Planta (Berl.) 95, 227-246 (1970) 9 by Springer-Verlag 1970
The Effect of Nitrate and Nitrite on Oxygen Evolution and Carbon-Dioxide Assimilation and the Reduction of Nitrate and Nitrite by Intact Chloroplasts* B. 1%. GUAZ~T** a n d D. T. CAz~vI~ Department of Biology, Queen's University, Kingston, Ontario, Canada Received June 22 / August 31, 1970
Summary. Intact chloroplasts isolated from spinach reduced N0~- and NOs- in the light without the addition of either co-factors or added enzymes. The maximum rate observed, however, for the reduction of NOa- was approximately 3 ~Moles hr -~ mg -1 (chlorophyll) and for N 0 s - 6 tzMoles hr -1 mg -1 (chlorophyll). These rates were consistent with the enzyme content of whole chloroplasts, but much lower than those found in whole leaf extracts. The addition of both NO3- and N Q - in low concentrations resulted in transient increases in both 02 evolution and COs fixation. The increases in oxygen evolution were not consistent in amount and bore no relation to the amount of substrate reduced. Similar transients were observed in a number of experiments when NaC1 or NHaC1 were added. The addition of NO2- at concentrations of 10-t M and above resulted in marked inhibition of both Os evolution and COs fixation. NO s- appears to inhibit by blocking the reduction of NADP. NO 3- at similar concentrations had no such effect. An increase in the soluble amino nitrogen content of the chloroplasts was observed when N O a or :NOs- was reduced. There was, however, no increase in the incorporation of sac from 14COs into amino acids under these conditions. Even with the addition of ammonia the amount of 14C incorporated into the amino acids was not changed from less than 5 % of the total saC fixed. We conclude that while intact chloroplasts do have the ability to reduce both NO~- and NO s- at low rates, they do not synthesize appreciable amounts of amino acid directly, and this fact must be considered when formulating any pathways for nitrogen metabolism during photosynthesis. Introduction C h l o r o p l a s t s , w h e n i l l u m i n a t e d , will r e d u c e n i t r i t e to a m m o n i a ( I t u z i s i g e a n d S a t o h , 1961 ; P a n e q u e et al., 1964) if t h e p r e p a r a t i o n s are supplemented with both nitrite reductase and ferredoxin. Under similar e x p e r i m e n t a l c o n d i t i o n s , b u t w i t h t h e f u r t h e r a d d i t i o n of n i t r a t e r e d u c t a s e it is possible to d e m o n s t r a t e t h a t n i t r a t e is r e d u c e d t o e i t h e r N O 2 or NH~ + ( L o s a d a et al., 1965; P a n e q u e et al., 1969). T h e s e e x p e r i m e n t s * Supported in part by the ~ational Research Council of Canada. ** On leave from CSIRO Marine Laboratory, Cronulla, Australia.
228
B. 1% Grant and D. T. Canvin:
d e m o n s t r a t e clearly t h a t t h e reducing power g e n e r a t e d in p h o t o s y n t h e s i s can be coupled via t h e a p p r o p r i a t e enzymes a n d cofactors to reduce b o t h NO~- a n d NO2-. These e x p e r i m e n t s were carried out, however, with chloroplasts p r e p a r e d in high-sMt osmotica, a m e t h o d k n o w n to cause considerable loss of s t r o m a p r o t e i n (Jacobi, 1963; W a l k e r , 1967), a n d therefore p r o v i d e only indirect evidence for t h e role t h a t chloroplasts p l a y in NO 2- or N O s - r e d u c t i o n in vivo, in light. Chloroplasts isolated u n d e r conditions which r e t a i n e d m o s t of t h e s t r o m a p r o t e i n c o n t a i n e d some n i t r a t e a n d n i t r i t e r e d u c t a s e ( G r a n t et al., 1970) b u t t h e larger p o r t i o n of b o t h enzymes was l o c a t e d outside of t h e chloroplast, a result in general a g r e e m e n t w i t h t h e findings of other workers (l~itenour et al., 1967). I n t h e p r e s e n t p a p e r we p r e s e n t evidence to show t h a t these i n t a c t chloroplasts will assimilate b o t h NO 3- a n d NO 2in t h e light w i t h o u t a d d i t i o n of either enzymes or cofaetors. The rates o b s e r v e d are low, b u t c o m p a r a b l e w i t h those p r e d i c t e d from the e n z y m e content. W h i l e t h e r e d u c t i o n of N Q - or NO C b y t h e chloroplast resulted in increases in a m i n o nitrogen, such r e d u c t i o n was n o t a c c o m p a n i e d b y an increase in t h e flow of carbon from CO 2 f i x a t i o n into t h e a m i n o acids. The results d e m o n s t r a t e t h a t t h e isolated chloroplast is n e i t h e r a m a j o r site for t h e r e d u c t i o n of oxidized forms of N or a m a j o r site of de-novo amino acid formation.
Materials and Methods Plant Material. Spinach (Spinacia oleraeea L. cv. Brookwood) was grown as previously described (Grant et al., 1970). The photosynthetic rate of the leaves was determined using the leaf chamber described by Atkins (1969) and the apparatus described by Ludwig (1968). Chloroplast Isolation. The methods described in a previous paper (Grant et al., 1970) were used and differ only slightly from the method of Jensen and Bassham (1966). In all of these experiments chloroplasts sedimenting at 500• g in 90 sec (designated P1) were used. Each preparation was examined under the phase microscope at the beginning of a series of experiments and in several experiments samples of chloroplasts from the incubation medium after I0 and 20 rain in the light were also examined, l~epresentative preparations were fixed in 5% glutaraldehyde and electron micrographs prepared of them after dehydration and embedding in Epon (Leech, 1964). Chlorophyll Determination. An aliquot of chloroplast suspension was sedimented at 1000 • g for 10 rain and the chlorophyll content determined by the method of Arnon (1949). Reaction Conditions. A modification of Medium C of Jensen and Bassham (1966) was used in all experiments and contained sorbitol, 0.33 M; HEPES (N-2-hydroxyethyl piperazine-N-2-ethanesulfonie acid) buffer, pI-I 7.6, 0.05 M; K 2 EDTA, 2.0 mM (Na2 EDTA was occasionally used without adverse results) ; MgC12, 1.0 raM; MnC12, 1.0 m ~ ; KH~PO~ 0.5 raM; N%P20~, 5 raM; dextran (M. W. 34000-43000) 2%, w/v. In a number of experiments pyrophosphate was omitted without any decrease in the rate of O2 evolution.
The Effect of Nitrate and Nitrite on Oxygen Evolution
229
The reaction mixture contained 1.85-2.0 ml Medium C, 0.2 ml chloroplast suspension (25-100 ~xg chlorophyll), and additions as specified in Results in a total volume of 2.2 ml. The reaction mixture was flushed with N 2 before the addition of the chloroplast suspension to reduce the 02 concentration to approximately 20% of air saturation. Other reaction conditions were as specified earlier (Grant et al., 1970), or as specified in Results. Oxygen Evolution and CO2 Fixation. Oe evolution and C02 fixation were measured as described previously (Grant et al., 1970). Nitrite and Nitrate Assimilation. Chloroplasts were centrifuged from an aliquot of incubation mixture, at 1000 • g for 10 rain at 4~ and the NO 2- or N Q - concentration of the supernatant measured. NO2- was measured directly by diazotization with sulfanilamide and coupling to N-l-naphthylethylenediamine as described in Hewitt and Nicholas (1964). NO a- was estimated as nitrite after reduction on a cadmium-copper reductor (Wood et al., 1967) with the ammonia-ammonium ehloride buffer of Grasshof (1964). No NO2- or NO3- could be detected in the chloroplast preparations used in these experiments. Amino Nitrogen. Samples of chloroplasts were removed following incubation, frozen and thawed, and the ruptured chloroplasts centrifuged out at 3 000 • g for 10 rain at 4~ Amino-N was determined by the method of Yemm and Cocking (1955), using a DL-alanine standard. Products o/Carbon Fixation. At the termination of the incubation period, and while the sample remained in the light, 1 ml of the reaction mixture was withdrawn and added to 0.5 ml of 90 % formic acid. The mixture was extracted and fraetionated into neutral, basic and acidic fractions, using Dowex ion-exchange resins as described by Atkins (1969). Recoveries of radioactivity ranged from 87 to 105%. The products of 14C fixation agreed closely with those observed by Walker (1967) and Bassham et al. (1968a).
Results Chloroplasts isolated as described appeared to be i n t a c t when viewed i n the phase contrast microscope, either a t the beginning of the experim e n t or following i n c u b a t i o n i n the reaction vessel. The m a j o r i t y of the chloroplasts h a d i n t a c t m e m b r a n e s (Figs. 1 a, 2 a) a n d appeared to have a n almost identical morphology to those seen in sections of i n t a c t leaf (Fig. 2 b). Chloroplasts with i n t a c t m e m b r a n e s were readily distinguished from chloroplasts which have lost their outer m e m b r a n e (Fig. 1 b). The chloroplasts exhibited rates of CO s fixation a n d O~ evolution t h a t ranged from 20 to 70 ~SIoles hr -1 mg -1 (chlorophyl]), with the most freq u e n t rates between 40 a n d 50 (Fig. 3). Such values were in good agreem e n t with those reported b y Coekburn et al. (1968). CO s fixation was equal to 03 evolution over the time course of most experiments (Fig. 3), a finding i n agreement with the results of Walker et al. (1968). The CO s fixation rate of the i n t a c t leaves (measured at s a t u r a t i n g light i n t e n s i t y , 300 p p m COs, 25 ~ was between 78 a n d 82 ~Noles hr -1 mg -1 (chlorophyll). A s s i m i l a t i o n o / N i t r i t e . Chloroplast p r e p a r a t i o n s assimilated NO2- in the light a t a rate between 2.5 a n d 6 ~Moles hr -~ mg -1 (chlorophyll)
230
B. 1~. G r a n t and D. T. Canvin:
Fig. 1 a and b. a P1 chloroplast fraction isolated from spinach leaves, b P2 fraction isolated from spinach leaves. (500 • g for 20 rain after removal of P1)
Fig. 2 a und b. a Typical chloroplast from P1 fraction, b Typical chloroplast in intact leaf
The Effect of Nitrate and Nitrite on Oxygen Evolution
Fig. 2 a and b
231
232
B. R. Gr~nt and D. T. Canvin: 400 -air saturation 360 --
320 --
-5 2 8 0 E E
-o 2 4 0 -
~ 200
The Effect of Nitrate and Nitrite on Oxygen Evolution and Carbon-Dioxide Assimilation and the Reduction of Nitrate and Nitrite by Intact Chloroplasts* B. 1%. GUAZ~T** a n d D. T. CAz~vI~ Department of Biology, Queen's University, Kingston, Ontario, Canada Received June 22 / August 31, 1970
Summary. Intact chloroplasts isolated from spinach reduced N0~- and NOs- in the light without the addition of either co-factors or added enzymes. The maximum rate observed, however, for the reduction of NOa- was approximately 3 ~Moles hr -~ mg -1 (chlorophyll) and for N 0 s - 6 tzMoles hr -1 mg -1 (chlorophyll). These rates were consistent with the enzyme content of whole chloroplasts, but much lower than those found in whole leaf extracts. The addition of both NO3- and N Q - in low concentrations resulted in transient increases in both 02 evolution and COs fixation. The increases in oxygen evolution were not consistent in amount and bore no relation to the amount of substrate reduced. Similar transients were observed in a number of experiments when NaC1 or NHaC1 were added. The addition of NO2- at concentrations of 10-t M and above resulted in marked inhibition of both Os evolution and COs fixation. NO s- appears to inhibit by blocking the reduction of NADP. NO 3- at similar concentrations had no such effect. An increase in the soluble amino nitrogen content of the chloroplasts was observed when N O a or :NOs- was reduced. There was, however, no increase in the incorporation of sac from 14COs into amino acids under these conditions. Even with the addition of ammonia the amount of 14C incorporated into the amino acids was not changed from less than 5 % of the total saC fixed. We conclude that while intact chloroplasts do have the ability to reduce both NO~- and NO s- at low rates, they do not synthesize appreciable amounts of amino acid directly, and this fact must be considered when formulating any pathways for nitrogen metabolism during photosynthesis. Introduction C h l o r o p l a s t s , w h e n i l l u m i n a t e d , will r e d u c e n i t r i t e to a m m o n i a ( I t u z i s i g e a n d S a t o h , 1961 ; P a n e q u e et al., 1964) if t h e p r e p a r a t i o n s are supplemented with both nitrite reductase and ferredoxin. Under similar e x p e r i m e n t a l c o n d i t i o n s , b u t w i t h t h e f u r t h e r a d d i t i o n of n i t r a t e r e d u c t a s e it is possible to d e m o n s t r a t e t h a t n i t r a t e is r e d u c e d t o e i t h e r N O 2 or NH~ + ( L o s a d a et al., 1965; P a n e q u e et al., 1969). T h e s e e x p e r i m e n t s * Supported in part by the ~ational Research Council of Canada. ** On leave from CSIRO Marine Laboratory, Cronulla, Australia.
228
B. 1% Grant and D. T. Canvin:
d e m o n s t r a t e clearly t h a t t h e reducing power g e n e r a t e d in p h o t o s y n t h e s i s can be coupled via t h e a p p r o p r i a t e enzymes a n d cofactors to reduce b o t h NO~- a n d NO2-. These e x p e r i m e n t s were carried out, however, with chloroplasts p r e p a r e d in high-sMt osmotica, a m e t h o d k n o w n to cause considerable loss of s t r o m a p r o t e i n (Jacobi, 1963; W a l k e r , 1967), a n d therefore p r o v i d e only indirect evidence for t h e role t h a t chloroplasts p l a y in NO 2- or N O s - r e d u c t i o n in vivo, in light. Chloroplasts isolated u n d e r conditions which r e t a i n e d m o s t of t h e s t r o m a p r o t e i n c o n t a i n e d some n i t r a t e a n d n i t r i t e r e d u c t a s e ( G r a n t et al., 1970) b u t t h e larger p o r t i o n of b o t h enzymes was l o c a t e d outside of t h e chloroplast, a result in general a g r e e m e n t w i t h t h e findings of other workers (l~itenour et al., 1967). I n t h e p r e s e n t p a p e r we p r e s e n t evidence to show t h a t these i n t a c t chloroplasts will assimilate b o t h NO 3- a n d NO 2in t h e light w i t h o u t a d d i t i o n of either enzymes or cofaetors. The rates o b s e r v e d are low, b u t c o m p a r a b l e w i t h those p r e d i c t e d from the e n z y m e content. W h i l e t h e r e d u c t i o n of N Q - or NO C b y t h e chloroplast resulted in increases in a m i n o nitrogen, such r e d u c t i o n was n o t a c c o m p a n i e d b y an increase in t h e flow of carbon from CO 2 f i x a t i o n into t h e a m i n o acids. The results d e m o n s t r a t e t h a t t h e isolated chloroplast is n e i t h e r a m a j o r site for t h e r e d u c t i o n of oxidized forms of N or a m a j o r site of de-novo amino acid formation.
Materials and Methods Plant Material. Spinach (Spinacia oleraeea L. cv. Brookwood) was grown as previously described (Grant et al., 1970). The photosynthetic rate of the leaves was determined using the leaf chamber described by Atkins (1969) and the apparatus described by Ludwig (1968). Chloroplast Isolation. The methods described in a previous paper (Grant et al., 1970) were used and differ only slightly from the method of Jensen and Bassham (1966). In all of these experiments chloroplasts sedimenting at 500• g in 90 sec (designated P1) were used. Each preparation was examined under the phase microscope at the beginning of a series of experiments and in several experiments samples of chloroplasts from the incubation medium after I0 and 20 rain in the light were also examined, l~epresentative preparations were fixed in 5% glutaraldehyde and electron micrographs prepared of them after dehydration and embedding in Epon (Leech, 1964). Chlorophyll Determination. An aliquot of chloroplast suspension was sedimented at 1000 • g for 10 rain and the chlorophyll content determined by the method of Arnon (1949). Reaction Conditions. A modification of Medium C of Jensen and Bassham (1966) was used in all experiments and contained sorbitol, 0.33 M; HEPES (N-2-hydroxyethyl piperazine-N-2-ethanesulfonie acid) buffer, pI-I 7.6, 0.05 M; K 2 EDTA, 2.0 mM (Na2 EDTA was occasionally used without adverse results) ; MgC12, 1.0 raM; MnC12, 1.0 m ~ ; KH~PO~ 0.5 raM; N%P20~, 5 raM; dextran (M. W. 34000-43000) 2%, w/v. In a number of experiments pyrophosphate was omitted without any decrease in the rate of O2 evolution.
The Effect of Nitrate and Nitrite on Oxygen Evolution
229
The reaction mixture contained 1.85-2.0 ml Medium C, 0.2 ml chloroplast suspension (25-100 ~xg chlorophyll), and additions as specified in Results in a total volume of 2.2 ml. The reaction mixture was flushed with N 2 before the addition of the chloroplast suspension to reduce the 02 concentration to approximately 20% of air saturation. Other reaction conditions were as specified earlier (Grant et al., 1970), or as specified in Results. Oxygen Evolution and CO2 Fixation. Oe evolution and C02 fixation were measured as described previously (Grant et al., 1970). Nitrite and Nitrate Assimilation. Chloroplasts were centrifuged from an aliquot of incubation mixture, at 1000 • g for 10 rain at 4~ and the NO 2- or N Q - concentration of the supernatant measured. NO2- was measured directly by diazotization with sulfanilamide and coupling to N-l-naphthylethylenediamine as described in Hewitt and Nicholas (1964). NO a- was estimated as nitrite after reduction on a cadmium-copper reductor (Wood et al., 1967) with the ammonia-ammonium ehloride buffer of Grasshof (1964). No NO2- or NO3- could be detected in the chloroplast preparations used in these experiments. Amino Nitrogen. Samples of chloroplasts were removed following incubation, frozen and thawed, and the ruptured chloroplasts centrifuged out at 3 000 • g for 10 rain at 4~ Amino-N was determined by the method of Yemm and Cocking (1955), using a DL-alanine standard. Products o/Carbon Fixation. At the termination of the incubation period, and while the sample remained in the light, 1 ml of the reaction mixture was withdrawn and added to 0.5 ml of 90 % formic acid. The mixture was extracted and fraetionated into neutral, basic and acidic fractions, using Dowex ion-exchange resins as described by Atkins (1969). Recoveries of radioactivity ranged from 87 to 105%. The products of 14C fixation agreed closely with those observed by Walker (1967) and Bassham et al. (1968a).
Results Chloroplasts isolated as described appeared to be i n t a c t when viewed i n the phase contrast microscope, either a t the beginning of the experim e n t or following i n c u b a t i o n i n the reaction vessel. The m a j o r i t y of the chloroplasts h a d i n t a c t m e m b r a n e s (Figs. 1 a, 2 a) a n d appeared to have a n almost identical morphology to those seen in sections of i n t a c t leaf (Fig. 2 b). Chloroplasts with i n t a c t m e m b r a n e s were readily distinguished from chloroplasts which have lost their outer m e m b r a n e (Fig. 1 b). The chloroplasts exhibited rates of CO s fixation a n d O~ evolution t h a t ranged from 20 to 70 ~SIoles hr -1 mg -1 (chlorophyl]), with the most freq u e n t rates between 40 a n d 50 (Fig. 3). Such values were in good agreem e n t with those reported b y Coekburn et al. (1968). CO s fixation was equal to 03 evolution over the time course of most experiments (Fig. 3), a finding i n agreement with the results of Walker et al. (1968). The CO s fixation rate of the i n t a c t leaves (measured at s a t u r a t i n g light i n t e n s i t y , 300 p p m COs, 25 ~ was between 78 a n d 82 ~Noles hr -1 mg -1 (chlorophyll). A s s i m i l a t i o n o / N i t r i t e . Chloroplast p r e p a r a t i o n s assimilated NO2- in the light a t a rate between 2.5 a n d 6 ~Moles hr -~ mg -1 (chlorophyll)
230
B. 1~. G r a n t and D. T. Canvin:
Fig. 1 a and b. a P1 chloroplast fraction isolated from spinach leaves, b P2 fraction isolated from spinach leaves. (500 • g for 20 rain after removal of P1)
Fig. 2 a und b. a Typical chloroplast from P1 fraction, b Typical chloroplast in intact leaf
The Effect of Nitrate and Nitrite on Oxygen Evolution
Fig. 2 a and b
231
232
B. R. Gr~nt and D. T. Canvin: 400 -air saturation 360 --
320 --
-5 2 8 0 E E
-o 2 4 0 -
~ 200
