The effect of CAP on the induction of N A R b y nitrate is n o t clear in itself. Afridi and H e w i t t (1965) f o u n d t h a t the induction of N A g was inhibited b y CItI, an inhibitor of protein synthesis on cytoplasmic * Abbreviations: CAP, ehloramphenicol; CHI, eycloheximide; G-6-P(-dh), glucose6-phosphate (dehydrogenase) ; NAI~, nitrate reductase; NIR, nitrite reductase.
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ribosomes (Smillie et al., 1968), b u t n o t b y CAP. This is in agreement with the results of Schrader et al. (1967), mentioned above, and also with those of Stewart (1968) and J o y (1969). On the other hand, several other authors reported t h a t the induction of N A R was inhibited b y CAP (Beevers et al., 1965) and b y both C A P and C H I (Ingle, 1965; Oji and Izawa, 1969; Shen, 1969; Sawhney and Naik, 1972). Etiolated leaves have low levels of N A R . During light-induced greening of the leaves the activity of NAI~ increases. This rise in activity is inhibited b y CAP (Beevers et al., 1965; Shibata et al., 1969). Travis et al. (1970) and Travis and K e y (1971) suggested t h a t the role of light in the induction of N A R m a y be attributed to the production of a high a m o u n t of polyribosomes. Recently it was found t h a t N I R , like N A R , is synthesized upon illumination of etiolated rice seedlings (Sawhney and Naik, 1972). The synthesis of b o t h enzymes was inhibited b y CAP and CHI. Sawhney and Naik assumed a close link between the synthesis of N A R and N I R and the development of functional chloroplasts. I n the present paper we report t h a t in green leaves of Phaseolus vulgaris the induction of both N A R and N I R is affected b y C H I only. No inhibitory effect of CAP on the induction of the enzymes during the light-induced greening of the leaves was observed when the developm e n t of the chloroplasts was allowed to proceed for 24 h before the application of the inhibitor.
Material and Methods Plant Material. One batch of seeds of Phaseolus vulgaris L. cv. Prelude, obtained from Pannevis en West-Friesland, Enkhuizen, The Netherlands, was used for all experiments. Greening Experiments. Seedlings were grown in darkness in the presence of 0.01 M KN03 for 10 d and transferred to light without addition of extra nitrate as reported previously (Slniters-Scholten et al., 1973). CAP and CHI were given to the attached leaves 1.5 h before the onset of illumination by wetting the lower sides of each leaf pair with about 0.25 ml of a solution of 500/~g/ml CAP and 50 #g/ml CHI under dim, green safelight. When the inhibitors were given after 24 li of illumination both sides of the leaves were wetted. When plants were grown in the absence of nitrate, KN03 was omitted from the nutrient medium. Inductlon Experiments with Nitrate in Green Leaves. Plants were grown on the same nutrient medium as described previously (Slniters-Scholten et al., 1973), except that the pit was adjusted to 7.0 rather than 4.0 and that nitrate was omitted, in a controlled environment room under 16 h light (7000-8000 lux, 6 Philips fluorescent lamps 65 W/29) at 26~ and 8 h dark at 24~ at a relative humidity of 90%. To induce the enzymes by nitrate, 25 leaf discs, diameter 1 cm, from 7- to 10day-old plants were floated on 50 ml of the same nutrient medium (pH 4.0) in Petri dishes in the presence of the concentration KNO 8 indicated.
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231
W h e n CAP a n d C t t I were given, the leaf discs were preincubated for 1.5 h in the presence of the inhibitor before nitrate was added. After the induction period the discs were washed with t a p water a n d with deionized water, dried between filter paper, a n d homogenized. Lea/Extract. For the greening experiments, 10 leaf pairs were homogenized in a solution containing 0.025 M KH2PO ~ a n d 0.005 M EDTA, p i t 8.5; (1 : 10, w/v) during 2 rain in a Virtis 45 homogenizer a t medium speed. For the induction experiments, 25 leaf discs were homogenized in 6 ml of the same solution. The homogenate was pressed through 4 layers of cheese cloth. There was no difference in the activity of N A R a n d N I R a n d in the stability of NAI~ when the leaves were homogenized at p H 7.5 or at pI-I 8.5. No cysteine was added, for eysteine in the extraction medium inhibited the activity of N A R (at 5 X 10 -3 M b y 11%, at 10 -3 M b y 59 %). This is in agreement with the behavior of the enzyme from Perilla leaves (Kannangara a n d Woolhouse, 1967), b u t in contrast to the hehavior of the enzyme from m a n y other sources (Beevers a n d Hageman, 1969). Enzyme Assays. The activity of N A R was measured b y the estimation of the a m o u n t of nitrite formed. Because N A R is very labile, the incubation was started within 5 m i n after the onset of the homogenization. The incubation mixture consisted of 140/tmol potassium-phosphate buffer (ptI 7.0), 20/~mol KNOs, 1.5 ~mol NADH, 100/~mol acetaldehyde, 0.4 ml leaf extract in a final volume of 2.1 ml. The reaction was started b y adding N A D H immediately followed b y the leaf extract. A control without N A D H was incubated at the same time. After 30 m i n incubation a t 30 ~ the reaction was stopped b y adding 25 units of alcohol dehydrogenase (E.C. 1.1.1.1) in 0.1 ml of 0.1 M potassium phosphate buffer, p H 7.0. The reaction was stopped b y alcohol dehydrogenase because acetaldehyde was included in the incubation mixture. Acetaldehyde has no effect on the assay. After 1 min, 1 m] of 1% sulfanilamide in 1.5 N HC1 a n d 1 ml of 0.02% N-(1-naphthyl)ethylenediamine dihydroehloride were added. 5 min later the tubes were centrifuged for 10 m i n a t 3000 X g to remove turbidity. The absorbance was read at 540 rim. The reaction rate was constant for at least 40 min and proportional to the a m o u n t of leaf extract added. W h e n the reaction was stopped b y sulfanilamide instead of b y removing N A D t t , much less nitrite was found, depending on the concentration of N A D H present (see also Medina and Nicholas, 1957). The high concentration of N A D H was necessitated b y the low affinity of the enzyme to N A D H in a crude extract of bean leaves (Km = 0.4 raM). No reduction of nitrite b y N A D H was found; thus, there was no interference of N I R in the assay of NAIl. The activity of M R was measured in the same leaf extract with reduced benzyl viologen as the electron donor. The a m o u n t of nitrite t h a t disappeared was determined. The incubation mixture consisted of 120/~mol potassium phosphate buffer, p g 7.0, 1.0 gmol benzyl viologen, 0.5 gmol KN02, 0.4 ml leaf extract in a final volume of 2.4 ml. The reaction was started b y adding 15 ~mol Na-dithionite in 0.6 ml of 0.2 M potassium phosphate buffer, p H 7.0. The tubes were closed with a r u b b e r stopper, gently stirred, a n d incubated a t 30 ~ for 20 min. The reaction was stopped b y shaking the tubes vigorously on a Vortex whirlmix until a t lea@ 5 s after the blue color of the reduced benzyl viologen h a d disappeared. For the zero-time controls the tubes were shaken immediately after the addition of dithionite. To measure the a m o u n t of nitrite left, 1.6 ml H~O was added to 0.4 ml of the incubation medium, followed b y the reagents. After 20 min the absorbanee was read at 540 nm. U p to 20 rain the reaction rate was constant a n d was proportional to the a m o u n t of leaf extract added. W h e n nitrite was replaced b y 20 ~mol KNOs no production of nitrite was measured. I n m a n y plants N A R activity with reduced benzyl viologen is found (Beevers a n d Hageman, 1969). I t is possible t h a t in our case the nitrite formed is reduced immediately b y NIR.
232
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Table 1. Induction of nitrate reductase and nitrite reductase by nitrate in green leaves of Phaseolus vulgaris plants grown in the absence of nitratea KNOa cone. (M) 0 0.01 0.05 0.1 0.2
Enzyme activityb
Protein content (mg/25 leaf discs)
NAR
NIR
G-6-P-dh
0.03 0.88 1.39 1.54 0.21
4.6 14.9 28.4 31.4 ~ 11.3
11.4 -15.1 15.5 9.4
12.3 12.3 13.1 13.8 8.4
a Nitrate was added at the beginning of the induction period. Induction for 18 h in the light. b NAR = /~mol NO 2- produeed/h 25 leaf discs; NIt~ ~ #mol NO 2- reduced/h 25 leaf discs; G-6-P-dh = /~mol NADPH/h 25 leaf discs.
G.6-P-dh (E.C. 1.1.4.49) was measured in the 15 rain 15000 • g supernatant of the above leaf extract. The reduction of NADP was followed at 340 nm according to Fiiner and Klein (1968). Other Assays. Chlorophyll was determined in 80O/o aqueous acetone (Arnon, 1949) and protein by the Folin method (Lowry et at., 1951) after precipitation of the protein with 5O/o trichloroacetic acid and redissolving in 0.1 N NaOH. Sources o/ Chemicals. Yeast alcohol dehydrogenase, G-6-P, NADH, NADP: Boehringer, Mannheim, Germany; benzyl viologen and Folin and Ciocalteu's phenol reagent: British Drug House, Poole, U.K. ; CAP: de Watermolen, Zaandam, The Netherlands; CHI: Calbiochem, Los Angeles, Calif., USA.
Reslflts Induction o / N A R and N I R by Nitrate P l a n t s grown i n light on a n u t r i e n t m e d i u m w i t h o u t n i t r a t e have low levels of N A R a n d N I R activity. I n c r e a s i n g c o n c e n t r a t i o n s of n i t r a t e lead to a n i n d u c t i o n of b o t h enzymes with a m a x i m u m a t 0.1 M. A t higher c o n c e n t r a t i o n s of n i t r a t e the i n d u c t i o n is m u c h lower, p r o b a b l y because of the high osmotic value of the i n d u c t i o n m e d i u m (Table 1). A t the same time as the increase i n a c t i v i t y of N A R a n d N I R a slight increase of the p r o t e i n c o n t e n t as well as an increase of the a c t i v i t y of a reference enzyme, G-6-P-dh, were also observed. The time-course of i n d u c t i o n is shown i n Fig. 1. The i n d u c t i o n of N A R starts a t once, the i n d u c t i o n of N I R shows a lag phase of a b o u t 3h. As is seen i n Table 2, C H I i n h i b i t s the i n d u c t i o n of b o t h N A R a n d N I R . Also the a m o u n t of p r o t e i n a n d the a c t i v i t y of G-6-P-dh decreases i n the presence of CHI.
Induction of Nitrate and Nitrite Reduetase
233
60 u m / / c~ .d
/
/ /
u
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Fig. 1. Time-course of the induction of NAR (x) and NIR (o) by nitrate in green leaves of Phaseolus vulgaris plants grown in the absence of nitrate. The induction was carried out in the light in the presence of 0.1 M KNOa
Table 2. Effect of cycloheximide on the induction of nitrate reductase and nitrite reduetase by nitrate in green leaves of P. vulgarls plants grown in the absence of nitratea CHI cone. (gg/ml) 0 0.2 2 20
Enzyme activityb
Protein content (rag/25 leaf discs)
NAI~
NIR
G-6-P-dh
1.37 1.13 0.61 0.11
27.0 16.5 7.0 3.4
19.0 15.7 10.2 5.6
15.0 14.2 12.3 12.4
a The leaf discs were preineubated for 1.5 h in the presence of the concentration CHI indicated, before adding 0.1 M I ~ O a. Induction in the presence of nitrate for 18 h in the light. b NAR = #tool NO~- produced/h 25 leaf discs; NII~ = #tool NO 2- reduced/h 25 leaf discs; G-6-P-dh = ~mol NADPH/h 25 leaf discs.
The effect of CAP on the i n d u c t i o n of N A R a n d N I R is v e r y different. Fig. 2 shows t h a t a t 0.1 m g / m l there is a clear s t i m u l a t i o n of the i n d u c t i o n of N A R a n d N I R . The increase of G-6-P-dh u p o n the a d d i t i o n of n i t r a t e is also s t i m u l a t e d b y CAP. The i n h i b i t i o n a t higher concent r a t i o n s is p r o b a b l y due to side effects. W i t h rising a m o u n t s of CAP there is a slow decline of t h e p r o t e i n c o n t e n t of the leaf discs. These results i n d i c a t e t h a t the i n d u c t i o n of the enzymes N A R a n d N I R b y n i t r a t e i n green leaves is i n h i b i t e d b y CHI, b u t n o t b y CAP.
234
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Fig. 3A--C. Effect of light and dark on chlorophyll content (A), activity ofNAR (B) and NIR (C) of leaves of 10-day-old, etiolated P. vulgaris seedlings grown in the presence of 0.01 M KNOB. Open symbols, light; closed symbols, dark
Induction o / N A R and NIR by Light During the tight-induced greening of leaves of etiolated bean seedtings an induction of N A R and N i l { takes place w h e n nitrate is present in the nutrient m e d i u m . N o increase of the a c t i v i t y of the e n z y m e s occurs in the dark (Fig. 3). I n the absence of nitrate the a c t i v i t y of the e n z y m e s does not increase although normal greening of the leaves is observed (Fig. 4). During the 10 days of darkness preceding the illumination period no distinct increase in a c t i v i t y of N A R and N i l { is found, in contrast to the e n z y m e G-6-P-dh (Fig. 5). The lack of increase of N A R and N i l { in the dark in leaves of etiolated bean seedlings is therefore not, due
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Induction of Nitrate and Nitrite Reductase
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Fig. 4. Effect of nitrate on the hght-induced increase of chlorophyll content (~), activity of NAR (~) and Nil% (o) of leaves of 10-day-old, etiolated P. vulgaris seedlings. Left, seedlings grown in the presence of 0.01 M KN03; right, seedlings grown in the absence of nitrate "C m
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Fig. 5. Activity of NAR (.), NII~ (,) and G-6-P-dh (o) and protein content (• in leaves of 0- to 10-day-old, etiolated P. vulgaris seedlings grown in the presence of 0.01 M KN03
to the absence of an active protein-synthesizing system, as was proposed b y Travis et al. (1970) and Travis and K e y (1971) for corn leaves. Both CAP, applied at 500 ~g/ml and CHI, applied at 50 ~g/ml, inhibit the increase in activity of N A R and N I R (Fig. 6B, C). The same holds true for the synthesis of chlorophyll (Fig. 6A). As reported previously, the increase of the activity of G-6-P-dh b y light is not influenced by this concentration of CAP. CHI at 50 Fg/ml inhibits the increase of the activity of G-6-P-dh b y almost 100% (Sluiters-Scholten et al., 1973). The experiments on induction of NAI% and N i l { b y nitrate in green leaves have shown that the increase in activity of both enzymes is in-
236
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Fig. 6A--C. Effect of CAP and CHI, applied at the beginning of the illumination period, on the light-induced increase of chlorophyll content (A), activity of NAR (B) and NIR (C) of leaves of 10-day-old, etiolated P. vulgarls seedlings grown in the presence of 0.01 M KNO 3. o light control; * CAP 500/*g/ml; , CHI 50/~g/ml
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Fig. 7A--C. Effect of CAP and CHI, applied after 24 h of illumination, on the lightinduced increase of chlorophyll content (A), activity of NAR (B) and NIR (C) of leaves of 10-day-old, etiolated P. vulgaris seedlings grown in the presence of 0.01 M KNO~. o light control; • CAP 500/~g/ml; ~ CHI 50 #g/ml
hibited by C H I only. W e therefore assume that during the light-induced greening of the leaves CAP affects the increase in a c t i v i t y of the e n z y m e s in an indirect way. To t e s t this assumption w e gave the inhibitors after a light period of 24 h, rather than before the exposure of the leaves to light. I n this case (Fig. 7) no inhibition of the increase in activity of N A R and N I R by CAP is observed. The lack of inhibition is n o t due to the i m p e r m e a b i l i t y of the leaves to CAP, for the further synthesis of chlorophyll is inhibited. C H I applied after 24 h of illumination stops the increase in a c t i v i t y of both e n z y m e s promptly.
Induction of Nitrate and Nitrite Reductase
237
These experiments show that CHI blocks the increase in activity of NAI% and N I R during the light-induced greening of leaves of etiolated seedlings in all circumstances. The inhibitory effect of CAP is not present when the inhibitor is applied after 24 h of illumination.
Discussion In leaves of Phaseolus vulgaris the induction of NAR and N I R is influenced by nitrate and by light, like in other plants. The lag phase in the induction of NII~ by nitrate was also ibund in radish cotyledons by Ingle et al. (1966). I t was concluded that nitrate induces NAIl, which reduces nitrate to nitrite; the nitrite thus formed induces I~IR in turn. This is in agreement with the induction of N I R by nitrite (Ingle et al., 1966; Chroboczek-Kelker and Filner, 1971 ; Hucklesby et al., 1973). However, the first two papers report an induction of NAR by nitrite, too. We did not succeed in obtaining a distinct induction of I~IR by nitrite. At 5 mM a slight induction of N I R as well as of NAR was observed. At higher concentrations, nitrite interfered with the estimation of nitrite in the assay of the activity of NAI~ and NIle. Besides, the inhibition by tungstate of the induction by nitrate of NAI~ but not of that of N I R in cultured tobacco cells (Chroboczek-Kelker and Filner, 1971) suggests that I~IR is induced directly by nitrate. Thus, the origin of the lag phase in the induction of NI]~ by nitrate is not yet clear. The induction of IqAR and 5IIR by nitrate is inhibited by CHI. CAP inhibits the induction only at high concentrations. At these high levels the activity of a reference enzyme, G-6-P-dh, is also affected. From this we conclude the inhibition by CAP to be due to side effects, such as inhibition of oxidative phosphorylation (Firkin and Linnane, 1968). In maize leaves Schrader et al. (1967) found an inhibition of the induction of NIR, but not that of NAR, by a concentration of CAP 10 times higher than used in our experiments. The reason for this lack of inhibition of NAR is obscure. I t appears questionable that it can be attributed only to the difference in material. At lower concentrations CAP stimulates the increase in activity of NAR and NIl%. Ireland and Bradbeer (1971) suppose that the stimulation of the cytoplasmic protein-synthesizing system by CAP is caused by the availability of extra energy and of amino acids because another protein-synthesizing system in the cell, namely that of the chloroplasts, is blocked. The concentration of CAP used in the greening experiments causes side effects in the induction experiments by nitrate. However, this concentration of CAP has no influence on the light-induced increase of the activity of G-6-P-dh (Sluiters-Seholten et al., 1973). The etiolated leaves were treated only once with the inhibitor whereas the leaf discs were floated on a solution with the inhibitor.
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Thus, the total amount of CAP offered to the leaf discs was much higher. Besides, leaf discs m a y be more penetrable to CAP than whole leaves. From the effects of CAP and C I I I on the induction of N A R and N I R it seems likely t h a t both enzymes are synthesized on cytoplasmic ribosomes. However, it cannot be ruled out t h a t cytoplasmic protein synthesis leads to activation of a latent enzyme. There is evidence t h a t in Chlorella NAR can exist in a latent form (Moreno et al., 1972). On the other hand, the labeling experiments of Zielke and Filner (1971) show t h a t the induction of N A R by nitrate in cultured tobacco cells is a result of de novo protein synthesis. The synthesis of N I R in the cytoplasm does not rule out its localization inside the chloroplast. I n our previous paper (Sluiters-Scholten et al., 1973) we found aminolevulinate dehydratase from bean leaves to be manufactured in the cytoplasm, although we and m a n y other authors have shown t h a t this enzyme is at least partly localized in the chloroplast. Before the induction of N A R and N I R can occur, some chloroplast development must have taken place. I n green leaves the induction of the enzymes is not affected by CAP. When the light-induced chloroplast development is inhibited b y CAP (Smillie et al., 1968) from the beginning of the illumination period no induction of NAR and N I R is observed. Applied after 24 h of illumination CAP has no effect on the induction of NAR and N I R . Sawhney and Naik (1972) suppose t h a t photosynthetic reactions are necessary for the synthesis of N A R and N I R . The results of their experiments with DCMU suggest t h a t a non-cyclic electron transport is required to allow the induction. I n our case, after 24 h of illumination the chloroplast development should be advanced far enough for electron transport. Gyldenholm and Whatley (1968) found between 15 and 25 h of illumination of 14- to 16-day-old leaves of ctiolated bean seedlings a distinct increase of the ability t o photorcduce N A D P with water as electron donor. I wish to thank Miss A. KSnst and Mrs. N. Stoter-Tims for their technical assistance. This investigation was supported by the Netherlands Organization for the Advancement of Pure Scientific Research (Z.W.O.), Grant 82003.
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