Planta (Berl.) 111, 47--56 (1973) 9 by Springer-Verlag 1973
Studies on Nitrite Reductase in Barley* W. F. Bourne** and B. J. Miflin Department of Plant Science, The University, Newcastle upon Tyne, NE1 7RU, U.K. Received Jannuary 2, 1973 Summary. Nitrite reductase from barley seedlings was purified 50-60 fold by ammonium sulphate precipitation and gel filtration. No differences were established in the characteristics of nitrite reductases isolated in this way from either leaf or root tissues. The root enzyme accepted electrons from reduced methyl viologen, ferredoxin, or an unidentified endogenous cofactor. Enzyme activity in both tissues was markedly increased by growth on nitrate. This activity was not associated with sulphite reductase activity. Microbial contamination could not account for the presence of nitrite reduetase activity in roots. Nitrite reductase assayed in vitro with reduced methyl viologen as the electron donor was inhibited by 2,4-dinitrophenol but not by arsenate.
Introduction Most information on the reduction of nitrate to ammonia b y higher plants has been derived from experiments using photosynthetic tissue, and, as discussed in a review by Beevers and Hageman (1969), N A D H and ferredoxin are generally considered to be the respective electron donors of the two enzymes of the reduction sequence--nitrate reductase and nitrite reductase. Roots are, however, also capable of reducing nitrate and exporting the reduced nitrogenous compounds to the aerial parts of the plant (Bollard, 1956; Wallace and Pate, 1965, 1967). I n several of the plants studied the relative concentrations of reduced and unreduced nitrogen in the bleeding xylem sap indicated that, in these cases, the majority of the nitrogen passing up the plant had been reduced in the roots. Studies on the nitrate and nitrite reductases of root tissues are relatively few, but it has been shown t h a t under certain conditions the extractable levels of these enzymes in roots is comparable to those in leaves (Miflin, 1967). Of the species t h a t have been investigated so far the cocklebur (Xanthium pennsylvanicum) appears to be unique in its inability to reduce nitrate in the root system and in its lack of a root nitrate reductase (Wallace and Pate, 1967). However, whilst there is * Abbreviations: DNP. 2,4-dinitrophenol, DEAE diethyl amino ethyl. ** Present address: Department of Biological Studies, Lanchester Polytechnic, Priory Street, Coventry, CVI 5FB, U.K.
48
W . F . Bourne and B. J. Miflin:
p l e n t y of evidence suggesting t h a t roots have a role i n n i t r a t e assimilation, little is k n o w n of t h e physiological system reducing n i t r i t e since there is n o t h i n g to indicate t h a t t h e y c o n t a i n ferredoxin a n d previous studies of this enzyme have i n v o l v e d the use of artificial electron d o n a t i n g systems such as reduced viologen dyes. The only physiologically likely system so far described i n root tissue is a p a r t i c u l a t e system capable of reducing n i t r a t e to a m m o n i a i n the presence of A T P (Bourne a n d Miflin, 1970). This paper reports a f u r t h e r i n v e s t i g a t i o n into the n i t r a t e reducing system of b a r l e y roots, with special reference to n i t r i t e reductase.
Methods Roots were routinely harvested from barley seedlings (var. Proctor) grown as previously described (Miflin, 1970a), while leaf material was from similar plants grown in natural daylight. Sterile roots were obtained by germinating surfacesterilised seeds on a stainless steel mesh supported half-way up a 250 • 120 mm glass vessel. The vessels were sterilised by autoclaving, filled to within 10 mm of the mesh with sterile nutrient medium containing 10 mM KNO3and, after the seeds were spread on the mesh, capped with a close-fitting sterilised glass lid. A positive pressure of sterile air was maintained by bubbling air into the medium via a bacteriological filter through a glass tube set in the wall of the vessel. After 7 days growth in the dark at 24~ the roots were harvested and the medium tested for microbial contamination. In this way sufficient sterile root material could be obtained for both enzyme studies and isolation of particulate sub-cellular fractions. Nitrite reductase was isolated from roots or leaves as follows: the plant material was washed in distilled water and homogenised in an equal amount (w/v) of isolation buffer (50 m_~ K2PO4, 5ml~ EDTA, 1 m_~ cysteine hydrochloride, pH 7.5) using a pestle and mortar. The brei was filtered through a 25 ~zmnylon mesh and the filtrate centrifuged at 10000 • g for 10 rain. Initial purification was by differential precipitation with either ammonium sulphate (added as an ice-cold saturated solution pH 7.5) or acetone (chilled to -15~ before use). In both cases the extracts were left at 0~ for 20 rain to precipitate, centrifuged at 5000 • g for 20 rain and the pellet redissolved in isolation buffer. Subsequent purification was by chromatography on either a 25 • 350 mm column of Sephadex G200 (elution buffer 50 ml~ KH2P04, 100 mlVI NaC1, 1 ml~ cysteine-hydrochloride, pH 7.5) or a 15 • 200 mm column of DEAE cellulose (Whatman DE 23) equilibrated with 5 m_~ tris-HC1, pH 7.5 (eluted with an increasing chloride ion concentration gradient between 0 and 0.5 M NaC1. Samples were dialysed against 5 mM tris-HC1 before chromatography on DEAE cellulose, and all extraction and purification procedures were carried out at 4~C, using precooled reagents and apparatus. Ferredoxin was isolated and purified from spinach leaves by a modification of the methods of Hill and Bendall (1960) and Tagawa and Amen (1964). Particulate fractions of barley roots were prepared as described by l~Iiflin (1967 and 1970b). Nitrate and nitrite reductase activities were assayed by the methods employed by ]Kiflin (1967). Ammonia production was determined by the alkaline phenol colourmetrie technique of Russell (1944) following mierodiffusion in Conway units. Sulphide was measured by Siegel's method (1965), and protein by that of Lowry et al. (1951).
Nitrite Reduetase in Barley
49
Results Tables 1 a n d 2 show t h e increases in specific a c t i v i t y o b t a i n e d b y f r a c t i o n a t i n g crude n i t r i t e r e d u c t a s e e x t r a c t s w i t h a m m o n i u m s u l p h a t e Table 1. Distribution of nitrite reductase in fractions obtained by differential precipitation of barley root extracts with acetone. Activities are those of proteins precipitated within the acetone concentrations stated % acetone
Nitrite reductase
% recovery
Specific activity Total activity (nmoles NO~- reduced/ (nmoles NO~/min) mg protein/rain) 0 0-33 33-66 66-80
21.6 16.0 123.3 82.5
7166 300 4200 1050
100 4.2 58.6 14.6
Table 2. Distribution of nitrite reductase in fractions from barley roots obtained by differential precipitation with ammonium sulphate. Activities are those of proteins precipitated within the % saturations stated % saturation
Nitrite reductase
% recovery
Total activity Specific activity (nmoles NO2- reduced/ (nmoles NOe/min) mg protein/rain) 0 0-25 25-50 50-75 75-100
47.5 54.5 9.1 313.3 145.8
9750 235 185 5373 525
100 2.4 1.9 57.0 5.4
a n d acetone respectively. F u r t h e r p u r i f i c a t i o n was carried o u t on t h e p r o t e i n p r e c i p i t a t i n g a t b e t w e e n 50% a n d 75% of s a t u r a t i o n w i t h a m m o n i u m sulphate, a n d t h e results of c h r o m a t o g r a p h i n g this f r a c t i o n on either D E A E cellulose or S e p h a d e x G200 are given in T a b l e 3. T h e r e was no evidence for two p e a k s of n i t r i t e r e d u c t a s e a c t i v i t y eluting from t h e D E A E cellulose column as has r e c e n t l y been r e p o r t e d for m a i z e scutellum p r e p a r a t i o n s ( H u e k l e s b y et al., 1972). T h e a c t i v i t y p e a k from t h e S e p h a d e x c o l u m n was used for c h a r a c t e r i s a t i o n of b a r l e y r o o t n i t r i t e r e d u e t a s e , a n d t h e leaf e n z y m e was purified in t h e s a m e w a y for comparison. B o t h e n z y m e s were f o u n d t o h a v e an o p t i m u m p H of 7.1 for t h e in vitro assay, using r e d u c e d m e t h y l viologen as electron d o n o r (Fig. 1). 4
P l a n t a (Berl.), Bd. 111
50
W.F. Bourne and ]3. J. Miflin: 3.2
2.4
1.6
/~ 7
0.8
5.5
6.0
6.5
7.0
7.5
8.0
pH of assay
Fig. I. pH optima for barley nitrife reductases. Leaf enzyme ~--., root enzyme 9--.. Components of assay: 0.04 mg methyl viologen, 1.6 mg Na2S204, 1.6 mg NatICOa, 400 nmoles NaN0~ all in 0.6 ml H20. 0.4 ml of enzyme added in 0.1 M phosphate buffer of the indicated pH
Table 3. Purification of barley root nitrite reduetase by ion-exchange or molecular exclusion chromatography Nitrite reductase specific activity (nmoles NO2/mg/min)
DEAE Sephadex G200
Crude extract
(NHa)2SO4 Activity ppt. peak from column
60.0 56.6
235.0 265.0
383.3 2350.0
% recovery of sample from column
69 65
Methyl viologen, reduced by NaHCO 3 and Na~S~04, becomes increasingly oxidised with decreasing pH, and is completely decolourised at pH 4.5, so values for the activity of nitrite reductase in this assay below pH 6.0 could reflect the decreasing reduction state of the electron donor. No differences between root and leaf enzymes in their affinity for nitrite could be established, both had a K m for nitrite, as determined by Lineweaver-Burke reciprocal plots, of about 2 mM. Similarly, incubation at 50 ~C, followed by rapid cooling in ice before assay, failed to demonstrafe any difference in the heat stability of the two proteins. Both were denatured at the same rate, with a half-life at this temperature of 4 min (Fig. 2). Further similarity of root and leaf nitrite rcduetases was shown during chromatography on Sephadex G200 of samples containing both
Nitrite Reductase in Barley
51
100 8O "~ 60
40 2O
I 4
g
12
16
20
24
28
g2
Incubation time (min.} Fig. 2. I n a c t i v a t i o n of barley nitrite reductases at 50 ~ C. L e a f e n z y m e enzyme ,---,
o - - ~ , root
~176
2.0
II
-i.-~ zE
1.5
=E
? IN 0 z
o
E
0.5
I
I
I
I
I
4
8
12
16
20
Assay time (rain.)
Fig. 3. Nitrite reduction and ammonia production by purified barley root nitrite reductase. Nitrite reduction o--~, ammonia production . - - .
enzymes. The activity was recovered from the column as a single peak, indicating that both proteins were of similar molecular size. The rates of nitrite loss and concomitant ammonia production by purified root nitrite reductase, with reduced methyl viologen as the electron donor, were linear over 10-12 min, with ammonia being produced in amounts equivalent to the nitrite loss from the assay (Fig. 3). No sulphite reductase activity could be detected in the purified root nitrite reductase extracts when nitrite was replaced in the assay by 1 ~mole of Na~SO a. While this indicates that sulphite reduction is not the function of the enzyme, both sulphite and nitrite reduetases have 4*
52
W.F. Bourne and B. g. Miflin:
been shown to be associated with particulate fractions of barley root extracts (Mayer, 1967; Miflin, 1970b) and in bacteria a single protein catalyses both reactions (Kemp et al., 1963). Accordingly a study was made of the distribution of both activities in barley roots. A particulate fraction was obtained from roots by the method previously described by Mifhn (1970b). The roots were homogenised in isotonic medium, using a chilled pestle and mortar, and filtered through 25 ~m nylon mesh. The filtrate (Fraction A) was fractionated by centrifugation at 18000 • g for 15 min in two parts to give a supernatant (Fraction B) and two pellets, one of which was resuspended in isotonic medium (Fraction C) and the other in the same medium made 1% w/v with Triton X-100 (Fraction D). This latter fraction was further centrifuged at 18000 • g for 15 min to remove particle fragments. All operations were carried out at 4 ~C, and the fractions assayed for sulphite and nitrite reductase activities. The results (Table 4) show t h a t while a part of both activities is associated with a particulate fraction, the ratio of the two changes with Triton X-100 treatment, which appears to be necessary before m a x i m u m rates of sulphite reductase activity are observed, an effect previously noted b y Mayer (1967). Table 4. Distribution of sulphite and nitrite reductase activities in barley root extracts. Activities as nmoles sulphide produced or nitrite lost/ml extraet/min Fraction
A B C D
% recovery
Activity
S.R~ as %
S.R.
NiR
Sulphite reductase
NO~ reductase
NiR
100 65.1 54.4 244.1
100 81.9 14.7 10.0
1.91 1.18 2.08 9.36
187.00 153.00 55.00 37.50
1.03 0.82 3.71 23.40
of
Although methyl viologen reduced by Na~S204 and N a H C 0 s was used as the electron donor in routine assays of nitrite reductase, some activity was detectable with Na2S~O~/NaHCO 3 alone. The proportion of this activity was increased to about a quarter of the total when the crude extracts were purified b y fractional precipitation with acetone (between 33% and 75%). Subsequent chromatography of the resuspended acetone-precipitated protein on D E A E cellulose rendered the enzyme completely dependent on the presence of methyl viologen. These results, summarised in Table 5, indicate t h a t an endogenous cofaetor is present in crude extracts which can be removed b y methods similar to those used by J o y and Hageman (1966) to separate ferredoxJn from
Nitrite Reductase in Barley
53
t00 8O
r
6o
~ 4o 20 0 0
I
I
I
I
0.4
0.8
1.2
1.6
2.0
Inhibitor concentration (mM)
Fig. 4. Inhibition of barley root nitrite reductase by DNP (.--.) and arsenate (~
Table 5. Cofactor requirements of barley root nitrite reductase Nitrite reductase activity (nmoles NO2 reduced/ml/min)
Crude extract 33-75% acetone precipitated extract Enzyme from DEAE column Boiled crude extract Minus enzyme
Methyl viologen
1~a2S204 Ferredoxin only
106.6 173.6 38.5 1.5 0.5
5.6 48.5 0.1 1.3 0.1
-148.5 15.6 -0.6
spinach-leaf n i t r i t e reductase. Spinach-leaf f e r r e d o x i n a d d e d to assays of t h e D E A E - t r e a t e d b a r l e y - r o o t n i t r i t e r e d u c t a s e can s u b s t i t u t e for t h e endogenous cofactor, b u t a t t e m p t s to recover such a cofactor from t h e column were unsuccessful. The lack of a c t i v i t y in t h e absence of t h e enzyme, or in t h e presence of boiled enzyme, show t h a t t h e concent r a t i o n s of s o d i u m d i t h i o n i t e used are n o t causing a n o n - e n z y m a t i c loss of nitrite. D N P a n d arsenate, i n h i b i t o r s of n i t r a t e a s s i m i l a t i o n b y algae in vivo ( A h m a d a n d Morris, 1968; H a t t o r i a n d Myers, 1966; Kessler a n d Czygan, 1963) are considered to act a t t h e level of n i t r i t e reduction. A r s e n a t e h a d no effect on b a r l e y - r o o t n i t r i t e r e d u c t a s e in vitro, a l t h o u g h D N P i n h i b i t e d a t 10-3M (Fig. 4). I t is p r o b a b l e t h a t this effect was caused b y diversion of electrons from t h e r e d u c e d m e t h y l viologen from n i t r i t e r e d u c t i o n t o D N P reduction.
54
W.F. Bourne and B. J. ~iflin:
Only minimal levels (0.6 nmoles NO~ reduced/rag of protein/min) of nitrite reductase activity are detectable in the roots of barley seedlings grown in nitrate-free medium. On transfer to a medium containing 10 mM KNO 3the activityincreases, after a 4-5 h lag, to reach a maximum of 50-60 nmoles NOg reduced/mg protein/mAn after 30-35 h. The majority of the experiments were done with roots grown in open culture and, while every precaution was taken to keep microbial contamination to as low a level as possible, the possibility exists that contamination was contributing to the levels of activity assayed. Accordingly nitrate and nitrite reductase activities were measured, both of roots grown under sterile conditions and of the bacteria contaminating roots grown in open culture. The bacteria were obtained by inoculating normal root growth medium containing sucrose as a carbon source, with an aliquot of the medium in which roots had been growing. After the bacteria had grown they were harvested by centrifugation. The bacterial pellet was extracted and assayed for activity in the same way as the sterile and non-sterile root tissue. As shown in Table 6 no activity could be detected in this extract, while roots grown under sterile conditions had levels of nitrate and nitrite reductase activity comparable to those of normally grown roots. Table 6. Nitrite reductase activities of roots grown in open culture, under sterile conditions and of the contaminating micro-organisms.Activities expressed as nmoles NO~ lost or formed/mg protein/min
Sterile roots Non-sterile roots Bacteria
Nitrate reductase
Nitrate reductase
2.2 2.6 0.1
21.1 21.8 0.3
Discussion Nitrite reductase, isolated from barley roots and purified 50-60 fold, produced ammonia from nitrite in eqnimolar amounts, indicating that in roots the enzyme catalyses the entire 6-electron reduction sequence, a similar situation to that which exists in leaf systems (Hageman et al., 1962). No differences could be established in the characteristics of nitrite reduetases from leaf or root tissues of barley seedlings, and both were dependent for activity in vitro on electron donors of high redox potential, such as reduced methyl viologen or ferredoxin. While no evidence exists as yet for the presence of ferredoxin in roots, the presence of a
Nitrite Reductase in Barley
55
low but significant level of activity in the absence of added cofaetor in the unpurified and partially purified root extracts suggests t h a t an endogenous eofactor was present. This activity was particularly apparent when acetone was used to fractionate the extract (Table 5). If a ferredoxin-like eofactor is present in barley roots, nitrite reduction in roots could proceed b y a system analogous to t h a t elaborated b y Paneque et al. (1964) for nitrite reduction in the dark b y leaves, involving N A D P H and NADPferredoxin oxidoreductase. Some support for such a system is provided b y the results of B u t t and Beevers (1961) study of carbohydrate metabolism in maize roots, which demonstrated t h a t nitrite stimulated the pentose phosphate pathway, an effect which they suggested might be due to re-oxidation of N A D P H b y nitrite. Several lines of evidence have been produced b y Kessler and his coworkers (Kessler, 1964) to suggest t h a t nitrite reduction, particularly in the dark, is dependent on ATP. Although our results with the particulate system from barley roots (Bourne and Miflin, 1970) support this contention, the effect of D N P in vivo could be due to its ability to inhibit nitrite reductase directly as well as its uncoupling effect. D N P has been shown b y Wessels (1959) to act as an alternative electron acceptor in the photosynthetic electron transport chain and it is also likely t h a t it can compete with nitrite for electrons in the reduced methyl viologen assay system. I n contrast arsenate, which inhibits in vivo nitrite reduction, has no effect on the isolated enzyme. Results shown here with roots grown under sterile conditions preclude the possibility t h a t microbial contamination is the source of enzyme activity in studies of root nitrate or nitrite reduetases. Similarly the induction of activity by nitrate and the dissociation of nitrite and sulphite reduetase activities, indicate t h a t a non-specific reaction is not responsible for nitrite reduction b y barley root extracts.
References Ahmad, J., Morris, I. : The effects of 2, 4-dinitrophenol and other uncoupling agents on the assimilation of nitrate and nitrite by Chlorella. Bioehim. biophys. Acta (Amst.) 162, 32-38 (1968). Beevers, L., Hageman, R. H. : Nitrate reduction in higher plants. Ann. Rev. Plant Physiol. 20, 495-522 (1969). Bollard, E. G.: Nitrogenous compounds in plant xylem sap. Nature (Lond.) 178, 1189-1190 (1956). Bourne, W. F., Miflin, B. J. : An ATP dependent reduction of nitrate to ammonia by a cell free particulate system from barley roots. Biochem. biophys. Res. Commun. 40, 1305-1310 (1970). Butt, V. S., Beevers, H.: The regulation of pathways of glucose catabolism in maize roots. Biochem. J. 80, 21-27 (1961). Hageman, R.H., Cresswell, C. 1~., Hewitt, E.g.: Reduction of nitrate, nitrite and hydroxylamine to ammonia by enzymes from higher plants. Nature (Lond.) 198, 247-250 (1962).
56
W . F . Bourne and B. g. Miflin: Nitrite Reductase in Barley
Hattori, A., Myers, J.: Reduction of nibrate and nitrite by subcellular preparations of Anabaena cydlindrlca. Plant Physiol. 41, 1031-1036 (1966). Hill, R., Bendall, F.: Crystallization of a photosynthetic reduetase from a green plant. Nature (Lond.) 187, 417 (1960). Hucklesby, D. P., Dalling, M. J., Hageman, R. H. : Some properties of two forms of nitrite reductase from corn (Zea mays L.) Scutellum. Planta (Berl.) 104, 220-233 (1972). Joy, K. W., Hageman, R. H.: The purification of nitrite reductase from higher plants and its dependence on ferredoxin. Biochem. J. 100, 263-273 (1966). Kemp, g. D., Atkinson, D. E., Ehret, A., Lazzarini, R. A.: Evidence for the identity of the NADH phosphate specific sulphite and nitrite rednctases of E. coli. J. biol. Chem. 238, 3466-3471 (1963). Kessler, E.: Nitrate assimilation by plants. Ann. Rev. Plant Physiol. 15, 57-72 (1964). Kessler, E., Czygan, F. C. : Seasonal changes in the nitrate-reducing activity of a green algae (Ankistrodesmus braunii). Experientia (Basel) 19, 89-90 (1963). Lowry, O. H., Rosenbrough, N. J., Farr, A. L., Randall, R. S. : Protein measurement with the Folin phenol reagent. J. biol. Chem. 198, 265-275 (1951). Mayer, A.M.: Subcellular location of snlphite reductase in plant tissues. Plant Physiol. 42, 324-326 (1967). Miflin, B. J.: Distribution of nitrate and nitrite reductase in barley. Nature (Lond.) 214, 1133-1134 (1967). Miflin, B. J.: Nitrate and nitrite reductase systems in barley roots. Rev. roum. Biochim. 7, 53-60 (1970a). Miflin, B. J.: Studies on the subcellular location of particulate nitrate and nitrite reductase, ghtamic dehydrogenase and other enzymes in barley roots. Planta (Berl.) 93, 160-170 (1970b). Paneque, A., Ramirez, J. M., Del Campo, F. F., Losada, M.: Light and dark reduction of nitrite in a reconstituted enzyme system. J. biol. Chem. 259, 1737-1741 (1964). Russel, J. A. : The estimation of small amounts of ammonia by the phenol-hypochlorite reaction. J. biol. Chem. 156, 457-465 (1944). Siegel, L. M.: A direct microdetermination for sulphide. Analyt. Bioehem. 11, 126-132 (1965). Tagawa, K., Arnon, D. I. : Ferredoxins as electron carriers in photosynthesis and in the biological production of hydrogen gas. Nature (Lond.) 195, 537-543 (1962). Wallace, W., Pate, J. S. : Nitrate reductase in the field pea. Ann. Botany 29, 655 (1965). Wallace, W., Pate, J. S. : Nitrate assimilation in higher plants with special reference to the Cocklebnrr. Ann. Botany 81, 213-228 (1967). Wessels, J. S. C. : DNP as a catalyst of photosynthetic phosphorylation. Biochem. biophys. Acta (Amst.) 36, 264-265 (1959).
Studies on Nitrite Reductase in Barley* W. F. Bourne** and B. J. Miflin Department of Plant Science, The University, Newcastle upon Tyne, NE1 7RU, U.K. Received Jannuary 2, 1973 Summary. Nitrite reductase from barley seedlings was purified 50-60 fold by ammonium sulphate precipitation and gel filtration. No differences were established in the characteristics of nitrite reductases isolated in this way from either leaf or root tissues. The root enzyme accepted electrons from reduced methyl viologen, ferredoxin, or an unidentified endogenous cofactor. Enzyme activity in both tissues was markedly increased by growth on nitrate. This activity was not associated with sulphite reductase activity. Microbial contamination could not account for the presence of nitrite reduetase activity in roots. Nitrite reductase assayed in vitro with reduced methyl viologen as the electron donor was inhibited by 2,4-dinitrophenol but not by arsenate.
Introduction Most information on the reduction of nitrate to ammonia b y higher plants has been derived from experiments using photosynthetic tissue, and, as discussed in a review by Beevers and Hageman (1969), N A D H and ferredoxin are generally considered to be the respective electron donors of the two enzymes of the reduction sequence--nitrate reductase and nitrite reductase. Roots are, however, also capable of reducing nitrate and exporting the reduced nitrogenous compounds to the aerial parts of the plant (Bollard, 1956; Wallace and Pate, 1965, 1967). I n several of the plants studied the relative concentrations of reduced and unreduced nitrogen in the bleeding xylem sap indicated that, in these cases, the majority of the nitrogen passing up the plant had been reduced in the roots. Studies on the nitrate and nitrite reductases of root tissues are relatively few, but it has been shown t h a t under certain conditions the extractable levels of these enzymes in roots is comparable to those in leaves (Miflin, 1967). Of the species t h a t have been investigated so far the cocklebur (Xanthium pennsylvanicum) appears to be unique in its inability to reduce nitrate in the root system and in its lack of a root nitrate reductase (Wallace and Pate, 1967). However, whilst there is * Abbreviations: DNP. 2,4-dinitrophenol, DEAE diethyl amino ethyl. ** Present address: Department of Biological Studies, Lanchester Polytechnic, Priory Street, Coventry, CVI 5FB, U.K.
48
W . F . Bourne and B. J. Miflin:
p l e n t y of evidence suggesting t h a t roots have a role i n n i t r a t e assimilation, little is k n o w n of t h e physiological system reducing n i t r i t e since there is n o t h i n g to indicate t h a t t h e y c o n t a i n ferredoxin a n d previous studies of this enzyme have i n v o l v e d the use of artificial electron d o n a t i n g systems such as reduced viologen dyes. The only physiologically likely system so far described i n root tissue is a p a r t i c u l a t e system capable of reducing n i t r a t e to a m m o n i a i n the presence of A T P (Bourne a n d Miflin, 1970). This paper reports a f u r t h e r i n v e s t i g a t i o n into the n i t r a t e reducing system of b a r l e y roots, with special reference to n i t r i t e reductase.
Methods Roots were routinely harvested from barley seedlings (var. Proctor) grown as previously described (Miflin, 1970a), while leaf material was from similar plants grown in natural daylight. Sterile roots were obtained by germinating surfacesterilised seeds on a stainless steel mesh supported half-way up a 250 • 120 mm glass vessel. The vessels were sterilised by autoclaving, filled to within 10 mm of the mesh with sterile nutrient medium containing 10 mM KNO3and, after the seeds were spread on the mesh, capped with a close-fitting sterilised glass lid. A positive pressure of sterile air was maintained by bubbling air into the medium via a bacteriological filter through a glass tube set in the wall of the vessel. After 7 days growth in the dark at 24~ the roots were harvested and the medium tested for microbial contamination. In this way sufficient sterile root material could be obtained for both enzyme studies and isolation of particulate sub-cellular fractions. Nitrite reductase was isolated from roots or leaves as follows: the plant material was washed in distilled water and homogenised in an equal amount (w/v) of isolation buffer (50 m_~ K2PO4, 5ml~ EDTA, 1 m_~ cysteine hydrochloride, pH 7.5) using a pestle and mortar. The brei was filtered through a 25 ~zmnylon mesh and the filtrate centrifuged at 10000 • g for 10 rain. Initial purification was by differential precipitation with either ammonium sulphate (added as an ice-cold saturated solution pH 7.5) or acetone (chilled to -15~ before use). In both cases the extracts were left at 0~ for 20 rain to precipitate, centrifuged at 5000 • g for 20 rain and the pellet redissolved in isolation buffer. Subsequent purification was by chromatography on either a 25 • 350 mm column of Sephadex G200 (elution buffer 50 ml~ KH2P04, 100 mlVI NaC1, 1 ml~ cysteine-hydrochloride, pH 7.5) or a 15 • 200 mm column of DEAE cellulose (Whatman DE 23) equilibrated with 5 m_~ tris-HC1, pH 7.5 (eluted with an increasing chloride ion concentration gradient between 0 and 0.5 M NaC1. Samples were dialysed against 5 mM tris-HC1 before chromatography on DEAE cellulose, and all extraction and purification procedures were carried out at 4~C, using precooled reagents and apparatus. Ferredoxin was isolated and purified from spinach leaves by a modification of the methods of Hill and Bendall (1960) and Tagawa and Amen (1964). Particulate fractions of barley roots were prepared as described by l~Iiflin (1967 and 1970b). Nitrate and nitrite reductase activities were assayed by the methods employed by ]Kiflin (1967). Ammonia production was determined by the alkaline phenol colourmetrie technique of Russell (1944) following mierodiffusion in Conway units. Sulphide was measured by Siegel's method (1965), and protein by that of Lowry et al. (1951).
Nitrite Reduetase in Barley
49
Results Tables 1 a n d 2 show t h e increases in specific a c t i v i t y o b t a i n e d b y f r a c t i o n a t i n g crude n i t r i t e r e d u c t a s e e x t r a c t s w i t h a m m o n i u m s u l p h a t e Table 1. Distribution of nitrite reductase in fractions obtained by differential precipitation of barley root extracts with acetone. Activities are those of proteins precipitated within the acetone concentrations stated % acetone
Nitrite reductase
% recovery
Specific activity Total activity (nmoles NO~- reduced/ (nmoles NO~/min) mg protein/rain) 0 0-33 33-66 66-80
21.6 16.0 123.3 82.5
7166 300 4200 1050
100 4.2 58.6 14.6
Table 2. Distribution of nitrite reductase in fractions from barley roots obtained by differential precipitation with ammonium sulphate. Activities are those of proteins precipitated within the % saturations stated % saturation
Nitrite reductase
% recovery
Total activity Specific activity (nmoles NO2- reduced/ (nmoles NOe/min) mg protein/rain) 0 0-25 25-50 50-75 75-100
47.5 54.5 9.1 313.3 145.8
9750 235 185 5373 525
100 2.4 1.9 57.0 5.4
a n d acetone respectively. F u r t h e r p u r i f i c a t i o n was carried o u t on t h e p r o t e i n p r e c i p i t a t i n g a t b e t w e e n 50% a n d 75% of s a t u r a t i o n w i t h a m m o n i u m sulphate, a n d t h e results of c h r o m a t o g r a p h i n g this f r a c t i o n on either D E A E cellulose or S e p h a d e x G200 are given in T a b l e 3. T h e r e was no evidence for two p e a k s of n i t r i t e r e d u c t a s e a c t i v i t y eluting from t h e D E A E cellulose column as has r e c e n t l y been r e p o r t e d for m a i z e scutellum p r e p a r a t i o n s ( H u e k l e s b y et al., 1972). T h e a c t i v i t y p e a k from t h e S e p h a d e x c o l u m n was used for c h a r a c t e r i s a t i o n of b a r l e y r o o t n i t r i t e r e d u e t a s e , a n d t h e leaf e n z y m e was purified in t h e s a m e w a y for comparison. B o t h e n z y m e s were f o u n d t o h a v e an o p t i m u m p H of 7.1 for t h e in vitro assay, using r e d u c e d m e t h y l viologen as electron d o n o r (Fig. 1). 4
P l a n t a (Berl.), Bd. 111
50
W.F. Bourne and ]3. J. Miflin: 3.2
2.4
1.6
/~ 7
0.8
5.5
6.0
6.5
7.0
7.5
8.0
pH of assay
Fig. I. pH optima for barley nitrife reductases. Leaf enzyme ~--., root enzyme 9--.. Components of assay: 0.04 mg methyl viologen, 1.6 mg Na2S204, 1.6 mg NatICOa, 400 nmoles NaN0~ all in 0.6 ml H20. 0.4 ml of enzyme added in 0.1 M phosphate buffer of the indicated pH
Table 3. Purification of barley root nitrite reduetase by ion-exchange or molecular exclusion chromatography Nitrite reductase specific activity (nmoles NO2/mg/min)
DEAE Sephadex G200
Crude extract
(NHa)2SO4 Activity ppt. peak from column
60.0 56.6
235.0 265.0
383.3 2350.0
% recovery of sample from column
69 65
Methyl viologen, reduced by NaHCO 3 and Na~S~04, becomes increasingly oxidised with decreasing pH, and is completely decolourised at pH 4.5, so values for the activity of nitrite reductase in this assay below pH 6.0 could reflect the decreasing reduction state of the electron donor. No differences between root and leaf enzymes in their affinity for nitrite could be established, both had a K m for nitrite, as determined by Lineweaver-Burke reciprocal plots, of about 2 mM. Similarly, incubation at 50 ~C, followed by rapid cooling in ice before assay, failed to demonstrafe any difference in the heat stability of the two proteins. Both were denatured at the same rate, with a half-life at this temperature of 4 min (Fig. 2). Further similarity of root and leaf nitrite rcduetases was shown during chromatography on Sephadex G200 of samples containing both
Nitrite Reductase in Barley
51
100 8O "~ 60
40 2O
I 4
g
12
16
20
24
28
g2
Incubation time (min.} Fig. 2. I n a c t i v a t i o n of barley nitrite reductases at 50 ~ C. L e a f e n z y m e enzyme ,---,
o - - ~ , root
~176
2.0
II
-i.-~ zE
1.5
=E
? IN 0 z
o
E
0.5
I
I
I
I
I
4
8
12
16
20
Assay time (rain.)
Fig. 3. Nitrite reduction and ammonia production by purified barley root nitrite reductase. Nitrite reduction o--~, ammonia production . - - .
enzymes. The activity was recovered from the column as a single peak, indicating that both proteins were of similar molecular size. The rates of nitrite loss and concomitant ammonia production by purified root nitrite reductase, with reduced methyl viologen as the electron donor, were linear over 10-12 min, with ammonia being produced in amounts equivalent to the nitrite loss from the assay (Fig. 3). No sulphite reductase activity could be detected in the purified root nitrite reductase extracts when nitrite was replaced in the assay by 1 ~mole of Na~SO a. While this indicates that sulphite reduction is not the function of the enzyme, both sulphite and nitrite reduetases have 4*
52
W.F. Bourne and B. g. Miflin:
been shown to be associated with particulate fractions of barley root extracts (Mayer, 1967; Miflin, 1970b) and in bacteria a single protein catalyses both reactions (Kemp et al., 1963). Accordingly a study was made of the distribution of both activities in barley roots. A particulate fraction was obtained from roots by the method previously described by Mifhn (1970b). The roots were homogenised in isotonic medium, using a chilled pestle and mortar, and filtered through 25 ~m nylon mesh. The filtrate (Fraction A) was fractionated by centrifugation at 18000 • g for 15 min in two parts to give a supernatant (Fraction B) and two pellets, one of which was resuspended in isotonic medium (Fraction C) and the other in the same medium made 1% w/v with Triton X-100 (Fraction D). This latter fraction was further centrifuged at 18000 • g for 15 min to remove particle fragments. All operations were carried out at 4 ~C, and the fractions assayed for sulphite and nitrite reductase activities. The results (Table 4) show t h a t while a part of both activities is associated with a particulate fraction, the ratio of the two changes with Triton X-100 treatment, which appears to be necessary before m a x i m u m rates of sulphite reductase activity are observed, an effect previously noted b y Mayer (1967). Table 4. Distribution of sulphite and nitrite reductase activities in barley root extracts. Activities as nmoles sulphide produced or nitrite lost/ml extraet/min Fraction
A B C D
% recovery
Activity
S.R~ as %
S.R.
NiR
Sulphite reductase
NO~ reductase
NiR
100 65.1 54.4 244.1
100 81.9 14.7 10.0
1.91 1.18 2.08 9.36
187.00 153.00 55.00 37.50
1.03 0.82 3.71 23.40
of
Although methyl viologen reduced by Na~S204 and N a H C 0 s was used as the electron donor in routine assays of nitrite reductase, some activity was detectable with Na2S~O~/NaHCO 3 alone. The proportion of this activity was increased to about a quarter of the total when the crude extracts were purified b y fractional precipitation with acetone (between 33% and 75%). Subsequent chromatography of the resuspended acetone-precipitated protein on D E A E cellulose rendered the enzyme completely dependent on the presence of methyl viologen. These results, summarised in Table 5, indicate t h a t an endogenous cofaetor is present in crude extracts which can be removed b y methods similar to those used by J o y and Hageman (1966) to separate ferredoxJn from
Nitrite Reductase in Barley
53
t00 8O
r
6o
~ 4o 20 0 0
I
I
I
I
0.4
0.8
1.2
1.6
2.0
Inhibitor concentration (mM)
Fig. 4. Inhibition of barley root nitrite reductase by DNP (.--.) and arsenate (~
Table 5. Cofactor requirements of barley root nitrite reductase Nitrite reductase activity (nmoles NO2 reduced/ml/min)
Crude extract 33-75% acetone precipitated extract Enzyme from DEAE column Boiled crude extract Minus enzyme
Methyl viologen
1~a2S204 Ferredoxin only
106.6 173.6 38.5 1.5 0.5
5.6 48.5 0.1 1.3 0.1
-148.5 15.6 -0.6
spinach-leaf n i t r i t e reductase. Spinach-leaf f e r r e d o x i n a d d e d to assays of t h e D E A E - t r e a t e d b a r l e y - r o o t n i t r i t e r e d u c t a s e can s u b s t i t u t e for t h e endogenous cofactor, b u t a t t e m p t s to recover such a cofactor from t h e column were unsuccessful. The lack of a c t i v i t y in t h e absence of t h e enzyme, or in t h e presence of boiled enzyme, show t h a t t h e concent r a t i o n s of s o d i u m d i t h i o n i t e used are n o t causing a n o n - e n z y m a t i c loss of nitrite. D N P a n d arsenate, i n h i b i t o r s of n i t r a t e a s s i m i l a t i o n b y algae in vivo ( A h m a d a n d Morris, 1968; H a t t o r i a n d Myers, 1966; Kessler a n d Czygan, 1963) are considered to act a t t h e level of n i t r i t e reduction. A r s e n a t e h a d no effect on b a r l e y - r o o t n i t r i t e r e d u c t a s e in vitro, a l t h o u g h D N P i n h i b i t e d a t 10-3M (Fig. 4). I t is p r o b a b l e t h a t this effect was caused b y diversion of electrons from t h e r e d u c e d m e t h y l viologen from n i t r i t e r e d u c t i o n t o D N P reduction.
54
W.F. Bourne and B. J. ~iflin:
Only minimal levels (0.6 nmoles NO~ reduced/rag of protein/min) of nitrite reductase activity are detectable in the roots of barley seedlings grown in nitrate-free medium. On transfer to a medium containing 10 mM KNO 3the activityincreases, after a 4-5 h lag, to reach a maximum of 50-60 nmoles NOg reduced/mg protein/mAn after 30-35 h. The majority of the experiments were done with roots grown in open culture and, while every precaution was taken to keep microbial contamination to as low a level as possible, the possibility exists that contamination was contributing to the levels of activity assayed. Accordingly nitrate and nitrite reductase activities were measured, both of roots grown under sterile conditions and of the bacteria contaminating roots grown in open culture. The bacteria were obtained by inoculating normal root growth medium containing sucrose as a carbon source, with an aliquot of the medium in which roots had been growing. After the bacteria had grown they were harvested by centrifugation. The bacterial pellet was extracted and assayed for activity in the same way as the sterile and non-sterile root tissue. As shown in Table 6 no activity could be detected in this extract, while roots grown under sterile conditions had levels of nitrate and nitrite reductase activity comparable to those of normally grown roots. Table 6. Nitrite reductase activities of roots grown in open culture, under sterile conditions and of the contaminating micro-organisms.Activities expressed as nmoles NO~ lost or formed/mg protein/min
Sterile roots Non-sterile roots Bacteria
Nitrate reductase
Nitrate reductase
2.2 2.6 0.1
21.1 21.8 0.3
Discussion Nitrite reductase, isolated from barley roots and purified 50-60 fold, produced ammonia from nitrite in eqnimolar amounts, indicating that in roots the enzyme catalyses the entire 6-electron reduction sequence, a similar situation to that which exists in leaf systems (Hageman et al., 1962). No differences could be established in the characteristics of nitrite reduetases from leaf or root tissues of barley seedlings, and both were dependent for activity in vitro on electron donors of high redox potential, such as reduced methyl viologen or ferredoxin. While no evidence exists as yet for the presence of ferredoxin in roots, the presence of a
Nitrite Reductase in Barley
55
low but significant level of activity in the absence of added cofaetor in the unpurified and partially purified root extracts suggests t h a t an endogenous eofactor was present. This activity was particularly apparent when acetone was used to fractionate the extract (Table 5). If a ferredoxin-like eofactor is present in barley roots, nitrite reduction in roots could proceed b y a system analogous to t h a t elaborated b y Paneque et al. (1964) for nitrite reduction in the dark b y leaves, involving N A D P H and NADPferredoxin oxidoreductase. Some support for such a system is provided b y the results of B u t t and Beevers (1961) study of carbohydrate metabolism in maize roots, which demonstrated t h a t nitrite stimulated the pentose phosphate pathway, an effect which they suggested might be due to re-oxidation of N A D P H b y nitrite. Several lines of evidence have been produced b y Kessler and his coworkers (Kessler, 1964) to suggest t h a t nitrite reduction, particularly in the dark, is dependent on ATP. Although our results with the particulate system from barley roots (Bourne and Miflin, 1970) support this contention, the effect of D N P in vivo could be due to its ability to inhibit nitrite reductase directly as well as its uncoupling effect. D N P has been shown b y Wessels (1959) to act as an alternative electron acceptor in the photosynthetic electron transport chain and it is also likely t h a t it can compete with nitrite for electrons in the reduced methyl viologen assay system. I n contrast arsenate, which inhibits in vivo nitrite reduction, has no effect on the isolated enzyme. Results shown here with roots grown under sterile conditions preclude the possibility t h a t microbial contamination is the source of enzyme activity in studies of root nitrate or nitrite reduetases. Similarly the induction of activity by nitrate and the dissociation of nitrite and sulphite reduetase activities, indicate t h a t a non-specific reaction is not responsible for nitrite reduction b y barley root extracts.
References Ahmad, J., Morris, I. : The effects of 2, 4-dinitrophenol and other uncoupling agents on the assimilation of nitrate and nitrite by Chlorella. Bioehim. biophys. Acta (Amst.) 162, 32-38 (1968). Beevers, L., Hageman, R. H. : Nitrate reduction in higher plants. Ann. Rev. Plant Physiol. 20, 495-522 (1969). Bollard, E. G.: Nitrogenous compounds in plant xylem sap. Nature (Lond.) 178, 1189-1190 (1956). Bourne, W. F., Miflin, B. J. : An ATP dependent reduction of nitrate to ammonia by a cell free particulate system from barley roots. Biochem. biophys. Res. Commun. 40, 1305-1310 (1970). Butt, V. S., Beevers, H.: The regulation of pathways of glucose catabolism in maize roots. Biochem. J. 80, 21-27 (1961). Hageman, R.H., Cresswell, C. 1~., Hewitt, E.g.: Reduction of nitrate, nitrite and hydroxylamine to ammonia by enzymes from higher plants. Nature (Lond.) 198, 247-250 (1962).
56
W . F . Bourne and B. g. Miflin: Nitrite Reductase in Barley
Hattori, A., Myers, J.: Reduction of nibrate and nitrite by subcellular preparations of Anabaena cydlindrlca. Plant Physiol. 41, 1031-1036 (1966). Hill, R., Bendall, F.: Crystallization of a photosynthetic reduetase from a green plant. Nature (Lond.) 187, 417 (1960). Hucklesby, D. P., Dalling, M. J., Hageman, R. H. : Some properties of two forms of nitrite reductase from corn (Zea mays L.) Scutellum. Planta (Berl.) 104, 220-233 (1972). Joy, K. W., Hageman, R. H.: The purification of nitrite reductase from higher plants and its dependence on ferredoxin. Biochem. J. 100, 263-273 (1966). Kemp, g. D., Atkinson, D. E., Ehret, A., Lazzarini, R. A.: Evidence for the identity of the NADH phosphate specific sulphite and nitrite rednctases of E. coli. J. biol. Chem. 238, 3466-3471 (1963). Kessler, E.: Nitrate assimilation by plants. Ann. Rev. Plant Physiol. 15, 57-72 (1964). Kessler, E., Czygan, F. C. : Seasonal changes in the nitrate-reducing activity of a green algae (Ankistrodesmus braunii). Experientia (Basel) 19, 89-90 (1963). Lowry, O. H., Rosenbrough, N. J., Farr, A. L., Randall, R. S. : Protein measurement with the Folin phenol reagent. J. biol. Chem. 198, 265-275 (1951). Mayer, A.M.: Subcellular location of snlphite reductase in plant tissues. Plant Physiol. 42, 324-326 (1967). Miflin, B. J.: Distribution of nitrate and nitrite reductase in barley. Nature (Lond.) 214, 1133-1134 (1967). Miflin, B. J.: Nitrate and nitrite reductase systems in barley roots. Rev. roum. Biochim. 7, 53-60 (1970a). Miflin, B. J.: Studies on the subcellular location of particulate nitrate and nitrite reductase, ghtamic dehydrogenase and other enzymes in barley roots. Planta (Berl.) 93, 160-170 (1970b). Paneque, A., Ramirez, J. M., Del Campo, F. F., Losada, M.: Light and dark reduction of nitrite in a reconstituted enzyme system. J. biol. Chem. 259, 1737-1741 (1964). Russel, J. A. : The estimation of small amounts of ammonia by the phenol-hypochlorite reaction. J. biol. Chem. 156, 457-465 (1944). Siegel, L. M.: A direct microdetermination for sulphide. Analyt. Bioehem. 11, 126-132 (1965). Tagawa, K., Arnon, D. I. : Ferredoxins as electron carriers in photosynthesis and in the biological production of hydrogen gas. Nature (Lond.) 195, 537-543 (1962). Wallace, W., Pate, J. S. : Nitrate reductase in the field pea. Ann. Botany 29, 655 (1965). Wallace, W., Pate, J. S. : Nitrate assimilation in higher plants with special reference to the Cocklebnrr. Ann. Botany 81, 213-228 (1967). Wessels, J. S. C. : DNP as a catalyst of photosynthetic phosphorylation. Biochem. biophys. Acta (Amst.) 36, 264-265 (1959).
