Planl a 9 by Springer-Verlag 1978
Planta 141, 183- 189 (1978)
Nitrate Reductase Activity in Paul's Scarlet Rose Suspension Cultures and the Differential Role of Nitrate and Molybdenum in Induction R.W. Jones,* A.J. Abbott,** E.J. Hewitt, G.R. Best, and E.F. Watson Long Ashton Research Station, University of Bristol, Bristol BS18 9AF, U.K.
Abstract. Induction of nitrate reductase (EC 1.6.6.1) activity was measured in Paul's Scarlet rose cell suspensions cultured in media containing nitrate (NO~-) or urea (U) as nitrogen source, and with ( + M o ) or without m o l y b d e n u m ( - M o ) . There was a lag of 30 min during induction by NO~- in + M o cultures but no lag occurred during induction after adding M o to N O 3 - M o or to U - M o cultures preincubated with N O 3 . Actinomycin D, cycloheximide, and puromycin completely blocked induction by NO~-, but had no effect on the initial rate of induction by Mo. Cycloheximide and puromycin blocked induction by NO~- more quickly than actinomycin D. Induction by N O j appeared to involve m R N A - d e p e n d e n t synthesis of apoprotein followed by rapid activation with m o l y b d e n u m in intact cells independently of protein synthesis. Nitrate-induced apoprotein appeared less stable than the holoenzyme. When induced by N O 3 in the absence of Mo, apoprotein concentration was about half the a m o u n t of maximally induced nitrate reductase. Cycloheximide stabilised preformed nitrate reductase which disappeared steadily in the presence o f p u r o m y c i n . Apoprotein was not stabilised by either antimetabolite. Key words: Antimetabolites - Cell culture - Enzyme induction - M o l y b d e n u m - Nitrate reductase - Rosa, Paul's Scarlet.
Present address: Dept. of Land Resource Science, University of Guelph, Ontario N1G 2WI, Canada ** To whom reprint requests should be made Abbreviations." Mo=molybdenum; NO 3 + Mo = standard MX1 culture medium; NO 3 - Mo = MX t medium purified of Mo ; NR = nitrate reductase; PSR=Paul's Scarlet rose; U=urea; U+Mo= MX1 medium with NO 3 replaced by urea; U-Mo=MXt medium with NO~- replaced by urea and also purified of Mo *
Introduction Nitrate reductase (NR) (EC 1.6.6.1.) is a molybdoprotein whose activity is increased (induced) by adding nitrate or molybdate to intact cells of plants or micro-organisms lacking one or other component; (Beevers and Hageman, 1969; Hewitt, 1957, 1958, 1959, 1975; Hewitt and Nicholas, 1964; Spencer and Wood, 1954). Nitrate-dependent N R in higher plants is usually suppressed by inhibitors of R N A or protein synthesis (Beevers and Hageman, 1969; Hewitt and Afridi, 1959; Afridi and Hewitt, 1964, 1965; Hewitt, 1975). In tobacco suspension cultures nitrate induced de novo synthesis of N R (Zielke and Filner, 1971). In C h I o r e l l a some N R activity was induced by Mo (compared to normal cells) during inhibition of protein synthesis by cycloheximide (Vega et al., 1971). Similar results were obtained in cauliflower leaf discs in the presence of cycloheximide after intervals of 2 to 10 h (Hewitt and Afridi, 1959; Afridi and Hewitt, 1965) but no differences in the response to NO;- or Mo were reported (Afridi and Hewitt, 1964). These results were interpreted by Vega et al. (1971) as showing that protein synthesis was not necessary for N R formation, but Afridi and Hewitt (1965) concluded that it was necessary for N R formation in response to Mo. It is nevertheless generally found that reactivation of N R by molybdate in vitro is not possible (see Hewitt, 1974). Our previous work (Jones et al., 1976) with cultures of Paul's Scarlet rose (PSR) showed N R activity depended on the supply of N O 3 and Mo and suggested the use of these cultures for more precise studies on the kinetics and response to R N A and protein synthesis inhibitors during induction by NO;or Mo. We show here that induction of N R activity
0032-0935/78/0141/0183/$01.40
184
R.W. Jones et al.: Nitrate Reductase Induction by Nitrate and Molybdenum
by NO3 or by Mo are distinguishable processes with different dependence on the needs for m R N A and protein synthesis. The work was described earlier very briefly (Hewitt et al., 1974).
Materials and Methods Culture System and NR Assay Standard ( N O ~ + M o ) and Mo-deficient ( N O ~ - M o ) PSR cells were batch cultured in purified MX1 media as described previously (Jones et al., 1976). Cells grown for one 14-day passage in medium containing urea as sole nitrogen source, provided non-nitrate (U + Mo and U - M o ) inoculum for experimental cultures as previously described (Jones et al., 1976). Nitrate reductase was routinely estimated using an in vivo assay. An in vitro technique was occasionally used. The relevance and use of both these techniques and all other methods have been fully described (Jones et al., 1976). N R activity is expressed as gmoles NO2 produced per g. fresh weight per hour.
Induction and Inhibitor System The time course of induction and effects of inhibitors of m R N A and protein synthesis was followed in 5 ml samples of cell suspensions taken from cultures in logarithmic growth phase and incubated under non-sterile conditions in Erlenmeyer flasks in a shaking water bath (120 oscillations/rain, 10 cm travel) at 27 ~ C. Inhibitors were added to NO~ + M o , N O 3 - M o , and U + M o cultures 1 h before or after addition of the inducers, NO~- or Mo. The final concentrations (when present) were 10 .2 M NaNO3, 10 7 M Na2MoO4, 50 gg/ml actinomycin D or cycloheximide and 200 gg/ml puromycin and in each case the volume added was 0.05 ml. Actinomycin D (5 mg/ml) was dissolved in 50% (v/v) ethyl alcohol and its controls were 50% alcohol treatment (0.5% final concentration), other controls were water only. The first 0.2 ml sample for assay was taken immediately before addition of inducer. U-Mo cultures were simitarly supplemented with either NO~ or Mo alone added to the suspensions 3 h before treatment with an inhibitor and then given the second inducer (Mo or NO~-) 1 h later and the first sample taken immediately.
Results Time Course of Induction by Nitrate or Mo
Nitrate added to NO~ + M o and U + M o cell suspensions induced an increase in in vivo N R activity after a short lag period detectable by sampling 0.5 h after treatment (Fig. 1A). Maximum rates of increase in activity were attained 30-60 rain after induction in cultures which had initially substantial or negligible N R activity in NO~ + M o and U + M o cultures, respectively (Fig. 1A). In contrast, Mo added to NO3 - M o cultures having some N R activity stimulated N R activity without a detectable lag and the rate of increase in activity was maximal during the first 30 min of sampling (Fig. 1A). Molybdenum given to
NO3 + M o and U + M o cultures did not induce any change in N R activity, regardless of initial values and NO3 given to NO~ - M o cultures similarly had no effect. The addition of NO~ and Mo together to N O ~ - M o or U + M o cultures did not alter the time courses shown in Figure 1A for responses to the single inducers. In cultures grown with U-Mo which require both NO~ and Mo for production of N R (Jones et al., 1976), induction by NO;. when added after a 3 h preincubation with Mo (during which no N R activity appeared) followed a time course similar to U + Mo grown cells with a lag of about 30 min after giving NO;- (Fig. 1B). Similarly, the simultaneous addition of NO~ and Mo produced the nitrate-type response curve. Molybdenum added after preincubation with NO;., however, produced a response similar to that for induction by Mo in NO3 - M o cultures (Fig. 1B) without a lag, but larger and initially about 3 times faster. In the U-Mo culture, the greatest rate of increase of N R activity occurred in the first 12 min (Fig. 1B). The initial kinetics of the responses to Mo and NO~ were quite different. The initially greater response to Mo was consistent up to the maximal values in all U-Mo cultures tested, but after the lag phase the responses in U-Mo cells to nitrate given after or together with Mo or to NO~ alone in U + Mo cells were similar (see also Fig. 7B). The response to Mo in the first 30 min was usually about 30% of the maximum but the corresponding response to NO3 was only 0 to 6%. The initial rate of response to Mo was almost 4 times faster in U-Mo cultures induced with NO;. than in NO3- - M o cultures (Fig. 1A and B). The response to Mo in cells preinduced with nitrate was detectable after one minute with a linear increase after two rain (Fig. 2). Appreciable induction by Mo occurred in the presence of propan1-ol (1.5%) during the in vivo assay incubation period. The activity produced when no Mo was given (A at 540 nm) was 0.005 compared with 0.02 for an assay initiated simultaneously with Mo addition and incubated for 30 min. The rate of induction by Mo in the presence of propan-l-ol was only 8% of that with Mo alone. This is attributed to the integrated production of N R during the assay period. The effects of 1-24 h preincubation with NO 3 (10- 2 M) on the subsequent response to Mo are shown in Table 1. Responses were similar for preincubation periods of 1, 3 and 6 h, but after longer times there were marked decreases in activity suggesting that the nitrate-inducible component became limiting. The in vitro assay, though less satisfactory than the in vivo method (Jones et al., 1976) was used as an independent measure of N R activity produced during the initial phase in response to Mo or NO;. to
R.W. Jones et al. : Nitrate Reductase Induction by Nitrate and Molybdenum
185
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time afteraddingNO~ or No (mini Fig. 3. Nitrate reductase induction by NO3 (9 and Mo (o) in 4-day cultures preincubated with the alternative inducer and measured in viva ( ) and in vitro (. . . . . -)
Table 1. Effect of duration of NO~- preincubation on Mo-dependent induction of nitrate reductase in low Mo, urea-grown cultures (gmoles NO)-/g fresh wt/h)
c o n s t a n t (1.70-1.75 : 1) a n d identical rates of response to N O 2 or M o after 30 m i n are s h o w n by b o t h assay methods.
Time after Mo Application (min)
Duration of nitrate preincubation(h) 1
3
6
9
24
0 30 60
0.20 2.77 3.76
0.21 2.76 3.80
0.26 2.68 3.69
0.26 2.03 2.08
0.26 0.79 0.79
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c o n f i r m the validity of the in viva m e t h o d used here. F i g u r e 3 shows that r a p i d i n d u c t i o n by M o a n d slow i n d u c t i o n by N O 3 were readily detectable by in vitro assay, a l t h o u g h the in viva assay was m o r e sensitive. The ratio of the slopes for b o t h phases of increase in response to NO;- or M o for in v i v a : i n vitro are
Lyfects of Inhibitors of RNA and Protein Synthesis on Induction The possible roles of m R N A a n d p r o t e i n synthesis in i n d u c t i o n by N O ~ or M o were investigated u s i n g a c t i n o m y c i n D, cycloheximide or p u r o m y c i n a n d results were interpreted in terms of the accepted effects of these a n t i m e t a b o l i t e s (Gale et al., 1972). I n d u c t i o n by N O ; . in N O ~ + M o (Fig. 4) a n d U + M o (Fig. 5) cultures was completely i n h i b i t e d by a c t i n o m y c i n D, cycloheximide or p u r o m y c i n added either 1 h before NO;- or s i m u l t a n e o u s l y . T h e small b u t steady increase in activity f o u n d w h e n samples of N O ; . + M o cultures were s h a k e n in open vessels with a d d i t i o n a l N O 3 was also i n h i b i t e d by the a n t i m e t a b o l i t e s (Fig. 4). E n d o g e n o u s N R activity in
186
R.W. Jones et al. : Nitrate Reductase Induction by Nitrate and Molybdenum
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B
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NO;- + Mo cultures decreased in the presence of NO3 and puromycin but more rapidly when NO3 was not added. Endogenous activity did not change in the presence of actinomycin D and NO;- but decreased when only actinomycin D was added (Figs. 4 and
6). Cycloheximide and puromycin halted the progress of induction by NO~ in U + M o cultures within 30 min (Fig. 5) and their effects were detected within 6 min in
some experiments. Actinomycin D had little effect for 30rain but slowed induction by NO~ after 1 h and halted it after 2 h. In contrast to their effect on induction by NO~, none of the antimetabolites when added 1 h before Mo, affected the rate of induction by Mo in NO~ Mo cuRures during the first hour (Fig. 6). Actinomycin D had little effect during the second hour of incubation with Mo, but there was a rapid decline in activity after 3 h. In the alcohol control treatment NR activity also declined at about the same rate but not until after 5 h. Inhibition during the second hour by cycloheximide was less than by puromycin, and activity declined 3 h after adding Mo (Fig. 6B). Thereafter, the remaining enzyme was apparently stabilized in the presence of cycloheximide but continued to decline with puromycin. The differential effects of the inhibitors on induction by NO3 in NO;- + M o and U + M o or by Mo in NO3 - M o cultures was confirmed using NO;- and Mo together for induction in U-Mo cultures (Fig. 7A and B). Induction by NO;- was completely inhibited whereas there were no effects on induction by Mo during the first hour when NO3 was added before the antimetabolite but induction was then checked and ceased after 2 h. The enzyme was again apparently stable with cycloheximide but declined with puromycin as in the previous tests. In these U-Mo cultures peak N R activity did not decline with alcohol as in the N O ; - - M o but declined with actinomycin D. Peak enzyme activity induced by Mo after 2 h in the presence of cycloheximide and puromycin was about 50% of the uninhibited activity after 8 h, and
R.W. Jones et al. : Nitrate Reductase Induction by Nitrate and M o l y b d e n u m
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Table 2. Effects of cyclobeximide, puromycin or actinomycin D on the induction of nitrate reductase in cells of 7-day-old cultures grown in different nitrogen or m o l y b d e n u m regimes Culture regime ~
NO~- + M o
Time Increase in after control with antimetabolite water ethah nol b
Response to N O 3 as % of control Cycloheximide
Puromycin
Actinomycin D
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was over 70% with actinomycin D (Figs. 6 and 7) for both N O ~ - Mo and U-Mo treatments. As cultures for several experiments had been used after periods of growth varying from 5 to 8 days (Figs. 4, 5, 6 and 7) these experiments were repeated
188
R.W. Jones et al. : Nitrate Reductase Induction by Nitrate and Molybdenum
for NO3 + M o , U + M o and U - M o cultures of the same age (Table 2). Inhibition by actinomycin D was slower and less severe than shown in Fig. 5, and was incomplete in 2 h. All the antimetabolites showed greatest inhibition in U - Mo cultures, although control treatments given NO 7 and Mo together showed maximal responses to the inducers. The stability of the nitrate-inducible component was studied by measuring the response to Mo in nitrate-induced U - Mo cultures when exposed to cycloheximide or puromycin for varying times, after induction by nitrate. Figure 8 shows that Mo-dependent induction declined rapidly after 1 h contact with the inhibitor before which there was little effect. Simultaneous addition of nitrate and antimetabolites again completely suppressed induction by Mo. The rate of decline was slightly faster for puromycin than for cycloheximide.
Discussion Our experiments show that induction of N R by NOg or Mo are distinct processes in PSR cultures as shown by differences in their response kinetics during the initial 30 min and the differences in dependence on protein synthesis for up to 2 h. These results collectively suggest that whereas NO~ induces a protein synthesis-dependent reaction after a delay to allow for prior m R N A transcription, Mo appears to produce rapidly an in vivo component which reacts with pre-existing apoprotein induced by NO~-. Comparison of Figures 7 and 8 suggest that holoenzyme formed by combination of Mo and apoprotein is more stable than the apoprotein after induction by NO3. The formation of nitrate-induced N R which occurred after giving actinomycin D suggests that m R N A already formed in the presence of NO~ allowed translation to continue for a short time. A half life of about 30 min for this m R N A deduced from the results in Figure 5 is similar to that of 20 min deduced for maize roots (Oaks et al., 1972). The induction by NO~ of nitrate permease systems showed similar time courses to the induction of N R and was inhibited by R N A and protein synthesis inhibitors in tobacco and A c e r cells and maize roots (Heimer and Filner, 1971 ; Jackson et al., 1973 ; King, 1974; Behrend and Mateles, 1975; Neyra and Hageman, 1975). Progressive induction of N R by NO3 was soon inhibited by cycloheximide and puromycin when added during rapid N R induction by contrast with initially rapid and linear response to Mo after 3 rain. The presence of permease activity however would not affect the interpretation regarding the proposed roles of protein synthesis in induction
of N R activity by Mo or NO3. Whereas permease induction in maize roots was accelerated by increasing N O 3 , in PSR cells increasing concentrations between 1 txM and 500 IxM NaNO3 had no effect on initial rates of N R induction but prolonged its life (Jones et al., 1976). The apparent stabilising effect of cycloheximide for N R (Figs. 6 and 7) has been observed previously but was not shown with puromycin. Stabilisation of N R by cycloheximide may result from protection against proteolytic degradation by specific enzymes (Wallace, 1974) but apoprotein was not correspondingly stabilized (Fig. 8). The persistence of apoprotein for about 1 h before the rather sudden loss (Fig. 8) in the presence of either antimetabolite may reflect sudden release of a degrading enzyme. The yield of N R produced in response to Mo when protein synthesis was prevented (about 50% of the maximum value) compares well with estimates of apoprotein found in leaves of Mo-deficient spinach plants grown with ammonium nitrate of about 36% of normal antigenic activity (Notton et al., 1974). The production of N R in response to Mo in vivo in leaves of similar Mo-deficient spinach was about 40% of maximal production when cycloheximide was present (Rucklidge and Hewitt, 1975). The maximal reconstitution of nitrate reductase obtained by combination of the molybdenum cofactor complex with apoprotein in crude extracts of Modeficient NH4NO3-grown plants was about 36% of the normal N R activity in extracts of plants grown with Mo (Rucklidge et al., 1976; Hewitt, et al., 1977). The incorporation of radioactive tungsten into protein similar to nitrate reductase and the induction of N R by Mo in molybdenum-deficient cauliflower leaves were about 25% in the presence of puromycin compared with amounts found in its absence (Notton et al., 1974; Hewitt et al., 1967). We conclude that concentrations of apoprotein in Mo deficient plants induced by NO~ are generally between 25 and 50% of corresponding holoenzyme found in normal plants. This apoprotein deficiency explains the requirement for protein synthesis for continued induction of N R by Mo reported previously (Hewitt and Afridi, 1959; Afridi and Hewitt, 1965; Hewitt et al., 1967). In N . c r a s s a the apoprotein concentration in Mo-deficient mycelia may be 90% of the corresponding holoenzyme content (Subramanian and Sorger, 1972; Ketchum and Downey, 1975). We are grateful to Dr. M.E. Davies, University of Birmingham, U.K., for the gift of cultures of Paul's Scarlet rose cells from which our strains wereestablished, to the ScienceResearch Council for a Postgraduate Fellowship held by R.W.J. and to Mr. D.M. James for preparing the diagrams.
R.W. Jones etal. : Nitrate Reductase Induction by Nitrate and Molybdenum
References Afridi, M.M.R.K., Hewitt, E.J.: The inducible formation and stability of nitrate reductase in higher plants. I. Effects of nitrate and molybdenum on enzyme activity in cauliflower (Brass. oleracea var. Botrytis). J. exp. Bot. 15, 251-271 (1964) Afridi, M.M.R.K., Hewitt, E.J.: The inducible formation and stability of nitrate reductase in higher plants. II. Effects of environmental factors, antimetabolites, and amino acids on induction. J. exp. Bot. 16, 628-645 (t965) Beevers, L., Hageman, R.H.: Nitrate reduction in higher plants. Ann. Rev. Plant Physiol. 20, 495-522 (1969) Behrend, J., Mateles, R.I. : Nitrogen metabolism in plant cell suspension cultures. I. Effect of amino acids on growth. Plant Physiol. 56, 584 589 (1975) Gale, E.F, Cundliffe, E., Reynold, P.E., Richmond, M.H., Waring, M.J. : In : The molecular'basis of antibiotic action. London : Wiley 1972 Heimer, Y.M., Filner, P.: Regulation of nitrate assimilation pathway in cultured tobacco cells. III. The nitrate uptake system. Biochim. Biophys. Acta 230, 362-372 (1971) Hewitt, E.J.: Some aspects of micronutrient element metabolism in plants. Nature 180, 1020 (1957) Hewitt, E.J. : The role of mineral elements in the activity of plant enzyme systems. In: Handbook of plant physiology, vol. 4, pp. 427-481. Ruhland, W., ed. Berlin: Springer 1958 Hewitt, E.J. : The metabolism of micronutrient elements in plants. Biol. Rev. 34,, 333-377 (1959) Hewitt, E.J.: Assimilatory nitrate-nitrite reduction. Ann. Rev. Plant Physiol. 26, 73 100 (1975) Hewitt, E.J., Afi-idi, M.M.R.K. : Adaptive synthesis of nitrate reductase in higher plants. Nature 183, 57-58 (I959) Hewitt, E.J., Jones, R.W., Abbott, A.J., Best, G.R.: Effect of nitrate and molybdenum on in vivo formation of nitrate reductase in Paul's Scarlet rose cells, Tulecke strain. Abstr. no. 13I. 3rd International Congress in Plant Tissue Culture, University of Leicester, England, 1974 Hewitt, E.J., Nicholas, D.J.D.: Enzymes of inorganic nitrogen metabolism. In: Modern methods of plant analysis, vol. 7, pp. 6%122. Linskens, H.F., Sanwa/, B.D., Tracey, M.V., eds., Berlin: Springer 1964 Hewitt, E.J., Notton, B.A. : Inhibition by L-azetidine-2-carboxylic acid of induction of nitrate reductase in plants and its reversal by L-proline. Phytochem. 6, 1329-1335 (1967) Hewitt, E.J., Norton, B.A., Afridi, M.M.R.K. : Comparative effects of some antimetabolites on ribonucleic acid synthesis and induction of nitrate reductase in cauliflower leaf tissue. Plant Cell Physiol. 8, 385-397 (1967)
189
Hewitt, E.J., Notton, B.A., Rucklidge, G.J. : Formation of nitrate reductase by recombination of apoprotein fractions from molybdenum-deficient plants with a molybdenum containing complex. J. Less-Common Metals 54, 537-553 (1977) Ketchum, P.A., Downey, R.J. : In vitro restoration of nitrate reductase : investigation of Aspergillus nidu[ans and Neurospora crassa nitrate reductase mutants. Biochim. Biophys. Acta 385, 354-361 (1975) Jackson, W.A., Flesher, D., Hageman, R.H.: Nitrate uptake by dark-grown corn seedlings, some characteristics of apparent induction. Plant Physiol. 51, 120-127 (1973) Jones, R.W., Abbott, A.J., Hewitt, E.J., Best, G.R., James, D.M.: Nitrate reductase activity and growth in Paul~s Scarlet rose suspension cultures in relation to nitrogen source and molybdenum. Planta 133, 2%34 (1976) King, J. : The uptake of nitrate by sycamore cells. Abstr. no. 133. 3rd International Congress on Plant Tissue Culture, University of Leicester, England, i974 Neyra, C.A., Hageman, R.H.: Nitrate uptake and induction of nitrate reductase in excised corn roots. Plant Physiol. 56, 692-695 (1975) Notton, B.A., Graf, L., Hewitt, E.J., Povey, R.C.: The role of molybdenum in the synthesis of nitrate reductase in cauliflower (Brassica oleracea L. var. Boto'tis L.) and spinach (Spinacea oleracea L.). Biochim. Biophys. Acta 364, 45-58 (1974) Oaks, A., Wallace, W., Stevens, D.: Synthesis and turnover of nitrate reductase in corn roots. Plant Physiol. 50, 649-654 (1972) Rucklidge, G.J., Hewitt, E.J.: Rep. Long Ashton Res. Star. for 1974. pp. 66-67 (I975) Rucklidge, G., Norton: B., Hewitt, E.: Reconstitution in vitro of nitrate reductase from apoprotein of molybdenum-deficient spinach. Biochem. Soc. Proc. 4, 77-80 (1976) Spencer, D., Wood, J.G. : The role of molybdenum in nitrate reductase in higher plants. Aust. J. biol. Sci. 7, 425-434 (1954) Subramanian, K.N., Sorger, G.J.: The role of molybdenum in the synthesis of Neurospora nitrate reductase. Biochim. Biophys. Acta 256, 533-543 (1972) Vega, J.M., Herrera, J., Aparicio, P.J., Paneque, A., Losada, M.: Role of molybdenum in nitrate reduction by Chlorella. Plant Physiol. 48, 294-299 (1971) Wallace, W. : Purification and properties of a nitrate reductase inactivating enzyme. Biochim. Biophys. Acta 341, 265-276 (1974) Zielke, H.R., Filner, P. : Synthesis and turnover of nitrate reductase induced by nitrate in cultured tobacco cells. J. Biol. Chem. 246, 1772 1779 (1971)
Received 15 February; accepted 15March I978
Planta 141, 183- 189 (1978)
Nitrate Reductase Activity in Paul's Scarlet Rose Suspension Cultures and the Differential Role of Nitrate and Molybdenum in Induction R.W. Jones,* A.J. Abbott,** E.J. Hewitt, G.R. Best, and E.F. Watson Long Ashton Research Station, University of Bristol, Bristol BS18 9AF, U.K.
Abstract. Induction of nitrate reductase (EC 1.6.6.1) activity was measured in Paul's Scarlet rose cell suspensions cultured in media containing nitrate (NO~-) or urea (U) as nitrogen source, and with ( + M o ) or without m o l y b d e n u m ( - M o ) . There was a lag of 30 min during induction by NO~- in + M o cultures but no lag occurred during induction after adding M o to N O 3 - M o or to U - M o cultures preincubated with N O 3 . Actinomycin D, cycloheximide, and puromycin completely blocked induction by NO~-, but had no effect on the initial rate of induction by Mo. Cycloheximide and puromycin blocked induction by NO~- more quickly than actinomycin D. Induction by N O j appeared to involve m R N A - d e p e n d e n t synthesis of apoprotein followed by rapid activation with m o l y b d e n u m in intact cells independently of protein synthesis. Nitrate-induced apoprotein appeared less stable than the holoenzyme. When induced by N O 3 in the absence of Mo, apoprotein concentration was about half the a m o u n t of maximally induced nitrate reductase. Cycloheximide stabilised preformed nitrate reductase which disappeared steadily in the presence o f p u r o m y c i n . Apoprotein was not stabilised by either antimetabolite. Key words: Antimetabolites - Cell culture - Enzyme induction - M o l y b d e n u m - Nitrate reductase - Rosa, Paul's Scarlet.
Present address: Dept. of Land Resource Science, University of Guelph, Ontario N1G 2WI, Canada ** To whom reprint requests should be made Abbreviations." Mo=molybdenum; NO 3 + Mo = standard MX1 culture medium; NO 3 - Mo = MX t medium purified of Mo ; NR = nitrate reductase; PSR=Paul's Scarlet rose; U=urea; U+Mo= MX1 medium with NO 3 replaced by urea; U-Mo=MXt medium with NO~- replaced by urea and also purified of Mo *
Introduction Nitrate reductase (NR) (EC 1.6.6.1.) is a molybdoprotein whose activity is increased (induced) by adding nitrate or molybdate to intact cells of plants or micro-organisms lacking one or other component; (Beevers and Hageman, 1969; Hewitt, 1957, 1958, 1959, 1975; Hewitt and Nicholas, 1964; Spencer and Wood, 1954). Nitrate-dependent N R in higher plants is usually suppressed by inhibitors of R N A or protein synthesis (Beevers and Hageman, 1969; Hewitt and Afridi, 1959; Afridi and Hewitt, 1964, 1965; Hewitt, 1975). In tobacco suspension cultures nitrate induced de novo synthesis of N R (Zielke and Filner, 1971). In C h I o r e l l a some N R activity was induced by Mo (compared to normal cells) during inhibition of protein synthesis by cycloheximide (Vega et al., 1971). Similar results were obtained in cauliflower leaf discs in the presence of cycloheximide after intervals of 2 to 10 h (Hewitt and Afridi, 1959; Afridi and Hewitt, 1965) but no differences in the response to NO;- or Mo were reported (Afridi and Hewitt, 1964). These results were interpreted by Vega et al. (1971) as showing that protein synthesis was not necessary for N R formation, but Afridi and Hewitt (1965) concluded that it was necessary for N R formation in response to Mo. It is nevertheless generally found that reactivation of N R by molybdate in vitro is not possible (see Hewitt, 1974). Our previous work (Jones et al., 1976) with cultures of Paul's Scarlet rose (PSR) showed N R activity depended on the supply of N O 3 and Mo and suggested the use of these cultures for more precise studies on the kinetics and response to R N A and protein synthesis inhibitors during induction by NO;or Mo. We show here that induction of N R activity
0032-0935/78/0141/0183/$01.40
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R.W. Jones et al.: Nitrate Reductase Induction by Nitrate and Molybdenum
by NO3 or by Mo are distinguishable processes with different dependence on the needs for m R N A and protein synthesis. The work was described earlier very briefly (Hewitt et al., 1974).
Materials and Methods Culture System and NR Assay Standard ( N O ~ + M o ) and Mo-deficient ( N O ~ - M o ) PSR cells were batch cultured in purified MX1 media as described previously (Jones et al., 1976). Cells grown for one 14-day passage in medium containing urea as sole nitrogen source, provided non-nitrate (U + Mo and U - M o ) inoculum for experimental cultures as previously described (Jones et al., 1976). Nitrate reductase was routinely estimated using an in vivo assay. An in vitro technique was occasionally used. The relevance and use of both these techniques and all other methods have been fully described (Jones et al., 1976). N R activity is expressed as gmoles NO2 produced per g. fresh weight per hour.
Induction and Inhibitor System The time course of induction and effects of inhibitors of m R N A and protein synthesis was followed in 5 ml samples of cell suspensions taken from cultures in logarithmic growth phase and incubated under non-sterile conditions in Erlenmeyer flasks in a shaking water bath (120 oscillations/rain, 10 cm travel) at 27 ~ C. Inhibitors were added to NO~ + M o , N O 3 - M o , and U + M o cultures 1 h before or after addition of the inducers, NO~- or Mo. The final concentrations (when present) were 10 .2 M NaNO3, 10 7 M Na2MoO4, 50 gg/ml actinomycin D or cycloheximide and 200 gg/ml puromycin and in each case the volume added was 0.05 ml. Actinomycin D (5 mg/ml) was dissolved in 50% (v/v) ethyl alcohol and its controls were 50% alcohol treatment (0.5% final concentration), other controls were water only. The first 0.2 ml sample for assay was taken immediately before addition of inducer. U-Mo cultures were simitarly supplemented with either NO~ or Mo alone added to the suspensions 3 h before treatment with an inhibitor and then given the second inducer (Mo or NO~-) 1 h later and the first sample taken immediately.
Results Time Course of Induction by Nitrate or Mo
Nitrate added to NO~ + M o and U + M o cell suspensions induced an increase in in vivo N R activity after a short lag period detectable by sampling 0.5 h after treatment (Fig. 1A). Maximum rates of increase in activity were attained 30-60 rain after induction in cultures which had initially substantial or negligible N R activity in NO~ + M o and U + M o cultures, respectively (Fig. 1A). In contrast, Mo added to NO3 - M o cultures having some N R activity stimulated N R activity without a detectable lag and the rate of increase in activity was maximal during the first 30 min of sampling (Fig. 1A). Molybdenum given to
NO3 + M o and U + M o cultures did not induce any change in N R activity, regardless of initial values and NO3 given to NO~ - M o cultures similarly had no effect. The addition of NO~ and Mo together to N O ~ - M o or U + M o cultures did not alter the time courses shown in Figure 1A for responses to the single inducers. In cultures grown with U-Mo which require both NO~ and Mo for production of N R (Jones et al., 1976), induction by NO;. when added after a 3 h preincubation with Mo (during which no N R activity appeared) followed a time course similar to U + Mo grown cells with a lag of about 30 min after giving NO;- (Fig. 1B). Similarly, the simultaneous addition of NO~ and Mo produced the nitrate-type response curve. Molybdenum added after preincubation with NO;., however, produced a response similar to that for induction by Mo in NO3 - M o cultures (Fig. 1B) without a lag, but larger and initially about 3 times faster. In the U-Mo culture, the greatest rate of increase of N R activity occurred in the first 12 min (Fig. 1B). The initial kinetics of the responses to Mo and NO~ were quite different. The initially greater response to Mo was consistent up to the maximal values in all U-Mo cultures tested, but after the lag phase the responses in U-Mo cells to nitrate given after or together with Mo or to NO~ alone in U + Mo cells were similar (see also Fig. 7B). The response to Mo in the first 30 min was usually about 30% of the maximum but the corresponding response to NO3 was only 0 to 6%. The initial rate of response to Mo was almost 4 times faster in U-Mo cultures induced with NO;. than in NO3- - M o cultures (Fig. 1A and B). The response to Mo in cells preinduced with nitrate was detectable after one minute with a linear increase after two rain (Fig. 2). Appreciable induction by Mo occurred in the presence of propan1-ol (1.5%) during the in vivo assay incubation period. The activity produced when no Mo was given (A at 540 nm) was 0.005 compared with 0.02 for an assay initiated simultaneously with Mo addition and incubated for 30 min. The rate of induction by Mo in the presence of propan-l-ol was only 8% of that with Mo alone. This is attributed to the integrated production of N R during the assay period. The effects of 1-24 h preincubation with NO 3 (10- 2 M) on the subsequent response to Mo are shown in Table 1. Responses were similar for preincubation periods of 1, 3 and 6 h, but after longer times there were marked decreases in activity suggesting that the nitrate-inducible component became limiting. The in vitro assay, though less satisfactory than the in vivo method (Jones et al., 1976) was used as an independent measure of N R activity produced during the initial phase in response to Mo or NO;. to
R.W. Jones et al. : Nitrate Reductase Induction by Nitrate and Molybdenum
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time afteraddingNO~ or No (mini Fig. 3. Nitrate reductase induction by NO3 (9 and Mo (o) in 4-day cultures preincubated with the alternative inducer and measured in viva ( ) and in vitro (. . . . . -)
Table 1. Effect of duration of NO~- preincubation on Mo-dependent induction of nitrate reductase in low Mo, urea-grown cultures (gmoles NO)-/g fresh wt/h)
c o n s t a n t (1.70-1.75 : 1) a n d identical rates of response to N O 2 or M o after 30 m i n are s h o w n by b o t h assay methods.
Time after Mo Application (min)
Duration of nitrate preincubation(h) 1
3
6
9
24
0 30 60
0.20 2.77 3.76
0.21 2.76 3.80
0.26 2.68 3.69
0.26 2.03 2.08
0.26 0.79 0.79
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c o n f i r m the validity of the in viva m e t h o d used here. F i g u r e 3 shows that r a p i d i n d u c t i o n by M o a n d slow i n d u c t i o n by N O 3 were readily detectable by in vitro assay, a l t h o u g h the in viva assay was m o r e sensitive. The ratio of the slopes for b o t h phases of increase in response to NO;- or M o for in v i v a : i n vitro are
Lyfects of Inhibitors of RNA and Protein Synthesis on Induction The possible roles of m R N A a n d p r o t e i n synthesis in i n d u c t i o n by N O ~ or M o were investigated u s i n g a c t i n o m y c i n D, cycloheximide or p u r o m y c i n a n d results were interpreted in terms of the accepted effects of these a n t i m e t a b o l i t e s (Gale et al., 1972). I n d u c t i o n by N O ; . in N O ~ + M o (Fig. 4) a n d U + M o (Fig. 5) cultures was completely i n h i b i t e d by a c t i n o m y c i n D, cycloheximide or p u r o m y c i n added either 1 h before NO;- or s i m u l t a n e o u s l y . T h e small b u t steady increase in activity f o u n d w h e n samples of N O ; . + M o cultures were s h a k e n in open vessels with a d d i t i o n a l N O 3 was also i n h i b i t e d by the a n t i m e t a b o l i t e s (Fig. 4). E n d o g e n o u s N R activity in
186
R.W. Jones et al. : Nitrate Reductase Induction by Nitrate and Molybdenum
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B
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NO;- + Mo cultures decreased in the presence of NO3 and puromycin but more rapidly when NO3 was not added. Endogenous activity did not change in the presence of actinomycin D and NO;- but decreased when only actinomycin D was added (Figs. 4 and
6). Cycloheximide and puromycin halted the progress of induction by NO~ in U + M o cultures within 30 min (Fig. 5) and their effects were detected within 6 min in
some experiments. Actinomycin D had little effect for 30rain but slowed induction by NO~ after 1 h and halted it after 2 h. In contrast to their effect on induction by NO~, none of the antimetabolites when added 1 h before Mo, affected the rate of induction by Mo in NO~ Mo cuRures during the first hour (Fig. 6). Actinomycin D had little effect during the second hour of incubation with Mo, but there was a rapid decline in activity after 3 h. In the alcohol control treatment NR activity also declined at about the same rate but not until after 5 h. Inhibition during the second hour by cycloheximide was less than by puromycin, and activity declined 3 h after adding Mo (Fig. 6B). Thereafter, the remaining enzyme was apparently stabilized in the presence of cycloheximide but continued to decline with puromycin. The differential effects of the inhibitors on induction by NO3 in NO;- + M o and U + M o or by Mo in NO3 - M o cultures was confirmed using NO;- and Mo together for induction in U-Mo cultures (Fig. 7A and B). Induction by NO;- was completely inhibited whereas there were no effects on induction by Mo during the first hour when NO3 was added before the antimetabolite but induction was then checked and ceased after 2 h. The enzyme was again apparently stable with cycloheximide but declined with puromycin as in the previous tests. In these U-Mo cultures peak N R activity did not decline with alcohol as in the N O ; - - M o but declined with actinomycin D. Peak enzyme activity induced by Mo after 2 h in the presence of cycloheximide and puromycin was about 50% of the uninhibited activity after 8 h, and
R.W. Jones et al. : Nitrate Reductase Induction by Nitrate and M o l y b d e n u m
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Table 2. Effects of cyclobeximide, puromycin or actinomycin D on the induction of nitrate reductase in cells of 7-day-old cultures grown in different nitrogen or m o l y b d e n u m regimes Culture regime ~
NO~- + M o
Time Increase in after control with antimetabolite water ethah nol b
Response to N O 3 as % of control Cycloheximide
Puromycin
Actinomycin D
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177 203
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191 205
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was over 70% with actinomycin D (Figs. 6 and 7) for both N O ~ - Mo and U-Mo treatments. As cultures for several experiments had been used after periods of growth varying from 5 to 8 days (Figs. 4, 5, 6 and 7) these experiments were repeated
188
R.W. Jones et al. : Nitrate Reductase Induction by Nitrate and Molybdenum
for NO3 + M o , U + M o and U - M o cultures of the same age (Table 2). Inhibition by actinomycin D was slower and less severe than shown in Fig. 5, and was incomplete in 2 h. All the antimetabolites showed greatest inhibition in U - Mo cultures, although control treatments given NO 7 and Mo together showed maximal responses to the inducers. The stability of the nitrate-inducible component was studied by measuring the response to Mo in nitrate-induced U - Mo cultures when exposed to cycloheximide or puromycin for varying times, after induction by nitrate. Figure 8 shows that Mo-dependent induction declined rapidly after 1 h contact with the inhibitor before which there was little effect. Simultaneous addition of nitrate and antimetabolites again completely suppressed induction by Mo. The rate of decline was slightly faster for puromycin than for cycloheximide.
Discussion Our experiments show that induction of N R by NOg or Mo are distinct processes in PSR cultures as shown by differences in their response kinetics during the initial 30 min and the differences in dependence on protein synthesis for up to 2 h. These results collectively suggest that whereas NO~ induces a protein synthesis-dependent reaction after a delay to allow for prior m R N A transcription, Mo appears to produce rapidly an in vivo component which reacts with pre-existing apoprotein induced by NO~-. Comparison of Figures 7 and 8 suggest that holoenzyme formed by combination of Mo and apoprotein is more stable than the apoprotein after induction by NO3. The formation of nitrate-induced N R which occurred after giving actinomycin D suggests that m R N A already formed in the presence of NO~ allowed translation to continue for a short time. A half life of about 30 min for this m R N A deduced from the results in Figure 5 is similar to that of 20 min deduced for maize roots (Oaks et al., 1972). The induction by NO~ of nitrate permease systems showed similar time courses to the induction of N R and was inhibited by R N A and protein synthesis inhibitors in tobacco and A c e r cells and maize roots (Heimer and Filner, 1971 ; Jackson et al., 1973 ; King, 1974; Behrend and Mateles, 1975; Neyra and Hageman, 1975). Progressive induction of N R by NO3 was soon inhibited by cycloheximide and puromycin when added during rapid N R induction by contrast with initially rapid and linear response to Mo after 3 rain. The presence of permease activity however would not affect the interpretation regarding the proposed roles of protein synthesis in induction
of N R activity by Mo or NO3. Whereas permease induction in maize roots was accelerated by increasing N O 3 , in PSR cells increasing concentrations between 1 txM and 500 IxM NaNO3 had no effect on initial rates of N R induction but prolonged its life (Jones et al., 1976). The apparent stabilising effect of cycloheximide for N R (Figs. 6 and 7) has been observed previously but was not shown with puromycin. Stabilisation of N R by cycloheximide may result from protection against proteolytic degradation by specific enzymes (Wallace, 1974) but apoprotein was not correspondingly stabilized (Fig. 8). The persistence of apoprotein for about 1 h before the rather sudden loss (Fig. 8) in the presence of either antimetabolite may reflect sudden release of a degrading enzyme. The yield of N R produced in response to Mo when protein synthesis was prevented (about 50% of the maximum value) compares well with estimates of apoprotein found in leaves of Mo-deficient spinach plants grown with ammonium nitrate of about 36% of normal antigenic activity (Notton et al., 1974). The production of N R in response to Mo in vivo in leaves of similar Mo-deficient spinach was about 40% of maximal production when cycloheximide was present (Rucklidge and Hewitt, 1975). The maximal reconstitution of nitrate reductase obtained by combination of the molybdenum cofactor complex with apoprotein in crude extracts of Modeficient NH4NO3-grown plants was about 36% of the normal N R activity in extracts of plants grown with Mo (Rucklidge et al., 1976; Hewitt, et al., 1977). The incorporation of radioactive tungsten into protein similar to nitrate reductase and the induction of N R by Mo in molybdenum-deficient cauliflower leaves were about 25% in the presence of puromycin compared with amounts found in its absence (Notton et al., 1974; Hewitt et al., 1967). We conclude that concentrations of apoprotein in Mo deficient plants induced by NO~ are generally between 25 and 50% of corresponding holoenzyme found in normal plants. This apoprotein deficiency explains the requirement for protein synthesis for continued induction of N R by Mo reported previously (Hewitt and Afridi, 1959; Afridi and Hewitt, 1965; Hewitt et al., 1967). In N . c r a s s a the apoprotein concentration in Mo-deficient mycelia may be 90% of the corresponding holoenzyme content (Subramanian and Sorger, 1972; Ketchum and Downey, 1975). We are grateful to Dr. M.E. Davies, University of Birmingham, U.K., for the gift of cultures of Paul's Scarlet rose cells from which our strains wereestablished, to the ScienceResearch Council for a Postgraduate Fellowship held by R.W.J. and to Mr. D.M. James for preparing the diagrams.
R.W. Jones etal. : Nitrate Reductase Induction by Nitrate and Molybdenum
References Afridi, M.M.R.K., Hewitt, E.J.: The inducible formation and stability of nitrate reductase in higher plants. I. Effects of nitrate and molybdenum on enzyme activity in cauliflower (Brass. oleracea var. Botrytis). J. exp. Bot. 15, 251-271 (1964) Afridi, M.M.R.K., Hewitt, E.J.: The inducible formation and stability of nitrate reductase in higher plants. II. Effects of environmental factors, antimetabolites, and amino acids on induction. J. exp. Bot. 16, 628-645 (t965) Beevers, L., Hageman, R.H.: Nitrate reduction in higher plants. Ann. Rev. Plant Physiol. 20, 495-522 (1969) Behrend, J., Mateles, R.I. : Nitrogen metabolism in plant cell suspension cultures. I. Effect of amino acids on growth. Plant Physiol. 56, 584 589 (1975) Gale, E.F, Cundliffe, E., Reynold, P.E., Richmond, M.H., Waring, M.J. : In : The molecular'basis of antibiotic action. London : Wiley 1972 Heimer, Y.M., Filner, P.: Regulation of nitrate assimilation pathway in cultured tobacco cells. III. The nitrate uptake system. Biochim. Biophys. Acta 230, 362-372 (1971) Hewitt, E.J.: Some aspects of micronutrient element metabolism in plants. Nature 180, 1020 (1957) Hewitt, E.J. : The role of mineral elements in the activity of plant enzyme systems. In: Handbook of plant physiology, vol. 4, pp. 427-481. Ruhland, W., ed. Berlin: Springer 1958 Hewitt, E.J. : The metabolism of micronutrient elements in plants. Biol. Rev. 34,, 333-377 (1959) Hewitt, E.J.: Assimilatory nitrate-nitrite reduction. Ann. Rev. Plant Physiol. 26, 73 100 (1975) Hewitt, E.J., Afi-idi, M.M.R.K. : Adaptive synthesis of nitrate reductase in higher plants. Nature 183, 57-58 (I959) Hewitt, E.J., Jones, R.W., Abbott, A.J., Best, G.R.: Effect of nitrate and molybdenum on in vivo formation of nitrate reductase in Paul's Scarlet rose cells, Tulecke strain. Abstr. no. 13I. 3rd International Congress in Plant Tissue Culture, University of Leicester, England, 1974 Hewitt, E.J., Nicholas, D.J.D.: Enzymes of inorganic nitrogen metabolism. In: Modern methods of plant analysis, vol. 7, pp. 6%122. Linskens, H.F., Sanwa/, B.D., Tracey, M.V., eds., Berlin: Springer 1964 Hewitt, E.J., Notton, B.A. : Inhibition by L-azetidine-2-carboxylic acid of induction of nitrate reductase in plants and its reversal by L-proline. Phytochem. 6, 1329-1335 (1967) Hewitt, E.J., Norton, B.A., Afridi, M.M.R.K. : Comparative effects of some antimetabolites on ribonucleic acid synthesis and induction of nitrate reductase in cauliflower leaf tissue. Plant Cell Physiol. 8, 385-397 (1967)
189
Hewitt, E.J., Notton, B.A., Rucklidge, G.J. : Formation of nitrate reductase by recombination of apoprotein fractions from molybdenum-deficient plants with a molybdenum containing complex. J. Less-Common Metals 54, 537-553 (1977) Ketchum, P.A., Downey, R.J. : In vitro restoration of nitrate reductase : investigation of Aspergillus nidu[ans and Neurospora crassa nitrate reductase mutants. Biochim. Biophys. Acta 385, 354-361 (1975) Jackson, W.A., Flesher, D., Hageman, R.H.: Nitrate uptake by dark-grown corn seedlings, some characteristics of apparent induction. Plant Physiol. 51, 120-127 (1973) Jones, R.W., Abbott, A.J., Hewitt, E.J., Best, G.R., James, D.M.: Nitrate reductase activity and growth in Paul~s Scarlet rose suspension cultures in relation to nitrogen source and molybdenum. Planta 133, 2%34 (1976) King, J. : The uptake of nitrate by sycamore cells. Abstr. no. 133. 3rd International Congress on Plant Tissue Culture, University of Leicester, England, i974 Neyra, C.A., Hageman, R.H.: Nitrate uptake and induction of nitrate reductase in excised corn roots. Plant Physiol. 56, 692-695 (1975) Notton, B.A., Graf, L., Hewitt, E.J., Povey, R.C.: The role of molybdenum in the synthesis of nitrate reductase in cauliflower (Brassica oleracea L. var. Boto'tis L.) and spinach (Spinacea oleracea L.). Biochim. Biophys. Acta 364, 45-58 (1974) Oaks, A., Wallace, W., Stevens, D.: Synthesis and turnover of nitrate reductase in corn roots. Plant Physiol. 50, 649-654 (1972) Rucklidge, G.J., Hewitt, E.J.: Rep. Long Ashton Res. Star. for 1974. pp. 66-67 (I975) Rucklidge, G., Norton: B., Hewitt, E.: Reconstitution in vitro of nitrate reductase from apoprotein of molybdenum-deficient spinach. Biochem. Soc. Proc. 4, 77-80 (1976) Spencer, D., Wood, J.G. : The role of molybdenum in nitrate reductase in higher plants. Aust. J. biol. Sci. 7, 425-434 (1954) Subramanian, K.N., Sorger, G.J.: The role of molybdenum in the synthesis of Neurospora nitrate reductase. Biochim. Biophys. Acta 256, 533-543 (1972) Vega, J.M., Herrera, J., Aparicio, P.J., Paneque, A., Losada, M.: Role of molybdenum in nitrate reduction by Chlorella. Plant Physiol. 48, 294-299 (1971) Wallace, W. : Purification and properties of a nitrate reductase inactivating enzyme. Biochim. Biophys. Acta 341, 265-276 (1974) Zielke, H.R., Filner, P. : Synthesis and turnover of nitrate reductase induced by nitrate in cultured tobacco cells. J. Biol. Chem. 246, 1772 1779 (1971)
Received 15 February; accepted 15March I978
