Planta (Berl.) 123, 175--184 (1975) 9 by Springer-Verlag 1975

Photosynthesis and the Induction of Nitrate Reductase and Nitrite Reductase in Bean Leaves* C. M. Th. Sluiters-Scholten Department of Plant Physiology, University of Amsterdam, IJdijk 26, Amsterdam, The Netherlands Received 28 June; accepted 23 December 1974 Summary. In leaves of Phaseolus vulgaris L. ev. Prelude, the light-induced increase in activity of NADH-nitrate oxidoreductase (E.C.1.6.6.2; NAR) and reduced benzylviologennitrite oxidoreductase (E.C.1.6.6.4; NIle) starts at a certain stage in the development of the chloroplasts. In leaves with completely developed chloroplasts, a higher increase in activity of NAR and NIR is observed, after induction by the addition of nitrate, in the light than in the dark. DCMU inhibits the increase in activity of the two enzymes in the light. Both in the light in the presence of DCMU, and in the dark the increase in activity reaches a higher level by the addition of sucrose. Induction of NAI~, but not of NIR, can be observed in excised etiolated leaves. No induction is found in leaves of intact etiolated seedlings. The relation between photosynthetic reactions and the increase in activity of NAR and NIR is discussed. It is suggested that IqADtt, indirectly formed by photosynthesis, protects NAR and affects in this way the balance between synthesis and breakdown of the enzyme. The increase in activity of ~IR is possibly influenced by the presence of reduced ferredoxin.

Introduction Light is an important factor in the induction of N A R (NADH-nitrate oxidoreductase; E.C.1.6.6.2) and N I R (reduced benzylviologen-nitrite oxidoreductase, E.C.1.6.6.4) by nitrate (see Beevers and Hageman, 1969). Kannangara and Woolhouse (1967) suggest that the synthesis of N A R is dependent on active photosynthesis, for synthesis of the enzyme was dependent on both light and COs. According to Sawhney and Naik (1972) non-cyclic photosynthetic electron transport is important for the induction of NAR and NIR. Travis et al. (1970) assume that sugars formed by photosynthesis are essential in the induction of N A R by supplying energy for the protein-synthesizing system. I n bean leaves an increase in activity of NAR and N I R is found only when at least partly developed chloroplasts are present. This suggests that the chloroplast is involved in the induction of NAR and N I R (Sluiters-SchoIten, 1973). The synthesis of N A R and M R in bean leaves occurs on cytoplasmic ribosomes (Sluiters-Scholten, 1973). I t is unlikely that the only role of the chloroplast in the induction of N A R and N I R is to supply energy for protein synthesis in the cytoplasm. I n etiolated bean seedhngs some enzymes, such as aminolevalinate dehydratase and glucose-6-phosphate dehydrogenase, are formed during illumination in the cytoplasm when the light-induced chloroplast development is * Abbreviations: CAP, D-threo-chloramphenicol; DCMU, 3-(3,4-dichlorophenyl)-l,l-dimethylurea; NAR, nitrate reduetase; NIR, nitrite reductase.

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inhibited by CAP, an inhibitor of chloroplast protein synthesis. Thus, protein synthesis is possible in the absence of functional chloroplasts (Sluiters-Scholten et al., 1973). N A R and NIR, however, are only formed when CAP is not given before 24 h of illumination, a time at which considerable development of the chloroplasts has occurred (Shiiters-Scholten, 1973). This means that there is another way in which the chloroplast plays a role in the induction of N A R and N I R in bean leaves. Next to induction of N A R and NII~ by nitrate the activity of the enzymes is dependent upon photosynthesis. Klepper et al. (1971) suggest that sugars migrating from the chloroplast to the cytoplasm are the prime source of NADtt, the electron donor of NAR. N A D H itself is formed in the cytoplasm by the oxidation of glyceraldehyde-3-phosphate. For the reduction of nitrite, electrons are derived from the non-cyclic photosynthetic electron transport via ferredoxin, the natural electron donor of IqIR (Joy and Hageman, 1966; Ramirez et al., 1966). I n the experiments to be reported in this paper it was investigated in which way the chloroplast influences the levels of NAR and NIR.

Material and Methods Plant Material. All experiments were carried out with one batch of seeds of Phaseolus vulgaris L., cv. Prelude, obtained from Pannevis en West-Friesland, Enkhuizen, The Netherlands. The batch of seeds is different from that used in our previous experiments. With the present seeds the inhibition by CAP of the increase in activity of NIR during the light-induced chloroplast development is lower. Induction Experiments and Enzyme Assays. The greening experiments and the induction experiments with nitrate in green leaves were carried out as described previously (SluitersSeholten, 1973; Sluiters-Scholten et al., 1973); so were the homogenization of leaves or leaf discs, the NA1%assay with NADtt, the NIR assay with reduced benzyl viologen, and protein determination (Sluiters-Scholten, 1973). The greening experiments were carried out twice, the other experiments were repeated two times at least. Tables 3 and 5 give the mean values of 3 experiments. The other tables and the figures represent typical experiments. Sources o/Chemicals. Benzyl viologen and Folin and Ciocalteu's phenol reagent: British Drug House, Poote, U.K.; CAP: de Watermolen, Zaandam, The Netherlands; DCMU: K & K Labs., Plainview, N. Y., USA; yeast alcohol dehydrogenase and NADtt: Boehringer, Mannheim, Germany. Results

Induction o / N A R a n d / Y I R during Light-induced Chloroplast Development During the light-induced chloroplast development the increase in activity of N A R and N I R is inhibited by CAP when it is given at the beginning of the illumination period. When CAP is applied after 24 h of illumination no effect of the drug is found (Sluiters-Scholten, 1973). ]Fig. 1 shows She effect of CAP (500 ~zg]ml) when given at 0, 5, 10 or 20 h after the beginning of the illumination period. CAP addition does not affect the increase in activity during the first 24 h of illumination. When CAP is given at 0 and 5 h after the onset o~ illumination, an inhibitory effect is found after 24 h. When CAP is given after 10 and 20 h of illumination, an inhibitory effect is seen

Photosynthesis and Induction of Nitrate and Nitrite Reductase

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Fig. 1. Effect of CAP, applied after different periods of illumination, on the light-induced increase in activity of NAR (left) and NIR (right) of leaves of 10-day-old etiolated Phaseolus vulgaris seedlings grown in the presence of 0.01 M K-N08. • Light control; o CAP (500 t~g/ml) applied at the beginning of the illumination period; 9 CAP after 5 h of light; [] CAP after 10 h of light; ~ CAP after 20 h of light

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Fig. 2. Effect of different concentrations CAP, applied at the beginning of the illumination period, on the light-induced increase in activity of NAI~ (left) and NIR (right) of leaves of 10-day-old etiolated Phaseolus vulgaris seedlings grown in the presence of 0.0t M I~NOa. x Light control; 9 CAP, 100 t~g/ml; 9 CAP, 500 ~g/ml

b e t w e e n 24 a n d 48 h a f t e r t h e beginning of t h e illumination, a n d a s u d d e n increase is f o u n d a f t e r 48 h. W h e n C A P is given a t t h e s t a r t of t h e i l l u m i n a t i o n p e r i o d a t a c o n c e n t r a t i o n of 100 ~zg/ml r a t h e r t h a n 500 ~g/ml, t h e i n h i b i t o r y effect is no longer p r e s e n t a f t e r 48 h (Fig. 2). T h e s e results suggest t h a t some chloroplast d e v e l o p m e n t is n e e d e d before a n i n c r e a s e in a c t i v i t y of N A R a n d N I R occurs. I n t h e presence of 500 ~ g / m l of C A P t h e chIoroplast d e v e l o p m e n t is n o t i n h i b i t e d c o m p l e t e l y , b u t goes on, a l t h o u g h slowly, a n d some chlorophyll s y n t h e s i s occurs (Sluiters-Scholten, 1973; SluitersS e h o l t e n et al., 1973). W h e n C A P is a p p l i e d a f t e r 10 or 20 h of i l l u m i n a t i o n , t h e c h l o r o p l a s t has p r o b a b l y r e a c h e d a stage a t which a n increase in a c t i v i t y of N A R a n d ~ - I R is possible a f t e r 48 h. A t a c o n c e n t r a t i o n of 100 ~zg/ml C A P inhibits t h e

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Table 1. Effect of light on the induction of NAR and NII~ by nitrate in green leaves o Phaseolus vulgaris grown in the absence of nitrate a Enzyme activityb

Protein content

(rag/25 leaf discs) Light, Light, Dark, Dark,

-- nitrate -{- nitrate -- nitrate ~- nitrate

NAI~

NIR

0.13 2.14 0.i0 0.68

3.2 17.3 1.5 7.1

11.7 12.6 11.0 11.1

a Nitrate (0. ! M KN03) was added at the beginning of the induction period. Induction for 18 h in light or in darkness, as indicated. b NAR ~ ~mol NO 2- produced/h/25 leaf discs; NIR = ~mol NO 2- reduced/h/25 leaf discs.

Table 2. Effect of DCMU on the induction of NAR and ~/II~ by nitrate in green leaves of Phaseolus vulgaris grown in the absence of nitrate a DCMU (~M) 0 10 50 100

Enzyme activityb NAR

NIR

2.06 1.42 0.99 0.66

15.4 9.7 9.3 3.9

Protein content (rag/25 leaf discs) 16.4 13.9 12.0 14.6

a Nitrate (0.1 M KNOs) and DCMU were added at the beginning of the induction period. Induction for 18 h in the light. b NAR ----~mol N0a- produced/h/25 leaf discs; NIR ~ fzmol NO 2- reduced/h/25 leaf discs.

s y n t h e s i s of chlorophyll b y a b o u t 60%, a n d chloroplast d e v e l o p m e n t is f a s t e r t h a n a t t h e h i g h e r c o n c e n t r a t i o n so t h a t t h e c h l o r o p l a s t h a s r e a c h e d a stage a t w h i c h a n increase in a c t i v i t y of N A R a n d N I R is possible a f t e r 48 h of i l l u m i n a t i o n , e v e n w h e n C A P is a d d e d a t t h e s t a r t of t h e illumination.

Induction o / N A R and M R in Green Leaves F r o m t h e p r e v i o u s e x p e r i m e n t s i t is clear t h a t a c e r t a i n c h l o r o p l a s t developm e n t is n e e d e d for a m a r k e d increase in a c t i v i t y of N A R a n d lk~IR. This effect could be d u e t o p h o t o s y n t h e s i s or to t h e f o r m a t i o n of a s t r u c t u r a l c h l o r o p l a s t c o m p o n e n t . To t e s t t h i s a l t e r n a t i v e , i n d u c t i o n e x p e r i m e n t s were carried o u t w i t h l e a v e s in which chloroplasts h a d f u l l y developed. I n g r e e n leaves a n increase in a c t i v i t y of N A R a n d N I R is f o u n d a f t e r t h e a d d i t i o n of n i t r a t e . This increase in a c t i v i t y is m u c h higher in t h e l i g h t t h a n in t h e d a r k (Table 1), i n d i c a t i n g t h a t f u n c t i o n a l chloroplasts are needed. I f t h e effect of t h e c h l o r o p l a s t d e v e l o p m e n t was due t o t h e f o r m a t i o n of a s t r u c t u r a l c o m p o n e n t of t h e chloroplast, c o m p a r a b l e p a t t e r n s of e n z y m e activities were to be e x p e c t e d in light a n d d a r k . H o w e v e r , t h e r e is no a b s o l u t e r e q u i r e m e n t for light since a n increase in a c t i v i t y of t h e e n z y m e s is f o u n d also in t h e d a r k .

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Table 3. Effect of sucrose on the induction of NAR and NIR by nitrate in the dark in green leaves of Phaseolus vulgaris grown in the absence of nitrate a Sucrose

Enzyme activityb

Protein content (rag/25 leaf discs)

(%)

NAI~

NIR

0 0.5 1 2

0.76 4- 0.069 1.13 ~ 0.221 1.33 :j=0.051 1.64 g- 0.183

6.1 ~ 1.90 9.7 ::]=2.44 11.6 :j= 2.46 14.5 4- 3.58

15.5 :J=1.35 16.0 4- 2.17 16.9 :J=2.74 16.9 • 1.86

a Nitrate (0.1 M KNOa) and sucrose were added at the beginning of the induction period. Induction for 18 h in the dark. b NAR = ~mol NO2- produced/h/25 leaf discs; NIR = ~mol NO 2- reduced/h/25 leaf discs.

Table 4. Effect of sucrose on the induction of NAI~ and NIR by nitrate in the presence of DCMU in green leaves of Phaseolus vulgaris grown in the absence of nitrate a Additions

Enzyme activityb

None 2% sucrose 100 ~M DCMU 100 y2r DCMU~-2% sucrose

NAR

NII~

1.80 2.28 0.27 1.06

16.7 26.3 2.8 8.7

Protein content (mg/25 leaf discs) 14.8 18.4 13.4 15.3

a Nitrate (0.1 1~I KNOa), sucrose and DCMU were added at the beginning of the induction period. Induction for 18 h in the light. b NAR = ~mol NO2- produced~h/25 leaf discs; NIR = ~mol NO 2- reduced/h/25 leaf discs.

Non-cyclic p h o t o s y a t h e t i c electron t r a n s p o r t is i n h i b i t e d b y DCMU. I n t h e presence of D C M U t h e increase in a c t i v i t y of b o t h N A R a n d N I R in t h e l i g h t is i n h i b i t e d (Table 2). Thus, non-cyclic electron t r a n s p o r t is i m p o r t a n t in t h e increase in a c t i v i t y of N A R a n d N I R , b u t t h e r e q u i r e m e n t is a g a i n n o t absolute. To e x a m i n e t h e w a y in which t h e i a c r e a s e in activities of N A R a n d N I R is influenced b y t h e p h o t o s y n t h e t i c electron t r a n s p o r t , sucrose was a d d e d d u r i n g t h e i n d u c t i o n in t h e d a r k (Table 3) a n d d u r i n g t h e i n d u c t i o n in t h e light in t h e presence of D C M U (Table 4). I n b o t h cases a s t i m u l a t i o n of t h e increase in a c t i v i t y was found. These results suggest t h a t r e d u c t i o n equivalents, f o r m e d b y p h o t o synthesis, p l a y a role in t h e increase in a c t i v i t y of b o t h N A R a n d N I l e . T h e l a c k of a n a b s o l u t e r e q u i r e m e n t for p h o t o s y n t h e s i s is p r o b a b l y d u e t o t h e f a c t t h a t some sugars a r e still p r e s e n t in t h e leaf discs a t t h e s t a r t of t h e i n d u c t i o n period.

Induction o[ N A R and N I R in Etiolated Leaves N o increase in t h e activities of N A R a n d N I R is o b s e r v e d in leaves of i n t a c t , e t i o l a t e d b e a n seedlings, e v e n w h e n n i t r a t e was a v a i l a b l e (Sluiters-Scholten, 1973).

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Table 5. Induction of NAR and NIR in the dark in excised etiolated leaves of Phaseolus vulgaris seedlings grown in the presence of nitrate a Additions

None 2% sucrose 0.1 MKN03 0.1 ~ K N O a - ~ 2 % sucrose

Enzyme activityb NAR

NIR

0.03 • 0.017 0.21 :t: 0.081 1.02• 1.59•

3.2 ~: 1.25 3.4• 6.2• 8.7~2.69

Protein content (mg/i0 leaf pairs)

10.1 ~: 2.70 12.0 :~:4.06 10.0• 11.0=]=2.66

a 10 leaf pairs of 6-day-old etiolated seedlings, grown in the presence of 0.01 ~ KNO 3, were floated for 24 h in the dark in Petri dishes with 50 ml of the medium used in the induction experiments with nitrate, and treated in the same way as the leaf discs. b NAR = Ezmol NO z- produced/h/10 leaf pairs; NIR = y.mol NOz- reduced/h/10 leaf pairs.

H o w e v e r , in y o u n g e t i o l a t e d seedlings e n o u g h sugars a r e p r e s e n t to m a k e a n increase in a c t i v i t y of N A R a n d N I R possible. To e x a m i n e if a n y i n d u c t i o n of N A R a n d l q I R is possible in e t i o l a t e d leaves, t h e l e a v e s were excised f r o m t h e seedlings a n d f l o a t e d in t h e d a r k o n a m e d i u m w i t h v a r i o u s a d d i t i o n s (Table 5). T h e seedlings were g r o w n in t h e presence of n i t r a t e so t h a t t h e leaves c o n t a i n e d a c e r t a i n a m o u n t of n i t r a t e . I n t h e a b s e n c e of exogenous n i t r a t e a n increase in a c t i v i t y of N A R is o n l y f o u n d w h e n sucrose is a d d e d . W h e n exogenous n i t r a t e is a d d e d , t h e r e is a n increase in a c t i v i t y of N A R ; t h i s is e n h a n c e d b y t h e a d d i t i o n of sucrose. I n c o n t r a s t t o N A R , no clear increase in t h e a c t i v i t y of N I R is found. T h e s e results i n d i c a t e t h a t in e t i o l a t e d l e a v e s i n d u c t i o n of N A R is possible. T h e e x t e n t t o which t h e a c t i v i t y of t h e e n z y m e increases is d e p e n d e n t u p o n n i t r a t e a n d sucrose. T h e i n d u c t i o n of N I R seems t o be r e g u l a t e d in a different fashion.

EHect ol N A D H on the Activity of N A R in a Crude Extract T h e electron d o n o r of N A R is N A D H . Sugars f o r m e d b y p h o t o s y n t h e s i s a r e t h e p r i m e source of 5~ADH ( K l e p p e r et al., 1971). I n a crude e x t r a c t of b e a n l e a v e s N A R is, in c o n t r a s t t o N I R , v e r y labile. A f t e r 30 m i n a t 0 ~ a b o u t 50% of t h e a c t i v i t y of N A R h a s d i s a p p e a r e d . A f t e r 30 rain a t 20 ~ t h e a c t i v i t y d e c r e a s e d b y 64% (Table 6). H o w e v e r , d u r i n g t h e a s s a y of N A R a t 30 ~ t h e a c t i v i t y is for a t l e a s t 40 rain l i n e a r w i t h time. This i n d i c a t e s t h a t a c o m p o n e n t of t h e i n c u b a t i o n m i x t u r e h a s a stabilizing effect on N A R . To t e s t this, n i t r a t e a n d N A D H were a d d e d t o a p a r t of t h e leaf e x t r a c t i m m e d i a t e l y a f t e r t h e h o m o g e n i z a t i o n . T a b l e 7 shows t h a t in t h e presence of n i t r a t e t h e a c t i v i t y of t h e e n z y m e decreases t o t h e s a m e e x t e n t as in t h e absence of t h e s u b s t r a t e . H o w e v e r , w h e n N A D H is a d d e d t h e loss of a c t i v i t y is m u c h less, b o t h a t 0 ~ a n d a t 25 ~ I t seems t h a t in a crude e x t r a c t of b e a n leaves N A R is s t a b i l i z e d b y its e l e c t r o n d o n o r N A D H .

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Table 6. Stability of NAR and NIR in a crude extract of leaves of Phaseolus vulgaris a Time of enzyme activity determination

Enzyme activity (%)

Immediately after homogenization After 30 min at 0 ~ After 30 min at 25 ~

NAR

NIR

100

100

53 36

102

a The extract was made from leaves of 10-day-old etiolated seedlings, illuminated for 96 h. NAR: 100 % = 9.8 tzmo! NO2- produced/h/10 leaf pairs; NIR: 100 % = 184 ~zmolNO2- reduced/ h/10 leaf pairs.

Table 7. Effect of N i D H and nitrate on the activity of nitrate reductase in a crude extract of leaves of Phaseolus vulgaris a Timo of enzyme activity determination

Immediately after homogenization After 30 rain at 0 ~ After 30 rain at 25~

N A R activity (%)

No additions

+ NADH b

A- KNOa b

100

100

100

53 36

90 70

47

a The extract was made from leaves of 10-day-old etiolated seedlings, illuminated for 96 h. 100% = 9.8 ~mol NO2- produeed/h/lO leaf pairs. b NADH (1 mg/ml) and KNO a (0.1 M) were added to a part of the leaf extract immediately after homogenization.

Discussion T h e i n d u c t i o n of N A R a n d IXrIR b y n i t r a t e in b e a n leaves is influenced b y t h e presence of r e d u c t i o n equivalents. W h e n r e d u c t i o n e q u i v a l e n t s are f o r m e d b y p h o t o s y n t h e s i s , o r w h e n in t h e a b s e n c e of p h o t o s y n t h e s i s sucrose is a d d e d , a d i s t i n c t increase in a c t i v i t y of N A R a n d N I R is found. G y l d e n h o l m a n d W h a t l e y (1968) a n d others r e p o r t t h a t t h e different comp o n e n t s of t h e e l e c t r o n - t r a n s p o r t chain are n o t s y n t h e s i z e d s i m u l t a n e o u s l y d u r i n g l i g h t - i n d u c e d chloroplast d e v e l o p m e n t . R e d u c t i o n e q u i v a l e n t s can o n l y be f o r m e d w h e n t h e l a s t c o m p o n e n t of t h e e l e c t r o n - t r a n s p o r t chain h a s been synthesized. C A P blocks t h e f o r m a t i o n of some c o m p o n e n t s of t h e e l e c t r o n - t r a n s p o r t chain ( G r e g o r y a n d B r a d b e e r , 1973). W h e n C A P is a d d e d w h e n t h e electront r a n s p o r t c h a i n is n o t y e t c o m p l e t e d no increase in a c t i v i t y of N A R a n d N I R is found. A f t e r 24 h of i l l u m i n a t i o n t h e chloroplast has d e v e l o p e d f a r e n o u g h t o allow t h e f o r m a t i o n of r e d u c t i o n e q u i v a l e n t s ( G y l d e n h o l m a n d W h a t l e y , 1968). A c c o r d i n g l y , C A P a p p l i e d a f t e r 24 h h a s n o effect on t h e increase in a c t i v i t y of t h e enzymes.

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The question is how the supply of reduction equivalents regulates the increase in activity of N A R and N I R . N A R is a very labile enzyme. I t s turn-over in vivo is very high (see Beevers and Hageman, 1969). According to Travis et al. (1969) the level of the enzyme is determined b y continuous, balanced synthesis and breakdown. The latter process in itself is apparently dependent on the synthesis of protein, presumably of a "breakdown enzyme". I n vitro, N A R is very labile, too. I n a crude extract of bean leaves the presence of N A D H limits the decrease in activity of NAR. Gandhi et al. (1973) found t h a t addition of N A D H to a crude extract of rice seedlings caused after 1 h at both 0 ~ and 25 ~ a higher level of NAR. The initial level was not reported. These results can be explained b y assuming a protective effect of NADH, rather t h a n an activation of the enzyme. Possibly N A D H is a stabilizing factor for N A R also in rive, protecting the enzyme against the "breakdown enzyme". During the first 24 h of chloroplast development and in the presence of CAP, applied at an early stage, induction of N A R is possible, for nitrate is present. I n this case the rate of synthesis and the rate of breakdown of the enzyme are in balance and only a small increase in the activity of NAR, if any, is found. When reduction equivalents can be formed the rate of synthesis of N A R is greater t h a n the rate of breakdown. When leaves are excised from etiolated seedlings, an increase in activity of N A R in the dark is found under certain conditions. I n the absence of exogenous nitrate the rate of synthesis is equal to or smaller than the rate of breakdown of the enzyme, whereas in the presence of sucrose the breakdown is less. I n the presence of exogenous nitrate the rate of synthesis of the enzyme is higher. When in addition to exogenous nitrate sucrose is added, the difference between synthesis and breakdown becomes still greater. I n leaves of intact, etiolated bean seedlings no increase in activity of N A R is found (Shiiters-Scholten, 1973). According to Oaks et al. (1972) no induction of N A R occurs when no formation of amino acids is needed. Etiolated seedlings contain large amounts of reserve protein and amino acids. Besides, the induction of N A R is in m a n y cases inhibited b y amino acids (Boutard, 1966; Filner, 1966). The increase in activity of iNIR seems also to be influenced by the formation of reduction equivalents. The induction of N I R does not depend on the presence of an active N A R because N I R can be induced by nitrate in the presence of an inactive iNAR (Chroboczek-Kelker and Filner, 1971; Stewart, 1972). Reduced ferredoxin, the natural electron donor of N I R , is perhaps necessary for the induction of N I R . I t is improbable t h a t ferredoxin influences the stability of 1NIR. The turn-over of N I R is much lower t h a n t h a t of N A R (see Beevers and Hageman, 1969). I n vitro 1NIR is also much more stable t h a n NAR. I n the dark, and in the light in the presence of DCMU sucrose stimulates the induction of N I R . Here ferredoxin can be reduced b y N A D P H via N A D P H ferredoxin oxidoreductase (Losada et al., 1963). iNIR activity is also found in non-green tissues like roots. I n these tissues probably a ferredoxin-like substance is present, as is suggested b y Bourne and Miflin (1973). I n etiolated bean leaves, in which ferredoxin is scarcely present (Melandri et al., 1969), no 1NIR activity is observed, nor is it found in granaless chloroplasts of maize (Mellor and Tregunna, 1971).

Photosynthesis and Induction of Nitrate and Nitrite l~eductase

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T h e m o d e of r e g u l a t i o n of N I R a c t i v i t y b y r e d u c e d f e r r e d o x i n is n o t clear. I t is possible t h a t r e d u c e d f e r r e d o x i n influences t h e f o r m a t i o n of a r e g u l a t o r protein. I n leaves of Perilla/rutescens glucose can n o t r e p l a c e light in t h e i n d u c t i o n of N A R ( K a n n a n g a r a a n d Woolhouse, 1967). I n rice seedlings t h e i n h i b i t i o n of N A R a n d N I R i n d u c t i o n b y DCMU is n o t a b o l i s h e d b y sucrose ( S a w h n e y a n d N a i k , 1972). These a u t h o r s conclude t h a t r e d o x changes, as a r e s u l t of Hill a c t i v i t y , p l a y a role in t h e i n d u c t i o n of b o t h enzymes. H o w e v e r , in granaless chloroplasts Hill a c t i v i t y (Bishop et al., 1971) b u t no NIl% a c t i v i t y is found. F r o m t h e p r e s e n t results it seems l i k e l y t h a t in b e a n leaves p h o t o s y n t h e s i s r e g u l a t e s t h e a c t i v i t i e s of N A R a n d N I R b y t h e f o r m a t i o n of r e d u c t i o n equivalents. I t is possible t h a t in different p l a n t species t h e r e g u l a t i o n of N A R a n d N I R occurs along different ways. I wish to thank Miss A. KSnst for her 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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Mellor, G. E., Tregunna, E. B.: The localization of nitrate-assimilating enzymes in leaves of plants with the C4-pathway of photosynthesis. Canad. J. Bot. 49, 137-142 (1971) Oaks, A., Wallace, W., Stevens, I).: Synthesis and turnover of nitrate reduetase in corn leaves. Plant Physiol. 50, 649-654 (1972) Ramirez, J. )r del Campo, F . F . , Paneque, A., Losada, M.: Ferredoxin-nitrite reductase from spinach. Biochim. biophys. Aeta (Amst.) 118, 58-71 (1966) Sawhney, S. K., Naik, M. S. : Role of light in the synthesis of nitrate reductase and nitrite reductase in rice seedlings. Biochem. J. 180, 475-485 (1972) Sluiters-Scholten, C. M. Th.: Effect of ehloramphenicol and eyeloheximide on the induction of nitrate reductase and nitrite reductase in bean leaves. Planta (Berl.) 118, 229-240 (1973) Sluiters-Scholten, C. ~ . Th., Berg, F. M., van den, Stegwee, D. : Aminolaevulinatedehydratase in greening leaves of Phaseolus vulgaris L. Z. Pflanzenphysiol. 69, 217-227 (1973) Stewart, G. R. : Regulation of nitrite reductase level in Lemna minor. J. exp. Bot. 28, 171-183 (1972) 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., Jordan, W. R., Huffaker, R. C. : Evidence for an inactivating system of nitrate reduetase in Hordeum vulgare L. during darkness that requires protein synthesis. Plant Physiol. 44, 1150-1156 (1969)

Photosynthesis and the induction of nitrate reductase and nitrite reductase in bean leaves.

In laaves of Phaseolus vulgaris L. cv. Prelude, the light-induced increase in activity of NADH-nitrate oxidoreductase (E.C.1.6.6.2; NAR) and reduced b...
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