Planta (Berl.) 94, 60--72 (1970) 9 by Springer-Verlag 1970
Intracellular Location of Nitrate Reductase and Nitrite Reductase in Spinach and Sunflower Leaves* B. R. GRANT**, C. A. ATKrNS and D. T. CANv~ Department of Biology, Queen's University, Kingston, Ontario, Canada Received March 5 / May 28, 1970
Summary. Chloroplasts have been isolated from spinach and from sunflower which retain their outer membrane and their stroma protein as determined both by ability to fix CO2 and evolve 02 at high rates, and by appearance under the phase contrast microscope. Such chloroplasts contain both nitrate and nitrite reductase activity. However, calculations on the distribution of these enzymes, when compared with the distribution of pyruvate kinase and cytechrome c oxidase activity, demonstrate that the larger part of both nitrate and nitrite reductase is located outside of the chloroplast. Introduction Previous work on the intracellular distribution of nitrate and nitrite rcductase has yielded conflicting results. Chloroplasts isolated in nonaqueous media contain nitrite reductase (Ritenour etal., 1967; Slack et al., 1969). Chloroplasts isolated under similar conditions were found by one group of workers to contain nitrate reductase activity (Coupe et al., 1967) but this was not confirmed by Ritenour et al. (1967). Chloroplasts isolated in aqueous media, using low ionic strength osmotica which allow the retention of most of the stroma protein (Jacobi, 1963), were found to contain only 25 % of the total nitrite reductase activity and none of the nitrate reduetase activity (Ritenour et al., 1967). Chloroplasts isolated in aqueous media of high ionic strength, which allow loss of much of the stroma protein, were reported to contain demonstrable nitrate reduetase activity (Del Campo etal., 1963) and high nitrite reduetase activity (Ramirez et al., 1966). None of these workers demonstrated that the chloroplasts which they isolated were capable of fixing CO 2 or evolving O 3 at kigh rates. This is now known to be a useful criteria of intactness of chloroplasts. * Supported in part by the National Research Council of Canada. ** On leave from CSIRO Marine Laboratory, Cronulla, Australia.
Intraeellular Location of Nitrate Reductase and Nitrite Reductase
61
I n this p a p e r we report results which show t h a t b o t h n i t r a t e a n d n i t r i t e reductase a c t i v i t y is present i n morphologically i n t a c t chloroplasts, b u t t h a t b y far the larger p a r t of b o t h enzymes is outside of the plastids.
Materials and Methods Plant Material. The chloroplasts used in this work were obtained from young leaves of spinach (Spinacea oleracea L. cv. Brookwood) and sunflower (Helianthus annuus L. cv. CM90RR). The growth conditions must be closely controlled in order to produce leaves which consistently yield a high proportion of active chloroplasts (Walker, 1966; Vose and Spencer, 1969). Both spinach and sunflowers were grown in 7.5 em (3 in.) of soil or "Turfaee" in fiats in a growth chamber (Model PGR16, Controlled Environments Ltd., Winnipeg, Manitoba) at 22~ on a 12/12-hr light/dark cycle. Light intensity at the leaf surface was 6,000--7,000 lax and light was provided by a mixture of fluorescent and incandescent lamps (10 General Electric F48T10CW and 14 25 W incandescent). The plants were watered liberally twice weekly with Hoagland's solution (Arnon, 1940). For chloroplast preparations, leaves (2--5 cm long) were harvested 2--3 hr after start of the daily light period from 6--10-week-old plants. 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. Chloroplasts were isolated by a modification of the method of Jensen and Bassham (1966). Medium A, the breaking medium, contained the following: sorbitol, 0.33M; MES [2-(N-morpholino)ethanesulfonie acid] buffer, pH 6.2, 0.05 M; isoaseorbic acid, 2 mlVl; dextran (MW 40,000=1=3,000), 2% w/v; bovine serum albumin (BSA) 0.1% w/v; disodium EDTA, 2 mM; MgC12, 1 mM; MnCl2, 1 raM; NaC1, 20ram and K2HP04, 0.5 mM. Medium B, the suspending' medium, was identical to A except that Hepes (N-2-hydroxy-ethylpiperazineN-2-ethanesuifonic acid) buffer, pH 6.8, 0.05 M was used instead of MES, and isoascorbate was omitted. Freshly harvested leaves were washed twice in distilled water at 4~ blotted dry and de-ribbed. They were then ground for 30 sec in a mortar at 0~ or in a Waring blendor for 5 sec at 0~ with 4 ml Medium A per gram leaf. The brei was immediately filtered through four layers of cheese cloth (80 mesh) and two layers of bolting silk (175 mesh), and fractionatcd as shown in Fig. 1. All manipulations were carried out at 4~ All pellet fractions were gently resuspended in 0.1 ml Medium B per gram leaf material. Each pellet preparation was examined under the phase microscope at the beginning of the experiments and in some cases during the experiments. The suspensions of particles were divided int~ two parts, one of which was frozen immediately, as were samples of each supernatant fraction, for later assay of enzyme activity. All enzyme assays were completed within 48 hr of harvesting. The remainder of the pellet fractions were used immediately for assay of CO2 fixation and 02 evolution. Chlorophyll concentration was determined by the method of Arnon (1949). In order to provide further protection for the nitrate and nitrite reductases during isolation (Sanderson and Cocking, 1964b) subcellular fractions were prepared as above with the breaking and resuspension medium supplemented with 10 mM eysteine. Chloroplasts prepared in the presence of cysteine were not capable of 02 evolution or CO~_fixation.
62
B . R . Grant, C. A. Atkins and D. T. Canvin: Filtrate ] centrifuge 500 • g for 90 see
I Pellet-1 (1)1) 1 Pellet-2 (P2)
I Penet-3 (P~) I Pellet-4 (P4)
I Supernatant-1 (Sx) ] centrifuge 500 X g for 20 min
1 Supernatant-2 ($2) ] centrifuge 3,000 • 9 for 20 rain
I Supernatant-3 ($3) I centrifuge 20,000 X g for 20 rain
I Supernatant-4 (S~)
Fig. 1. Separation of particulate fractions from leaf homogenates by differential centrifugation
Reaction Conditions/or 02 Evolution and CO2 Fixation. The reaction medium was a modification of Medium C of Jensen and Bassham (1966) and contained sorbitol, 0.33 M; Hepes buffer, p H 7.6, 0.05 M; dextran, 2% ; BSA 0.1% ; disodium EDTA, 2 mM; MgC12, 1 mM; MnC12, 1 raM; ~aC1, 20 raM; and K2HPO4, 0.5 mM. The reaction mixture contained 1.85 ml Medium C, 0.2 ml chloroplast preparation (75--100 ~g chlorophyll) and additions as specified in Results to a total volume of 2.2 ml. The reaction mixture was flushed with nitrogen before the addition of the chloroplast suspension, to reduce the O 2 concentration to approximately 20% of air saturation. The reaction vessel (Yellow Springs electrode cuvette) was positioned in a constant temperature bath (20 ~ and illuminated through a glass port. Light was provided by a 500 W incandescent lamp in a 35-ram slide projector. The beam was focussed and filtered with a water-filled round-bottom flask. In most experiments light of wavelength greater than 610 nm was supplied to the reaction vessel by insertion of a light filter. Subsequent experiments showed, however, that there was no difference in reaction rate when filtered or unfiltered light of similar intensity was employed. Light intensity (measured with a Y S I Model 65 Radiometer, Yellow Springs Instrument Co., Yellow Springs, Ohio) at the center of the reaction vessel was 2.8 x 10~ ergs cm -2 see-1. With this experimental arrangement, only 2.0 ml of the total reaction mixture was illuminated and the results have been corrected to take this factor into account. 02 evolution and COz fixation by the isolated chloroplasts was measured by a modification of the methods used by Walker and Hill (1967) and will be described below. The addition of sugar phosphates to the spinach chloroplast assay stimulated rates of O 2 evolution and CO2 fixation by 20--50%, depending on the preparation, but had no effect on the distribution of 14C in the products of photosynthesis (Grant, Atkins and Canvin, unpublished data). The addition of 3-phosphoglycerie acid to the sunflower chloroplast assay was essential, as no O2 evolution or CO~ fixation occurred in its absence. Other sugar phosphates by themselves would not effect O 2 evolution or COz fixation by sunflower chloroplasts. Ferricyanide at a concentration of 0.5 ~ was added t o the reaction mixture as an additional electron accepter in order to determine the capacity of the electron-
Intracellular Location of Nitrate Reductase and Nitrite Reductase
63
transport system. With ferricyanide as electron acceptor the same rates of 02 evolution were obtained in the presence or absence of CO2. Oxygen Evolution. 02 concentration was determined using a Clark type 02 electrode (Model 5331, Yellow Springs Instrument Co., Yellow Springs, Ohio). The 02 electrode was calibrated by adding catalase to the reaction mixture without chloroplasts and adding known volumes of a standard solution of tI202 (Goldstein, 1968). The results obtained by this method agreed to within 10% of the values calculated assuming that the 02 content of an air-saturated reaction mixture was identical with that of an air-saturated solution of saturated KC1. Carbon.Dioxide Fixation. Total carbon fixed was measured by injecting laClabelled bicarbonate solution of known specific activity (to give a final concentration of 5.5 mM) into the reaction mixture, and then withdrawing aliquots of 10 ~l at intervals and spotting on cellulose-acetate membrane filters (Millipore filters are suitable). The filter absorbed the solution immediately and stopped any further reaction. Bicarbonate was then removed by placing the filter in a desiccator over concentrated ttC1 for 15 rain. The filter was placed in a scintillation counting vial, wetted with 0.2 ml water and dissolved in 10 ml of a dioxanc-base scintillation mixture (Atkins, 1969). During the course of the experiment several aliquots were withdrawn and injected directly into 0.2 ml of freshly distilled monoethanolamine; this was dissolved in scintillator and counted. The CO., fixed was calculated as dpm fixed per aliquot • total CO2 present in 2.2 ml. The total CO2 present and the total dpm in aliquot final CO2 concentration specified included dissolved CO2 present in the reaction medium in equilibrium with air as well as the added bicarbonate. Radioactivity was determined with a liquid scintillation counter. All counts were corrected for efficiency using the channels ratio method (Bush, 1963). Assay o/Enzyme Activity. Nitrite reductase was assayed using the dithionitemethyl viologen donor system described by Ramirez et al. (1966). Nitrate reductase was assayed by the method of Hageman and Flesher (1960), except that excess NADH was precipitated with barium acetate in ethanol as described in Hewitt and Nicholas (1964). I n separate experiments it was shown that the loss of nitrite under conditions of the nitrate-rednctase assay was usually negligible. In sunflower preparations to which 10 mM cystine had been added, approximately 25% of the nitrite was lost, and this loss was taken into account in computing the activity of the nitrate reductase in these fractions. Pyruvate kinase was assayed by coupling the reaction to NADH oxidation by the addition of lactic dehydrogcnase (Gibbs and Turner, 1964). Cytochrome-c-oxidase activity was measured by determing in a spectrophotometer the rate of reoxidatibn of reduced cytochrome c (reduced with dithionite), as described by Hackett (1964). Protein was determined by the Lowry method as modified by Layne (1957).
Results The isolated spinach chloroplasts evolved O 3 a n d fixed CO s a t high rates (Table 1). The spinach leaves from which this p r e p a r a t i o n was m a d e h a d a rate of CO S assimilation of 82 ~= 2 txmoles hr -1 (rag chlorophyll) -1 at a light i n t e n s i t y of 20,000 lux, a t e m p e r a t u r e of 250 a n d a COs c o n c e n t r a t i o n of 350 p p m . The P1 fraction activities i n 20 preparations r a n g e d from 20 to 80 ~,moles CO 2 fixed hr -1 (rag chlorophyll) -1,
64
B.R. Grant, C. A. Atkins and D. T. Canvin:
Table 1. Carbon dioxide /ixation and oxygen evolution by spinach and sunflower chtoroplas~ All values are in ~moles hr -1 (rag chlorophyll)-I. Fraction
CO2 fixation
PI P~ P~ P~
55 20 0 o
P1 P2 P~
18 0 0
02 evolution
Spinach b 47 15 0 0
Sunflower~ 23 0 0
02 evolution with ferrieyanide a 62 26 30
0
24 ND ND
a Ferricyanide was added to the above reaction mixtures to a concentration of 5 mM. ND not determined. b Reaction mixture contained 1.85 ml Medium C, 0.2 ml chloroplasts (75-100 tzg chlorophyll) and additions in a total volume of 2.2 ml. Final bicarbonate concentration was 5.5raM. Ribose-5-phosphato, ffuctose-6-phosphate and 3phosphoglyceric acid were added, each at 0.5 raM. Incubation time, 10 rain. c Reaction mixture as for spinach except that ribose-5-phosphate and fructose6-phosphato were omitted.
w i t h r a t e s m o s t f r e q u e n t l y b e t w e e n 25 a n d 40. T h e P1 f r a c t i o n c o n t a i n e d chloroplasts, all of which a p p e a r e d to be i n t a c t as j u d g e d b y t h e presence of a l u m i n e s c e n t halo when viewed u n d e r t h e p h a s e - c o n t r a s t microscope. T h e P2 fraction c o n t a i n e d a large p r o p o r t i o n of a p p a r e n t l y i n t a c t chloroplasts (always m o r e t h a n 50 %) a n d a n u m b e r of s l i g h t l y swollen chloroplasts, which a p p e a r e d d a r k when viewed u n d e r phase contrast. T h e P~ f r a c t i o n c o n t a i n e d c h l o r o p l a s t f r a g m e n t s a n d a large n u m b e r of m i t o c h o n d r i a , while t h e P~ f r a c t i o n c o n t a i n e d m i t o c h o n d r i a b u t no recognizable chloroplasts f r a g m e n t s . Sunflower chloroplasts, a l t h o u g h showing a p p a r e n t l y i n t a c t m e m branes u n d e r p h a s e microscopy, always g a v e lower r a t e s of CO~ fixation, b e t w e e n 5 a n d 35 ~moles CO~ h r -1 (rag chlorophyll) -1 (most f r e q u e n t l y b e t w e e n 10 a n d 15). A n e x a m p l e is shown in T a b l e 1. T h e leaves f r o m which this p r e p a r a t i o n (Table 1) was m a d e h a d a r a t e of CO 2 assimilat i o n of 58 [~moles h r -~ (mg chlorophyll) -1 a t a l i g h t i n t e n s i t y of 35,000 lux, a t e m p e r a t u r e of 250 a n d a COz c o n c e n t r a t i o n of 350 p p m . The relatively low COz-fixation rates of isolated sunflower chloroplasts seemed to be due to inhibition of plastid enzymes rather than to protein loss by mechanical damage during isolation. This conclusion was based on the following observations: 1) Sunflower chloroplast preparations which remained in contact with the Sx frae-
Intracellular Location of Nitrate Reductase and Nitrite t~eductase
65
tion lost all ability to evolve 02 or fix CO 2 within 5 rain, (e.g. Pe in Table 1). 2) The highest rates of O 2 evolution obtained from sunflower chloroplasts [35 ~xmoles hr-1 (rag chlorophyll) -1] were obtained from preparations which h a d been isolated in less t h a n 2 min a n d assayed immediately. 3) The addition of 20 ~zl of S 1 fraction from sunflower to a preparation of spinach chloroplasts evolving 02 at 30 ~xmoles hr -1 (rag chlorophyll) -1 caused the rate to decrease to 5 ~zmoles hr -1 (mg chlorophyll) -1 in 1 rain. 4) The ratios of protein to chlorophyll in the P1 and P2 fractions from sunflower chloroplasts (7.5 and 6.8) was not markedly different from those observed for the P1 and P2 fractions from spinach (10 a n d 7.8; Table 3). T h e d i s t r i b u t i o n of 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 i n t h e f i r s t t h r e e p e l l e t a n d s u p e r n a t a n t f r a c t i o n s is s h o w n i n T a b l e 2. Of t h e t h r e e p e l l e t fractions from spinach examined in this preparation the highest specific activity per unit chlorophyll for both nitrite and nitrate reductase was
Table 2. Nitrite and nitrate reductase activity in chloroplast-containing /ractions
/rom spinach and sun/lower Fraction
Total chlorophyll (mg)
Total NO D reductase a
Total NO2 reductase b
Specific activityc N02 reductase
NOa reductase
Spinach P1 P2 P3 S2 Sa
0.8 3.2 1.3 6.5 3.1 1.0
18.6 38.2 I7.7 372 274 292
P1 P~ Pa
0.7 1.8 0.9
7.8 15.9 3.8
S1 S2 Sa
4.6 2.2 1.3
S1
2.4 1.8 2.0 64 103 60
23.3 12.0 13.5 57 88 292
3.0 0.6 1.5 l0 33 60
Sun/lower
168 122 113
trace trace trace
10.3 8.7 4.2
----
5.0 5.2 2.3
36.4 55.5 87.0
1.1 2.3 1.8
a Nitrite reduetase (NO2R) units are given as izmoles of substrate transformed per hr. The assay mixture contained in 2.0ml, 0.1 ml enzyme, 100 ~moles phosphate buffer, p H 7 . 6 , 200m~zmoles NO2, 0.1 ~mole of methyl viologen, 2.0 ~zmoles of dithionite, with controls of boiled enzyme, no enzyme, and no dithionite. Reaction time was 10 minutes at 20 ~ b Nitrate reductase (NO,R) units are given as ~moles of substrate transformed per hr. The assay mixture contained in 2,0 ml, 0.5 ml of enzyme, 100 ~xmoles phosphate buffer, p H 7.6, 2.0 ~zmoles NOa and 1.0 ~zmole NADH, with controls of boiled enzyme and no enzyme. I n separate experiments additional controls were r u n in which 20m~moles of NO 2 replaced NO 3. Reaction time was 15 min a t 20 ~ c The specific activity is given in terms of units per mg chlorophyll. 5 Planta (Berl.), Bd. 94
Total chl. (rag)
2.1 12.9 1.4 2.1
16.9 2.3 1.9 0.0
1.0 4.2 0.3 0.0
5.4 1.2 0.5 0.0
Fraction
t)l
P~ Pa P~
S1 Sa Sa S4
P1 Pa P3
1:)4
S1 Sa Sa Sa
99 70 76 95
7.5 27.6 7.0 25.5
348 261 230 187
21.6 101.4 24.5 30.0
Total prot. (rag)
78 112 78 84
2.8 5.6 0 4.0
740 680 445 570
14.0 33.2 6.4 9.2
Total RO2R
24 19 15 16
0.7 1.5 0.8 2.2
199 174 185 162
0.8 1.2 1.9 5.7
Total ROaR
6.8 2,6 4.5 4,4
14.4 93 156 --
2.7 1.3 ---
Sun]lower
0.8 1.6 1.0 0.9
0.4 0.2 -0.2
2.2 2.6 2.0 3.0
0.68 0.33 0.26 0.30
-
0.6 0.4 3.0 -4.4 15.9 29.6 --
-
11.8 76 97
0.4 0.1 1.3 2.6
(rag ehl.) -1
( m g chl,) -1
( m g prof.) -1
ROaR
Specific a c t i v i t i e s NO2R NO2R
43.6 296 234 --
Spinach
All c o n d i t i o n s as for T a b l e 2, e x c e p t t h a t 10 m M c y s t e i n e i n c l u d e d in b r e a k i n g a n d s u s p e n d i n g m e d i a .
prepared in the presence o] cysteine
T a b l e 3. Nitrite and nitrate reductase activity in chloroplast containing/ractions
0.24 0.27 0.20 0.17
0.09 0.05 0.11 0.09
0.57 0.67 0.86 0.86
0.037 0.012 0.078 0.19
( m g prot.) -1
ROaR
~"
eV
Intracellular Location of Nitrate Reductase and Nitrite Reductase
67
in t h e t71 fraction. N i t r i t e r e d u c t a s e a c t i v i t y r e m a i n e d high in b o t h P2 a n d P1 fractions while n i t r a t e r e d u c t a s e a c t i v i t y fell to a m i n i m u m in the P2 f r a c t i o n a n d increased in P~. T h e g r e a t e s t p r o p o r t i o n of b o t h enzymes was in t h e s u p e r n a t a n t fractions. T h e s a m e is t r u e of t h e sunflower p r e p a r a t i o n s , b u t n i t r a t e - r e d u c t a s e a c t i v i t y was too low for a c c u r a t e m e a s u r e m e n t in t h e pellet fractions. T h e low a c t i v i t y o b s e r v e d for n i t r a t e r e d u e t a s e in b o t h tissues is m o s t l i k e l y due in p a r t to t h e low light i n t e n s i t y u n d e r which t h e p l a n t s were grown ( S c h r a d e r et al., 1965) a n d to t h e l a c k of a s u l f h y d r y l - p r o t e c t i v e a g e n t d u r i n g t h e isolation ( S a n d e r s o n a n d Cocking, 1964a). I n parallel e x p e r i m e n t s in which l 0 m M cysteine was a d d e d to t h e b r e a k i n g a n d s u s p e n d i n g m e d i u m , significant increases in n i t r a t e - r e d n c t a s e a c t i v i t y were o b s e r v e d in t h e p a r t i c u l a t e fractions from sunflowers (Table 3). T h e d i s t r i b u t i o n of n i t r a t e - a n d n i t r i t e - r e d u e t a s e a c t i v i t y in this p r e p a r a t i o n agrees w i t h t h a t p r e s e n t e d in t h e previous table. There was significant a m o u n t s of b o t h 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 a c t i v i t y in t h e i n t a c t chloroplasts, b u t t h e t o t a l a c t i v i t y was low. The specific activities expressed either in t e r m s of c h l o r o p h y l l or in t e r m s of p r o t e i n were m u c h higher in t h e s u p e r n a t a n t t h a n in t h e p a r t i c u l a t e fractions. The inclusion of t h e Pa fraction d e m o n s t r a t e s m o r e clearly t h a t n i t r a t e r e d u c t a s e of a r e l a t i v e l y high specific a c t i v i t y was associated w i t h p a r t i c u l a t e m a t e r i a l s e d i m e n t i n g a t high speed.
Table 4. Distribution o/ pyruvate kinase and cytochrome-c oxidase in pellet/ractions ]rom spinach Fraction
P1 P~ P3 Pa S4
Total pyruvate kinase a
4.2 12.2 9.4 4.6 1900
Total cytochrome-e oxidase b
0.0 20 180 120 0
Maximum percentage of total activity due to occlusion
NO2R
N0sR
9 l0 4O 15 --
25 60 3O 3 --
a Pyruvate kinase activity is given in units which represent a decrease in extinction of 1.0 unit cm-1 hr -1 at 340 nm at 20 ~ The reaction mixture contained in 3 mh tris-HC1 buffer, pH 7.6, 200 ~moles; MgC12, 7.5 ~moles; ADP, 1.0 ~mole; PEP, 2.5 ~moles; NADH, 0.45 ~moles; Lactic dehydrogenasc, Type I I (Sigma), 4 units; 0.01 to 0.2 ml enzyme. b Cytochromc-c oxidase activity is given in units which represent a decrease in extinction of 0.25 units cm-1 hr 1 at 550 nm at 20 ~ The reaction mixture contained in 3.0ml: cytochrome c (reduced with dithionite), 0.045~moles; phosphate buffer, pH 7.0, 200 ~moles; 0.05 to 0.2 ml enzyme. 5*
68
B. 1~. Gr~nt, C. A. Atkins ~nd D. T. Canvin:
The distribution of pyruvate-kinase and eytoehrome-e-oxidase aetivires in fractions from the spinach preparation used in Table 3 are given in Table 4. P y r u v a t e kinase has been used by other workers (Santarius and Stocking, 1969) as a measure of inclusion of cytoplasmic enzymes. From the distribution of the kinase we have calculated the amount of nitrate and nitrite reduetase which could be present in the pellets due to occlusion (Table 4). I t can be seen that with one exception (the nitrate-reductase activity eontMned in pellet 2) the amounts of activity present are too large to be accounted for by occlusion. Cytoehrome-eoxidase activity is considered to be a marker for mitoehondria, and the m a x i m u m amount of this enzyme occurs in the Pa fractions. The largest total amount and the highest specific activity of the particulate nitrate reductase occurs in the P4 fraction. I t is therefore unlikely that the particulate nitrate reduetase is located within the mitoehondrion. Attempts to further purify this fraction on sucrose gradients have not been successful. Discussion The greater part of the nitrate- and the nitrite-reduetase activity in both tissues occurs in the supernatant fraction. Taking the spinach data given in Table 2 as representative it can be seen that if all of the nitrite reduetase present in the extracts (SI-~P1) was contained within the chloroplasts (chlorophyll content of PI-~ $1) in the intact leaf, the specific activity must have been equal to at least 53 units per mg chlorophyll. The chloroplasts which sediment in the P1 fraction have lost only 32 % of their capacity to fix C02, when compared with the leaves from which they were taken. They have probably lost less than 25 % of such activity if the rate of 02 evolution with ferrieyanide is taken as a measure of the ability of the system to carry out photosynthetic electron transport under our experimental conditions. If the total nitrite-reduetase activity resides within the plastid in the intact leaf, the P1 chloroplasts must have lost 60 % of their capacity to reduce nitrite based on a comparison of the actual specific activity of P1, corrected for the occlusion of 10 % of cytoplasmic enzymes (21.3) and the theoretical specific activity calculated above (53). Centrifugation at higher centrifugal forces and for longer periods does cause loss of protein from the chloroplast, as shown in the changing ratio of protein to chlorophyll in the P1 and P~ fractions (Table 3), and they also lose some nitrate and nitrite reduetase (compare specific activities of P1 and P~; Table 3). However, the results indicate that these enzymes are lost less readily than are those of the Calvin cycle. This is shown by the more rapid loss of ability to fix CO 2 than ability to reduce nitrate or nitrite (compare Table 1 and Table 2). The data from sunflower leaves supports fully that obtained
Intraeellular Location of Nitrate I~eductase and Nitrite geduetase
69
with spinach, although the lower rates usually obtained make the argument less telling if considered alone. Although nitrate-reductase activity in both tissues is over-whelmingly outside of the chloroplast, there is, nevertheless, always a demonstrable component associated with the intact plastid. There is also an association with a particulate fraction which is sedimenting at higher speeds and is not associated with either chlorophyll or with mitochondria. I~owever, this may have originated as part of the intact chloroplast membrane and it is still low in total amount when contrasted to the activity present in the soluble component. We conclude that in the intact leaf cell, there are at least two possible sites of nitrate and nitrite reduction, inside the chloroplast, and outside of it. The ratios of enzyme distribution observed in these experiments argue strongly against the main site of nitrate and nitrite reduction being located within the chloroplast in intact leaves. The major objection which can be raised to the participation of a nitrite reductase external to the chloroplast, is the requirement for a suitable electron donor. Studies on the isolated enzyme have demonstrated that ferredoxin is required as a cofaetor and reduced pyridine nueleotides alone are ineffective (Ramirez et al., 1966; J o y and ttageman, 1966; Shin and Oda, 1966). To date, there is no evidence to show that ferredoxin occurs outside of the chloroplast in photosynthetic tissues (Hall and Evans, 1969). Itowever, both nitrite and nitrate reduction do take place in plant tissues which do not contain chlorophyll (Sanderson and Cocking, 1964b ; I-Iucklesby and Elsner, 1969; see Beevers and Hageman, 1969, for additional references). This provides clear evidence that a suitable electron donor, not specifically associated with the chloroplast, exists in plant tissues. The reduction of nitrate at a site outside of the ehloroplast has been generally accepted, although Losada and co-workers disagree with this view (Paneque st al., 1969). Our results support the contention that the main site of nitrate reduction is outside of the chloroplasts. The higher specific activity of nitrate reductase on a per-mg-protein basis in the material sedimenting at 20,000 X g may be an indication that the fraction of the enzyme located within the chloroplast is associated with a membrane, as was suggested by Hageman and co-workers (I~itenour et al., 1967). On the basis of these results, we Ieel that the stimulation of nitrate assimilation by light, in vivo, observed originally by Warburg (1920) and since confirmed and extended by other workers (see Kessler, 1964, for references) is probably indirect, and takes place via transfer of reducing power across the plastid membrane. Mechanisms by which
70
B. R. Grant, C. A. Atkins and D. T. Canvin:
this m i g h t t a k e place h a v e been discussed b y Stocking a n d L a r s o n (1969) a n d b y t t a g e m a n (1969). A similar s h u t t l e in m i t o c h o n d r i a has been discussed b y K r e b s (1966). T h e existence of this t y p e of transfer, d e p e n d e n t u p o n t h e p r o d u c t s of c a r b o n fixation, would e x p l a i n ~he d e p e n d e n c e of l i g h t - s t i m u l a t e d n i t r a t e r e d u c t i o n u p o n t h e presence of CO 2 (Davis, 1953; G r a n t , 1967). N i t r i t e r e d u c t i o n in light a p p e a r s to be less d e p e n d e n t u p o n t h e presence of CO 2 a l t h o u g h such a r e q u i r e m e n t was o b s e r v e d b y G r a n t (1967).
References Arnon, D. I. : Vitamin B 1 in relation to the growth of green plants. Science 92, 264--266 (1940). - - Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1--15 (1949). Atkins, C. A. : Intermediary metabolism of photosynthesis in relation to C02 evolution in light. Doct. dissert., Queen's University, Kingston, Ont., Canada, 1969. Beevers, L., I-Iageman, R. It. : Nitrate reduction in higher plants. Ann. Rev. Plant Physiol. 29, 495--522 (1969). Bush, E. : General applicability of the channels ratio method of measuring liquid scintillation counting efficiencies. Anal. Chem. 35, 1024--1029 (1963). Coupe, M., Champigny, M. L., Moyse, A.: Sur la localisation intracellulaire de la nitrate reductase dans les feuilles et les racines d'orge. Physiol. v6g. 5, 271--291 (1967). Davis, E. A. : Nitrate reduction by Chlorella. Plant Physiol. 28, 539--544 (1953). Del Campo, F. F., Paneque, A., Ramirez, J. M., Losada, M. : Nitrate reduction in the light by isolated chloroplasts. Biochim. biophys. Acta (Amst.) 55, 450~452 (1963). Gibbs, M., Turner, J. F. : Enzymes of glycolysis. In: Modern methods of plant analysis, I-I. Linskens, B.D. Sanwal and M.V. Tracey, eds., vol. 7, p. 543. Berlin-G6ttingen-Heidelberg-New York: Springer 1964. Goldstein, D. B. : A method for assay of catalase with the oxygen electrode. Anal. Biochem. 24, 431--437 (1968). Grant, B. R. : The action of light on nitrate and nitrite assimilation by the marine chlorophyte Dunaliella tertiolecta (Butcher). J. gen. Mierobiol. 48, 379--389 (1967). Hackett, D. P. : Enzymes of terminal respiration. In: Modern methods of plant analysis, H. Linskens, B.D. Sanwal and M.V. Tracey, eds., vol. 7, p. 647. Berlin-G6ttingen-Iteidelberg-New York: Springer 1964. ttageman, R. H. : Physiological responses to nitrogen-discussion. In: Physiological aspects of crop yield, J. E. Easton, F. A. ttaskins, C. Y. Sullivan and C. H. M. Van B&vel, eds., p. 260--262. Madison, Wise. : Amer. Soc. Agron. 1969. - - Flesher, D. : Nitrate reductase activity in corn seedlings affected by light and nitrate content of nutrient media. Plant Physiol. 35, 700--708 (1960). Hall, D. 0., Evans, M. C. W. : Iron-sulphur proteins. Nature (Lond.) 223, 1342-1348 (1969). Hewitt, E. J., Nicholas, D. J. D. : Enzymes of inorganic nitrogen metabolism. In: Modern methods of plant analysis, K. Paech and M. V. Tracey, eds., vol. 7, p. 67--172. Berlin-G6ttingen~Heidetberg~ew York: -Springer t
Intracellular Location of Nitrate Reductase and Nitrite Reductase in Spinach and Sunflower Leaves* B. R. GRANT**, C. A. ATKrNS and D. T. CANv~ Department of Biology, Queen's University, Kingston, Ontario, Canada Received March 5 / May 28, 1970
Summary. Chloroplasts have been isolated from spinach and from sunflower which retain their outer membrane and their stroma protein as determined both by ability to fix CO2 and evolve 02 at high rates, and by appearance under the phase contrast microscope. Such chloroplasts contain both nitrate and nitrite reductase activity. However, calculations on the distribution of these enzymes, when compared with the distribution of pyruvate kinase and cytechrome c oxidase activity, demonstrate that the larger part of both nitrate and nitrite reductase is located outside of the chloroplast. Introduction Previous work on the intracellular distribution of nitrate and nitrite rcductase has yielded conflicting results. Chloroplasts isolated in nonaqueous media contain nitrite reductase (Ritenour etal., 1967; Slack et al., 1969). Chloroplasts isolated under similar conditions were found by one group of workers to contain nitrate reductase activity (Coupe et al., 1967) but this was not confirmed by Ritenour et al. (1967). Chloroplasts isolated in aqueous media, using low ionic strength osmotica which allow the retention of most of the stroma protein (Jacobi, 1963), were found to contain only 25 % of the total nitrite reductase activity and none of the nitrate reduetase activity (Ritenour et al., 1967). Chloroplasts isolated in aqueous media of high ionic strength, which allow loss of much of the stroma protein, were reported to contain demonstrable nitrate reduetase activity (Del Campo etal., 1963) and high nitrite reduetase activity (Ramirez et al., 1966). None of these workers demonstrated that the chloroplasts which they isolated were capable of fixing CO 2 or evolving O 3 at kigh rates. This is now known to be a useful criteria of intactness of chloroplasts. * Supported in part by the National Research Council of Canada. ** On leave from CSIRO Marine Laboratory, Cronulla, Australia.
Intraeellular Location of Nitrate Reductase and Nitrite Reductase
61
I n this p a p e r we report results which show t h a t b o t h n i t r a t e a n d n i t r i t e reductase a c t i v i t y is present i n morphologically i n t a c t chloroplasts, b u t t h a t b y far the larger p a r t of b o t h enzymes is outside of the plastids.
Materials and Methods Plant Material. The chloroplasts used in this work were obtained from young leaves of spinach (Spinacea oleracea L. cv. Brookwood) and sunflower (Helianthus annuus L. cv. CM90RR). The growth conditions must be closely controlled in order to produce leaves which consistently yield a high proportion of active chloroplasts (Walker, 1966; Vose and Spencer, 1969). Both spinach and sunflowers were grown in 7.5 em (3 in.) of soil or "Turfaee" in fiats in a growth chamber (Model PGR16, Controlled Environments Ltd., Winnipeg, Manitoba) at 22~ on a 12/12-hr light/dark cycle. Light intensity at the leaf surface was 6,000--7,000 lax and light was provided by a mixture of fluorescent and incandescent lamps (10 General Electric F48T10CW and 14 25 W incandescent). The plants were watered liberally twice weekly with Hoagland's solution (Arnon, 1940). For chloroplast preparations, leaves (2--5 cm long) were harvested 2--3 hr after start of the daily light period from 6--10-week-old plants. 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. Chloroplasts were isolated by a modification of the method of Jensen and Bassham (1966). Medium A, the breaking medium, contained the following: sorbitol, 0.33M; MES [2-(N-morpholino)ethanesulfonie acid] buffer, pH 6.2, 0.05 M; isoaseorbic acid, 2 mlVl; dextran (MW 40,000=1=3,000), 2% w/v; bovine serum albumin (BSA) 0.1% w/v; disodium EDTA, 2 mM; MgC12, 1 mM; MnCl2, 1 raM; NaC1, 20ram and K2HP04, 0.5 mM. Medium B, the suspending' medium, was identical to A except that Hepes (N-2-hydroxy-ethylpiperazineN-2-ethanesuifonic acid) buffer, pH 6.8, 0.05 M was used instead of MES, and isoascorbate was omitted. Freshly harvested leaves were washed twice in distilled water at 4~ blotted dry and de-ribbed. They were then ground for 30 sec in a mortar at 0~ or in a Waring blendor for 5 sec at 0~ with 4 ml Medium A per gram leaf. The brei was immediately filtered through four layers of cheese cloth (80 mesh) and two layers of bolting silk (175 mesh), and fractionatcd as shown in Fig. 1. All manipulations were carried out at 4~ All pellet fractions were gently resuspended in 0.1 ml Medium B per gram leaf material. Each pellet preparation was examined under the phase microscope at the beginning of the experiments and in some cases during the experiments. The suspensions of particles were divided int~ two parts, one of which was frozen immediately, as were samples of each supernatant fraction, for later assay of enzyme activity. All enzyme assays were completed within 48 hr of harvesting. The remainder of the pellet fractions were used immediately for assay of CO2 fixation and 02 evolution. Chlorophyll concentration was determined by the method of Arnon (1949). In order to provide further protection for the nitrate and nitrite reductases during isolation (Sanderson and Cocking, 1964b) subcellular fractions were prepared as above with the breaking and resuspension medium supplemented with 10 mM eysteine. Chloroplasts prepared in the presence of cysteine were not capable of 02 evolution or CO~_fixation.
62
B . R . Grant, C. A. Atkins and D. T. Canvin: Filtrate ] centrifuge 500 • g for 90 see
I Pellet-1 (1)1) 1 Pellet-2 (P2)
I Penet-3 (P~) I Pellet-4 (P4)
I Supernatant-1 (Sx) ] centrifuge 500 X g for 20 min
1 Supernatant-2 ($2) ] centrifuge 3,000 • 9 for 20 rain
I Supernatant-3 ($3) I centrifuge 20,000 X g for 20 rain
I Supernatant-4 (S~)
Fig. 1. Separation of particulate fractions from leaf homogenates by differential centrifugation
Reaction Conditions/or 02 Evolution and CO2 Fixation. The reaction medium was a modification of Medium C of Jensen and Bassham (1966) and contained sorbitol, 0.33 M; Hepes buffer, p H 7.6, 0.05 M; dextran, 2% ; BSA 0.1% ; disodium EDTA, 2 mM; MgC12, 1 mM; MnC12, 1 raM; ~aC1, 20 raM; and K2HPO4, 0.5 mM. The reaction mixture contained 1.85 ml Medium C, 0.2 ml chloroplast preparation (75--100 ~g chlorophyll) and additions as specified in Results to a total volume of 2.2 ml. The reaction mixture was flushed with nitrogen before the addition of the chloroplast suspension, to reduce the O 2 concentration to approximately 20% of air saturation. The reaction vessel (Yellow Springs electrode cuvette) was positioned in a constant temperature bath (20 ~ and illuminated through a glass port. Light was provided by a 500 W incandescent lamp in a 35-ram slide projector. The beam was focussed and filtered with a water-filled round-bottom flask. In most experiments light of wavelength greater than 610 nm was supplied to the reaction vessel by insertion of a light filter. Subsequent experiments showed, however, that there was no difference in reaction rate when filtered or unfiltered light of similar intensity was employed. Light intensity (measured with a Y S I Model 65 Radiometer, Yellow Springs Instrument Co., Yellow Springs, Ohio) at the center of the reaction vessel was 2.8 x 10~ ergs cm -2 see-1. With this experimental arrangement, only 2.0 ml of the total reaction mixture was illuminated and the results have been corrected to take this factor into account. 02 evolution and COz fixation by the isolated chloroplasts was measured by a modification of the methods used by Walker and Hill (1967) and will be described below. The addition of sugar phosphates to the spinach chloroplast assay stimulated rates of O 2 evolution and CO2 fixation by 20--50%, depending on the preparation, but had no effect on the distribution of 14C in the products of photosynthesis (Grant, Atkins and Canvin, unpublished data). The addition of 3-phosphoglycerie acid to the sunflower chloroplast assay was essential, as no O2 evolution or CO~ fixation occurred in its absence. Other sugar phosphates by themselves would not effect O 2 evolution or COz fixation by sunflower chloroplasts. Ferricyanide at a concentration of 0.5 ~ was added t o the reaction mixture as an additional electron accepter in order to determine the capacity of the electron-
Intracellular Location of Nitrate Reductase and Nitrite Reductase
63
transport system. With ferricyanide as electron acceptor the same rates of 02 evolution were obtained in the presence or absence of CO2. Oxygen Evolution. 02 concentration was determined using a Clark type 02 electrode (Model 5331, Yellow Springs Instrument Co., Yellow Springs, Ohio). The 02 electrode was calibrated by adding catalase to the reaction mixture without chloroplasts and adding known volumes of a standard solution of tI202 (Goldstein, 1968). The results obtained by this method agreed to within 10% of the values calculated assuming that the 02 content of an air-saturated reaction mixture was identical with that of an air-saturated solution of saturated KC1. Carbon.Dioxide Fixation. Total carbon fixed was measured by injecting laClabelled bicarbonate solution of known specific activity (to give a final concentration of 5.5 mM) into the reaction mixture, and then withdrawing aliquots of 10 ~l at intervals and spotting on cellulose-acetate membrane filters (Millipore filters are suitable). The filter absorbed the solution immediately and stopped any further reaction. Bicarbonate was then removed by placing the filter in a desiccator over concentrated ttC1 for 15 rain. The filter was placed in a scintillation counting vial, wetted with 0.2 ml water and dissolved in 10 ml of a dioxanc-base scintillation mixture (Atkins, 1969). During the course of the experiment several aliquots were withdrawn and injected directly into 0.2 ml of freshly distilled monoethanolamine; this was dissolved in scintillator and counted. The CO., fixed was calculated as dpm fixed per aliquot • total CO2 present in 2.2 ml. The total CO2 present and the total dpm in aliquot final CO2 concentration specified included dissolved CO2 present in the reaction medium in equilibrium with air as well as the added bicarbonate. Radioactivity was determined with a liquid scintillation counter. All counts were corrected for efficiency using the channels ratio method (Bush, 1963). Assay o/Enzyme Activity. Nitrite reductase was assayed using the dithionitemethyl viologen donor system described by Ramirez et al. (1966). Nitrate reductase was assayed by the method of Hageman and Flesher (1960), except that excess NADH was precipitated with barium acetate in ethanol as described in Hewitt and Nicholas (1964). I n separate experiments it was shown that the loss of nitrite under conditions of the nitrate-rednctase assay was usually negligible. In sunflower preparations to which 10 mM cystine had been added, approximately 25% of the nitrite was lost, and this loss was taken into account in computing the activity of the nitrate reductase in these fractions. Pyruvate kinase was assayed by coupling the reaction to NADH oxidation by the addition of lactic dehydrogcnase (Gibbs and Turner, 1964). Cytochrome-c-oxidase activity was measured by determing in a spectrophotometer the rate of reoxidatibn of reduced cytochrome c (reduced with dithionite), as described by Hackett (1964). Protein was determined by the Lowry method as modified by Layne (1957).
Results The isolated spinach chloroplasts evolved O 3 a n d fixed CO s a t high rates (Table 1). The spinach leaves from which this p r e p a r a t i o n was m a d e h a d a rate of CO S assimilation of 82 ~= 2 txmoles hr -1 (rag chlorophyll) -1 at a light i n t e n s i t y of 20,000 lux, a t e m p e r a t u r e of 250 a n d a COs c o n c e n t r a t i o n of 350 p p m . The P1 fraction activities i n 20 preparations r a n g e d from 20 to 80 ~,moles CO 2 fixed hr -1 (rag chlorophyll) -1,
64
B.R. Grant, C. A. Atkins and D. T. Canvin:
Table 1. Carbon dioxide /ixation and oxygen evolution by spinach and sunflower chtoroplas~ All values are in ~moles hr -1 (rag chlorophyll)-I. Fraction
CO2 fixation
PI P~ P~ P~
55 20 0 o
P1 P2 P~
18 0 0
02 evolution
Spinach b 47 15 0 0
Sunflower~ 23 0 0
02 evolution with ferrieyanide a 62 26 30
0
24 ND ND
a Ferricyanide was added to the above reaction mixtures to a concentration of 5 mM. ND not determined. b Reaction mixture contained 1.85 ml Medium C, 0.2 ml chloroplasts (75-100 tzg chlorophyll) and additions in a total volume of 2.2 ml. Final bicarbonate concentration was 5.5raM. Ribose-5-phosphato, ffuctose-6-phosphate and 3phosphoglyceric acid were added, each at 0.5 raM. Incubation time, 10 rain. c Reaction mixture as for spinach except that ribose-5-phosphate and fructose6-phosphato were omitted.
w i t h r a t e s m o s t f r e q u e n t l y b e t w e e n 25 a n d 40. T h e P1 f r a c t i o n c o n t a i n e d chloroplasts, all of which a p p e a r e d to be i n t a c t as j u d g e d b y t h e presence of a l u m i n e s c e n t halo when viewed u n d e r t h e p h a s e - c o n t r a s t microscope. T h e P2 fraction c o n t a i n e d a large p r o p o r t i o n of a p p a r e n t l y i n t a c t chloroplasts (always m o r e t h a n 50 %) a n d a n u m b e r of s l i g h t l y swollen chloroplasts, which a p p e a r e d d a r k when viewed u n d e r phase contrast. T h e P~ f r a c t i o n c o n t a i n e d c h l o r o p l a s t f r a g m e n t s a n d a large n u m b e r of m i t o c h o n d r i a , while t h e P~ f r a c t i o n c o n t a i n e d m i t o c h o n d r i a b u t no recognizable chloroplasts f r a g m e n t s . Sunflower chloroplasts, a l t h o u g h showing a p p a r e n t l y i n t a c t m e m branes u n d e r p h a s e microscopy, always g a v e lower r a t e s of CO~ fixation, b e t w e e n 5 a n d 35 ~moles CO~ h r -1 (rag chlorophyll) -1 (most f r e q u e n t l y b e t w e e n 10 a n d 15). A n e x a m p l e is shown in T a b l e 1. T h e leaves f r o m which this p r e p a r a t i o n (Table 1) was m a d e h a d a r a t e of CO 2 assimilat i o n of 58 [~moles h r -~ (mg chlorophyll) -1 a t a l i g h t i n t e n s i t y of 35,000 lux, a t e m p e r a t u r e of 250 a n d a COz c o n c e n t r a t i o n of 350 p p m . The relatively low COz-fixation rates of isolated sunflower chloroplasts seemed to be due to inhibition of plastid enzymes rather than to protein loss by mechanical damage during isolation. This conclusion was based on the following observations: 1) Sunflower chloroplast preparations which remained in contact with the Sx frae-
Intracellular Location of Nitrate Reductase and Nitrite t~eductase
65
tion lost all ability to evolve 02 or fix CO 2 within 5 rain, (e.g. Pe in Table 1). 2) The highest rates of O 2 evolution obtained from sunflower chloroplasts [35 ~xmoles hr-1 (rag chlorophyll) -1] were obtained from preparations which h a d been isolated in less t h a n 2 min a n d assayed immediately. 3) The addition of 20 ~zl of S 1 fraction from sunflower to a preparation of spinach chloroplasts evolving 02 at 30 ~xmoles hr -1 (rag chlorophyll) -1 caused the rate to decrease to 5 ~zmoles hr -1 (mg chlorophyll) -1 in 1 rain. 4) The ratios of protein to chlorophyll in the P1 and P2 fractions from sunflower chloroplasts (7.5 and 6.8) was not markedly different from those observed for the P1 and P2 fractions from spinach (10 a n d 7.8; Table 3). T h e d i s t r i b u t i o n of 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 i n t h e f i r s t t h r e e p e l l e t a n d s u p e r n a t a n t f r a c t i o n s is s h o w n i n T a b l e 2. Of t h e t h r e e p e l l e t fractions from spinach examined in this preparation the highest specific activity per unit chlorophyll for both nitrite and nitrate reductase was
Table 2. Nitrite and nitrate reductase activity in chloroplast-containing /ractions
/rom spinach and sun/lower Fraction
Total chlorophyll (mg)
Total NO D reductase a
Total NO2 reductase b
Specific activityc N02 reductase
NOa reductase
Spinach P1 P2 P3 S2 Sa
0.8 3.2 1.3 6.5 3.1 1.0
18.6 38.2 I7.7 372 274 292
P1 P~ Pa
0.7 1.8 0.9
7.8 15.9 3.8
S1 S2 Sa
4.6 2.2 1.3
S1
2.4 1.8 2.0 64 103 60
23.3 12.0 13.5 57 88 292
3.0 0.6 1.5 l0 33 60
Sun/lower
168 122 113
trace trace trace
10.3 8.7 4.2
----
5.0 5.2 2.3
36.4 55.5 87.0
1.1 2.3 1.8
a Nitrite reduetase (NO2R) units are given as izmoles of substrate transformed per hr. The assay mixture contained in 2.0ml, 0.1 ml enzyme, 100 ~moles phosphate buffer, p H 7 . 6 , 200m~zmoles NO2, 0.1 ~mole of methyl viologen, 2.0 ~zmoles of dithionite, with controls of boiled enzyme, no enzyme, and no dithionite. Reaction time was 10 minutes at 20 ~ b Nitrate reductase (NO,R) units are given as ~moles of substrate transformed per hr. The assay mixture contained in 2,0 ml, 0.5 ml of enzyme, 100 ~xmoles phosphate buffer, p H 7.6, 2.0 ~zmoles NOa and 1.0 ~zmole NADH, with controls of boiled enzyme and no enzyme. I n separate experiments additional controls were r u n in which 20m~moles of NO 2 replaced NO 3. Reaction time was 15 min a t 20 ~ c The specific activity is given in terms of units per mg chlorophyll. 5 Planta (Berl.), Bd. 94
Total chl. (rag)
2.1 12.9 1.4 2.1
16.9 2.3 1.9 0.0
1.0 4.2 0.3 0.0
5.4 1.2 0.5 0.0
Fraction
t)l
P~ Pa P~
S1 Sa Sa S4
P1 Pa P3
1:)4
S1 Sa Sa Sa
99 70 76 95
7.5 27.6 7.0 25.5
348 261 230 187
21.6 101.4 24.5 30.0
Total prot. (rag)
78 112 78 84
2.8 5.6 0 4.0
740 680 445 570
14.0 33.2 6.4 9.2
Total RO2R
24 19 15 16
0.7 1.5 0.8 2.2
199 174 185 162
0.8 1.2 1.9 5.7
Total ROaR
6.8 2,6 4.5 4,4
14.4 93 156 --
2.7 1.3 ---
Sun]lower
0.8 1.6 1.0 0.9
0.4 0.2 -0.2
2.2 2.6 2.0 3.0
0.68 0.33 0.26 0.30
-
0.6 0.4 3.0 -4.4 15.9 29.6 --
-
11.8 76 97
0.4 0.1 1.3 2.6
(rag ehl.) -1
( m g chl,) -1
( m g prof.) -1
ROaR
Specific a c t i v i t i e s NO2R NO2R
43.6 296 234 --
Spinach
All c o n d i t i o n s as for T a b l e 2, e x c e p t t h a t 10 m M c y s t e i n e i n c l u d e d in b r e a k i n g a n d s u s p e n d i n g m e d i a .
prepared in the presence o] cysteine
T a b l e 3. Nitrite and nitrate reductase activity in chloroplast containing/ractions
0.24 0.27 0.20 0.17
0.09 0.05 0.11 0.09
0.57 0.67 0.86 0.86
0.037 0.012 0.078 0.19
( m g prot.) -1
ROaR
~"
eV
Intracellular Location of Nitrate Reductase and Nitrite Reductase
67
in t h e t71 fraction. N i t r i t e r e d u c t a s e a c t i v i t y r e m a i n e d high in b o t h P2 a n d P1 fractions while n i t r a t e r e d u c t a s e a c t i v i t y fell to a m i n i m u m in the P2 f r a c t i o n a n d increased in P~. T h e g r e a t e s t p r o p o r t i o n of b o t h enzymes was in t h e s u p e r n a t a n t fractions. T h e s a m e is t r u e of t h e sunflower p r e p a r a t i o n s , b u t n i t r a t e - r e d u c t a s e a c t i v i t y was too low for a c c u r a t e m e a s u r e m e n t in t h e pellet fractions. T h e low a c t i v i t y o b s e r v e d for n i t r a t e r e d u e t a s e in b o t h tissues is m o s t l i k e l y due in p a r t to t h e low light i n t e n s i t y u n d e r which t h e p l a n t s were grown ( S c h r a d e r et al., 1965) a n d to t h e l a c k of a s u l f h y d r y l - p r o t e c t i v e a g e n t d u r i n g t h e isolation ( S a n d e r s o n a n d Cocking, 1964a). I n parallel e x p e r i m e n t s in which l 0 m M cysteine was a d d e d to t h e b r e a k i n g a n d s u s p e n d i n g m e d i u m , significant increases in n i t r a t e - r e d n c t a s e a c t i v i t y were o b s e r v e d in t h e p a r t i c u l a t e fractions from sunflowers (Table 3). T h e d i s t r i b u t i o n of n i t r a t e - a n d n i t r i t e - r e d u e t a s e a c t i v i t y in this p r e p a r a t i o n agrees w i t h t h a t p r e s e n t e d in t h e previous table. There was significant a m o u n t s of b o t h 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 a c t i v i t y in t h e i n t a c t chloroplasts, b u t t h e t o t a l a c t i v i t y was low. The specific activities expressed either in t e r m s of c h l o r o p h y l l or in t e r m s of p r o t e i n were m u c h higher in t h e s u p e r n a t a n t t h a n in t h e p a r t i c u l a t e fractions. The inclusion of t h e Pa fraction d e m o n s t r a t e s m o r e clearly t h a t n i t r a t e r e d u c t a s e of a r e l a t i v e l y high specific a c t i v i t y was associated w i t h p a r t i c u l a t e m a t e r i a l s e d i m e n t i n g a t high speed.
Table 4. Distribution o/ pyruvate kinase and cytochrome-c oxidase in pellet/ractions ]rom spinach Fraction
P1 P~ P3 Pa S4
Total pyruvate kinase a
4.2 12.2 9.4 4.6 1900
Total cytochrome-e oxidase b
0.0 20 180 120 0
Maximum percentage of total activity due to occlusion
NO2R
N0sR
9 l0 4O 15 --
25 60 3O 3 --
a Pyruvate kinase activity is given in units which represent a decrease in extinction of 1.0 unit cm-1 hr -1 at 340 nm at 20 ~ The reaction mixture contained in 3 mh tris-HC1 buffer, pH 7.6, 200 ~moles; MgC12, 7.5 ~moles; ADP, 1.0 ~mole; PEP, 2.5 ~moles; NADH, 0.45 ~moles; Lactic dehydrogenasc, Type I I (Sigma), 4 units; 0.01 to 0.2 ml enzyme. b Cytochromc-c oxidase activity is given in units which represent a decrease in extinction of 0.25 units cm-1 hr 1 at 550 nm at 20 ~ The reaction mixture contained in 3.0ml: cytochrome c (reduced with dithionite), 0.045~moles; phosphate buffer, pH 7.0, 200 ~moles; 0.05 to 0.2 ml enzyme. 5*
68
B. 1~. Gr~nt, C. A. Atkins ~nd D. T. Canvin:
The distribution of pyruvate-kinase and eytoehrome-e-oxidase aetivires in fractions from the spinach preparation used in Table 3 are given in Table 4. P y r u v a t e kinase has been used by other workers (Santarius and Stocking, 1969) as a measure of inclusion of cytoplasmic enzymes. From the distribution of the kinase we have calculated the amount of nitrate and nitrite reduetase which could be present in the pellets due to occlusion (Table 4). I t can be seen that with one exception (the nitrate-reductase activity eontMned in pellet 2) the amounts of activity present are too large to be accounted for by occlusion. Cytoehrome-eoxidase activity is considered to be a marker for mitoehondria, and the m a x i m u m amount of this enzyme occurs in the Pa fractions. The largest total amount and the highest specific activity of the particulate nitrate reductase occurs in the P4 fraction. I t is therefore unlikely that the particulate nitrate reduetase is located within the mitoehondrion. Attempts to further purify this fraction on sucrose gradients have not been successful. Discussion The greater part of the nitrate- and the nitrite-reduetase activity in both tissues occurs in the supernatant fraction. Taking the spinach data given in Table 2 as representative it can be seen that if all of the nitrite reduetase present in the extracts (SI-~P1) was contained within the chloroplasts (chlorophyll content of PI-~ $1) in the intact leaf, the specific activity must have been equal to at least 53 units per mg chlorophyll. The chloroplasts which sediment in the P1 fraction have lost only 32 % of their capacity to fix C02, when compared with the leaves from which they were taken. They have probably lost less than 25 % of such activity if the rate of 02 evolution with ferrieyanide is taken as a measure of the ability of the system to carry out photosynthetic electron transport under our experimental conditions. If the total nitrite-reduetase activity resides within the plastid in the intact leaf, the P1 chloroplasts must have lost 60 % of their capacity to reduce nitrite based on a comparison of the actual specific activity of P1, corrected for the occlusion of 10 % of cytoplasmic enzymes (21.3) and the theoretical specific activity calculated above (53). Centrifugation at higher centrifugal forces and for longer periods does cause loss of protein from the chloroplast, as shown in the changing ratio of protein to chlorophyll in the P1 and P~ fractions (Table 3), and they also lose some nitrate and nitrite reduetase (compare specific activities of P1 and P~; Table 3). However, the results indicate that these enzymes are lost less readily than are those of the Calvin cycle. This is shown by the more rapid loss of ability to fix CO 2 than ability to reduce nitrate or nitrite (compare Table 1 and Table 2). The data from sunflower leaves supports fully that obtained
Intraeellular Location of Nitrate I~eductase and Nitrite geduetase
69
with spinach, although the lower rates usually obtained make the argument less telling if considered alone. Although nitrate-reductase activity in both tissues is over-whelmingly outside of the chloroplast, there is, nevertheless, always a demonstrable component associated with the intact plastid. There is also an association with a particulate fraction which is sedimenting at higher speeds and is not associated with either chlorophyll or with mitochondria. I~owever, this may have originated as part of the intact chloroplast membrane and it is still low in total amount when contrasted to the activity present in the soluble component. We conclude that in the intact leaf cell, there are at least two possible sites of nitrate and nitrite reduction, inside the chloroplast, and outside of it. The ratios of enzyme distribution observed in these experiments argue strongly against the main site of nitrate and nitrite reduction being located within the chloroplast in intact leaves. The major objection which can be raised to the participation of a nitrite reductase external to the chloroplast, is the requirement for a suitable electron donor. Studies on the isolated enzyme have demonstrated that ferredoxin is required as a cofaetor and reduced pyridine nueleotides alone are ineffective (Ramirez et al., 1966; J o y and ttageman, 1966; Shin and Oda, 1966). To date, there is no evidence to show that ferredoxin occurs outside of the chloroplast in photosynthetic tissues (Hall and Evans, 1969). Itowever, both nitrite and nitrate reduction do take place in plant tissues which do not contain chlorophyll (Sanderson and Cocking, 1964b ; I-Iucklesby and Elsner, 1969; see Beevers and Hageman, 1969, for additional references). This provides clear evidence that a suitable electron donor, not specifically associated with the chloroplast, exists in plant tissues. The reduction of nitrate at a site outside of the ehloroplast has been generally accepted, although Losada and co-workers disagree with this view (Paneque st al., 1969). Our results support the contention that the main site of nitrate reduction is outside of the chloroplasts. The higher specific activity of nitrate reductase on a per-mg-protein basis in the material sedimenting at 20,000 X g may be an indication that the fraction of the enzyme located within the chloroplast is associated with a membrane, as was suggested by Hageman and co-workers (I~itenour et al., 1967). On the basis of these results, we Ieel that the stimulation of nitrate assimilation by light, in vivo, observed originally by Warburg (1920) and since confirmed and extended by other workers (see Kessler, 1964, for references) is probably indirect, and takes place via transfer of reducing power across the plastid membrane. Mechanisms by which
70
B. R. Grant, C. A. Atkins and D. T. Canvin:
this m i g h t t a k e place h a v e been discussed b y Stocking a n d L a r s o n (1969) a n d b y t t a g e m a n (1969). A similar s h u t t l e in m i t o c h o n d r i a has been discussed b y K r e b s (1966). T h e existence of this t y p e of transfer, d e p e n d e n t u p o n t h e p r o d u c t s of c a r b o n fixation, would e x p l a i n ~he d e p e n d e n c e of l i g h t - s t i m u l a t e d n i t r a t e r e d u c t i o n u p o n t h e presence of CO 2 (Davis, 1953; G r a n t , 1967). N i t r i t e r e d u c t i o n in light a p p e a r s to be less d e p e n d e n t u p o n t h e presence of CO 2 a l t h o u g h such a r e q u i r e m e n t was o b s e r v e d b y G r a n t (1967).
References Arnon, D. I. : Vitamin B 1 in relation to the growth of green plants. Science 92, 264--266 (1940). - - Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1--15 (1949). Atkins, C. A. : Intermediary metabolism of photosynthesis in relation to C02 evolution in light. Doct. dissert., Queen's University, Kingston, Ont., Canada, 1969. Beevers, L., I-Iageman, R. It. : Nitrate reduction in higher plants. Ann. Rev. Plant Physiol. 29, 495--522 (1969). Bush, E. : General applicability of the channels ratio method of measuring liquid scintillation counting efficiencies. Anal. Chem. 35, 1024--1029 (1963). Coupe, M., Champigny, M. L., Moyse, A.: Sur la localisation intracellulaire de la nitrate reductase dans les feuilles et les racines d'orge. Physiol. v6g. 5, 271--291 (1967). Davis, E. A. : Nitrate reduction by Chlorella. Plant Physiol. 28, 539--544 (1953). Del Campo, F. F., Paneque, A., Ramirez, J. M., Losada, M. : Nitrate reduction in the light by isolated chloroplasts. Biochim. biophys. Acta (Amst.) 55, 450~452 (1963). Gibbs, M., Turner, J. F. : Enzymes of glycolysis. In: Modern methods of plant analysis, I-I. Linskens, B.D. Sanwal and M.V. Tracey, eds., vol. 7, p. 543. Berlin-G6ttingen-Heidelberg-New York: Springer 1964. Goldstein, D. B. : A method for assay of catalase with the oxygen electrode. Anal. Biochem. 24, 431--437 (1968). Grant, B. R. : The action of light on nitrate and nitrite assimilation by the marine chlorophyte Dunaliella tertiolecta (Butcher). J. gen. Mierobiol. 48, 379--389 (1967). Hackett, D. P. : Enzymes of terminal respiration. In: Modern methods of plant analysis, H. Linskens, B.D. Sanwal and M.V. Tracey, eds., vol. 7, p. 647. Berlin-G6ttingen-Iteidelberg-New York: Springer 1964. ttageman, R. H. : Physiological responses to nitrogen-discussion. In: Physiological aspects of crop yield, J. E. Easton, F. A. ttaskins, C. Y. Sullivan and C. H. M. Van B&vel, eds., p. 260--262. Madison, Wise. : Amer. Soc. Agron. 1969. - - Flesher, D. : Nitrate reductase activity in corn seedlings affected by light and nitrate content of nutrient media. Plant Physiol. 35, 700--708 (1960). Hall, D. 0., Evans, M. C. W. : Iron-sulphur proteins. Nature (Lond.) 223, 1342-1348 (1969). Hewitt, E. J., Nicholas, D. J. D. : Enzymes of inorganic nitrogen metabolism. In: Modern methods of plant analysis, K. Paech and M. V. Tracey, eds., vol. 7, p. 67--172. Berlin-G6ttingen~Heidetberg~ew York: -Springer t
