Mol Gen Genet (1982) 186:164-169 © Springer-Verlag 1982

Regulation of the Nitrate-Reducing System Enzymes in Wild-type and Mutant Strains of Chlamydomonas reinhardii Emilio Fernandez 1 and Jacobo Cfirdenas 2 1 Departamento de Bioquimica, Facultad de Biologia y C.S.I.C., Universidad de Sevilla, Sevilla, Spain 2 Departamento de Bioquimica, Facultad de Ciencias, Universidad de C6rdoba, Cdrdoba, Spain

Summary. Six mutant strains (301, 102, 203, 104, 305, and 307) affected in their nitrate assimilation capability and their corresponding parental wild-type strains (6145c and 21gr) from Chlamydomonas reinhardii have been studied on different nitrogen sources with respect to NAD(P)H- nitrate reductase and its associated activities (NAD(P)H- cytochrome c reductase and reduced benzyl viologen-nitrate reductase) and to nitrite reductase activity. The mutant strains lack NAD(P)H-nitrate reductase activity in all the nitrogen sources. Mutants 301,102, 104, and 307 have only NAD(P)H-cytochrome c reductase activity whereas mutant 305 solely has reduced benzyl viologen-nitrate reductase ac- tivity. Both activities are repressible by ammonia but, in contrast to the nitrate reductase complex of wild-type strains, require neither nitrate nor nitrite for their induction. Moreover, the enzyme from mutant 305 is always obtained in active form whereas nitrate reductase from wild-types needs to be reactivated previously with ferricyanide to be fully detected. Wild-type strains and mutants 301,102, 104, and 307, when properly induced, exhibit an NAD(P)H-cy- tochrome c reductase distinguishable electrophoretically from constitutive diaphorases as a rapidly migrating band. Nitrite reductase from wild-type and mutant strains is also repressible by ammonia and does not require nitrate or nitrite for its synthesis. These facts are explained in terms of a regulation of nitrate reductase synthesis by the enzyme itself.

Introduction

Nitrate assimilation by eukaryotic organisms is a highly regulated process. Although the possible mechanisms in- volved are still controversial, it is at present accepted that regulation may be exerted at three different levels: nitrate uptake, enzyme synthesis and enzyme activity (Beevers and Hageman 1969; Garrett and Amy 1978; Losada eta1. 1981). Ammonia, the end product of the reductive nitrate assimilation pathway, has clear effects at these three levels. Its addition to a culture of cells actively growing on nitrate causes an immediate blocking of nitrate uptake (Pistorius et al. 1978; Florencio et al. 1980), and a decrease in NR 1

Offprints request to : J. Cfirdenas

i Abbreviations: NR, nitrate reductase; NiR, nitrite reductase; BVH, reduced benzyl viologen; MVH, reduced methyl viologen; CR, cytochrome c reductase

activity after short treatments by reversible inactivation (Losada 1974) and after several hours because of enzyme repression (Morris and Syrett 1963; Losada et al. 1970).

The points of control of NR seem to be interconnected and to depend on environmental conditions such as light or nitrogen source (Losada et al. 1981), Recently, it has been proposed that the reversible deactivation of glutamine synthetase may play an important role in the regulation of nitrate assimilation either directly or via the products of its metabolism (Cullimore and Sims 1980, 1981; Flores et al. 1980). Modifications of the rate of NR turnover by external conditions have also been suggested to be of regu- latory significance in nitrate assimilation (Hipkin et al. 1980).

NiR synthesis and repression, usually but not always, follow that of NR (Vennesland and Guerrero 1979), al- though it is unclear whether or not their regulation systems are linked.

NR-deficient mutants from wild-type strains 614c and 21gr of Chlamydomonas reinhardii have been obtained (Sosa et al. 1978) and their enzymatic and physicochemical char- acteristics have been established (Fern/mdez 1981 ; Fern/m- dez and Cfirdenas 1981a, 1981b). All the mutants lack NAD(P)H-NR activity. Mutants 301, 102, 104, and 307 were described as having only NAD(P)H-CR activity whereas mutant 305 was solely able to reduce nitrate with viologens or flavins chemically reduced (Sosa et al. 1978).

In the present paper, the regulation of the enzymes of the nitrate-reducing system in wild-type and mutant strains of C. reinhardii is reported. The results indicate that, in the absence of ammonia, the integrity of the NR protein plays a very significant role in its own regulation. In addi- tion, NiR regulation seems to be independent of that of NR.

Materials and Methods

Chemicals. NADH, NADPH and cytochrome c (horse heart) were obtained from Boehringer, Mannheim, FRG; FAD from Sigma Chemical Co., St Louis, Mo., USA; ben- zyl viologen, methyl viologen and p-nitrobluetetrazolium from Serva, Heidelberg, FRG, and liquid nitrogen from Dpto. Quimica Inorgfinica (Universidad de Sevilla). All other reagents used were of analytical grade.

Strains and Culture Conditions. C. reinhardii wild-type pa- rental strains 6145c and 21gr (a gift from Dr. R. Sager, Hunter College, New York), and mutant strains 301, 102,

0026-8925/82/0186/0164/$01.20

203, 104, 305, and 307 (Sosa et al. 1978), were grown under conditions previously described (Fern/mdez and Citrdenas 1981 a).

Preparation of Extracts. Cell pellets, obtained by centrifuga- tion at 16,000xg for 10 rain from cultures harvested at a mid-exponential phase of growth, were broken by one of the following methods: (1) Cells were ground in a cold mortar with alumina (3 g alumina/g wet weight), and the resulting material was resuspended in 50 mM Tris-HC1 buffer, pH 7.5; 0.1 mM dithioerythritol; 0.1 mM EDTA; 20 gM FAD (5 ml/g wet weight) (buffer 1). The homoge- hate was then centrifuged at 27,000 × g, 15 rain, and the supernatant used as source of enzyme. (2) Celt pellets were resuspended in cold buffer 1 (7.5 ml/g wet weight) and bro- ken at 0 ° C with a Branson B-12 sonicator at 90 W, 15 s. The resulting homogenate was treated as above. (3) Cell pellets were frozen by immersion in liquid nitrogen ( - 190 ° C) or in the deep-freezer of a Westinghouse refrig- erator. The frozen material was thawed in buffer i (5 ml/g wet weight) with continuous and gentle stirring for 1 h. Then the suspension was centrifuged as in (1). Unless other- wise stated, ceils were always broken in this way.

Enzyme Assays and Analytical Methods. NAD(P)H-nitrate reductase activity (EC. 1.6.6.2., NAD(P)H: nitrate oxidore- ductase) was determined by measuring the nitrite formed by the enzymatic reduction of nitrate (Paneque and Losada 1966). NAD(P)H-cytochrome c reductase activity was esti- mated spectrophotometrically by following the reduction of cytochrome c at 550 nm (Zumft et al. 1969). BVH-nitrate reductase activity was assayed by the method of Paneque et al. (1965). When NAD(P)H or BVH-NR activity had to be reactivated, extracts were preincubated with 0.3 #tool ferricyanide for 1 rain, and the concentration of reductant was duplicated in the assay mixture (Losada et al. 1970). Nitrite reductase activity (EC. 1.7.7.1., ferredoxin: nitrite oxidoreductase) was assayed with MVH according to Ra- mirez et al. (1966). Activity units are expressed as gmol substrate transformed per rain.

Nitrite was determined colorimetrically by the method of Snell and Snell (1949). Protein was measured according to Lowry et al. (1951) as modified by Bailey (1967) using bovine serum albumin as a standard.

Detection of Diaphorase Activity on Polyacrylamide Gels. Crude extracts from C. reinhardii wild-type and mutant strains cells were subjected to analytical electrophoresis on 7.5% polyacrylamide gels according to the discontinuous gel system of Jovin et al. (1964). Diaphorase activity was detected directly on the gels by staining with a solution of 0.6 mM N A D H and 0.6 mM p-nitrobluetetrazolium in 0.5 M potassium phosphate buffer (pH 7.5) (Wang and Raper 1970).

R e s u l t s

Disruption of C. reinhardii Cells

The usual procedures of C. reinhardii cell disruption (by grinding in a mortar with alumina or by sonication) pro- duced extracts containing large amounts of pigments and membrane remnants which interfered with the analytical and enzymatic assays. C. reinhardii is an organism extreme-

E

uJ ~t9

U

@

165

Z

el

<

z

N NH4CI 2 0 I~L xN°3

IILX"o, Urea

Aspartate

l o

0 ~ . . . . . . . . . . . . . . . . . . w'~ h r l

301 102 203 104 305 307 6145_.£ 21g./

Fig. l. NADPH-NR activity levels in wild-type and mutant strains of C. reinhardii induced on different nitrogen sources. Strains were grown on ammonia media, harvested at mid-exponential phase of growth, and transferred into media containing 9.5 mM NH4C1, 4 mM KNO3, 3 mM KNO2, no nitrogen source, 9.5 mM urea or 0.5 mM aspartate, as indicated. After 5.5 h, cells were harvested and the activity of extracts determined as described in Materials and Methods

ly sensitive to freezing and thawing (Morris et al. 1979). When cells were frozen either in liquid nitrogen or at -20 ° C, 1 h, and then thawed in a buffer solution with

continuous and gentle stirring, 1 h, a clear supernatant de- void of pigments and membranes was obtained after contri- fuging the suspension at 27000 x g, 15 rain. Extracts from wild and mutant strains prepared in this manner exhibited the same enzymatic activity levels in the nitrate-reducing system as those obtained by the usual methods (results not shown) and were used in the experiments presented below.

Enzyme Levels in C. reinhardii on Different Nitrogen Sources

C. reinhardii cells grown on ammonia developed maximal NR levels 5-6 h after induction in media with nitrate (Thacker and Syrett 1972). Thus, 5.5 h was chosen as the optimal time for induction of the nitrate-reducing system enzymatic activities.

Wild-type strains 6145c and 21gr showed significant NADPH-NR levels when induced on nitrate under these conditions. Lower levels were observed with nitrite whereas, in the presence of ammonia, NR was nonexistent. Likewise, negligible levels were detected on urea, aspartate or N-free media. In addition, none of the mutants exhibited NADPH- NR activity in all the nitrogen sources studied (Fig. 1). The same results were obtained by using N A D H as electron donor (results not shown). The NR activity of wild-type strains was always about 60% active and had to be reacti- vated by oxidation with ferricyanide before assay (results not shown).

In the presence of ammonia, wild-type and mutant strains possessed basal levels of NADPH-CR of ca. 36 mU/ mg (Fig. 2). These levels increased in wild-type strains when induced in nitrate or nitrite media. In contrast, mutants 301, 102, 104, and 307 showed comparable higher levels of NADPH-CR when induced not only with nitrate or nitrite but also with aspartate, urea or N-free media. By contrast,

166

E

K N 0 3 N H4CI

"~ llll xmo 2 None ~ N e n . f l

- II ! !

z 301 102 203 104 305 307 6145£ 21~ 301 102 203 104 305 307 6145£ 21g_.r

Fig. 2. NADPH-CR activity levels in wild-type and mutant strains Fig. 4. MVH-NiR activity levels in wild-type and mutant strains of C. reinhardii induced on different nitrogen sources. Experimental of C. reinhardii induced on different nitrogen sources. Experimental conditions as for Fig. 1 conditions as for Fig. 1

E

E

U

LLJ

QC

2 D "r

6 0

3 0

0

KNO2 None

Urea

A s p a r t a t e

301 102 203 104 305 307 6145£ 219._r

Fig. 3. BVH-NR activity levels in wild-type and mutant strains of C. reinhardii induced on different nitrogen sources. Experimental conditions as for Fig. 1

A

B

mutants 203 and 305 showed only constitutive levels of d iaphorase in all the induct ion media tested. Identical re- sults were obta ined with N A D H (results not shown).

B V H - N R activity appeared exclusively in wild-type strains when induced on nitrate or nitrite media (Fig. 3). A unique mutan t strain, 305, exhibited B V H - N R on all the media used, a l though this activity was always repressed by ammonia . Like N A D ( P ) H - N R , B V R - N R from wild- type strains was only about 60% active, whereas in mu- tant 305 the enzyme was always found fully active and in higher amounts than in the wild-type strains (data from wild-type strains in Figs. 1 and 3 were those obta ined after enzyme react ivat ion with ferricyanide). As expected, N A D ( P ) H - C R and B V H - N R from wild-type strains dis- played the same regulatory propert ies as the N A D ( P ) H - N R complex. In contrast , the par t ia l activities o f the mutants have lost the ni t rate or nitrite requirement for their induc- t ion to take place.

On the other hand, M V H - N i R activity was similar in wild-type and mutan t strains (Fig. 4). In all cases, the en-

Fig. 5A, B. Visualization on polyacrylamide gels of NADH-dia- phorase activities of wild-type strain 21gr A and mutant102 B from C. reinhardii. Cells, grown on ammonia, were kept for 5.5 h in media containing 9.5 mM NHgCI (1), 4 mM KNO3 (2), 3 mM KNO2 (3), N-free (4), 9.5 mM urea (5), and 0.5 mM aspartate (6). 0.1q3.2 mg of the corresponding extracts were run and gels stained for NADH-diaphorase activity as described in Materials and Methods

167

zyme was repressed by ammonia but its synthesis did not need nitrate or nitrite in the induction media. Mutations affecting NR were without effect on the regulation of NiR, except in the 307 mutant that overinduced an increased capacity for nitrite reduction.

Occurrence of an Inducible Diaphorase

Extracts of the wild-type strain 21gr, grown on ammonia and derepressed 5.5 h on different nitrogen sources, were subjected to electrophoresis, and the gels stained for dia- phorase activity (Fig. 5, A). Bands corresponding to the constitutive diaphorase activities of C. reinhardii are seen in gel 1. When the cells were derepressed on N-free media, urea or aspartate, only bands of constitutive diaphorases were detected (gels 4, 5, and 6), in agreement with the data shown in Fig. 2. When induction took place in the presence of nitrate (gel 2) or nitrite (gel 3) a faster migrating new band of Rv=0.58 appeared (arrow) which did not corre- spond to the diaphorase activity of the NR complex, which has a much slower migration rate (R v = 0.20) and is masked by constitutive diaphorases (results not shown).

Similarly, a!l the mutant strains, when grown on ammo- nia, showed the same distribution pattern of constitutive diaphorases. In contrast, electrophoresis of extracts from mutant 102 (Fig. 5, B) induced with nitrate, nitrite, N-free media, urea or aspartate revealed the existence of the fast band of diaphorase, again in good agreement with the re- sults presented in Fig. 2. Wild-type 6145c behaved exactly like 21gr and 301, 104 and 307 mutants like t02 (results not shown). In the same conditions, 305 and 203 showed only the bands corresponding to constitutive diaphorases in all the media used (results not shown).

Discussion

In the wild-type strains 6145c and 21gr of C. reinhardii, NAD(P)H-NR and its partial activities NAD(P)H-CR and BVH-NR were absent when grown on ammonia, but were induced significantly on nitrate or nitrite media. However, in urea, aspartate or N-free media NR levels were practi- cally nonexistent. These findings are in agreement with others previously described for C. reinhardii (Herrera et al. 1972; Nichols et al. 1978; Hipkin et al. 1980), although in aspartate media NR was never detected in contrast to the finding of Herrera et al. (1972). NAD(P)H-CR from mu- tants 301, 102, 104, and 307, and BVH-NR from mu- tant 305 were also repressed by ammonia but, unlike the corresponding activities of wild-type strains, they also ap- peared in cells derepressed on aspartate, urea or N-free media.

Wild-type and mutant strains of C. reinhardii grown on ammonia have a constitutive NAD(P)H-CR activity that can be visualized by electrophoresis on polyacrylamide gels. The existence of diaphorases not related to N R is not exclu- sive to C. reinhardii and has been described in fungi (Subra- manian and Sorger 1972; MacDonald et al. 1974) and high- er plants (Wray and Filner 1970; Wallace and Johnson 1978; Mendel and Mfiller 1979; Maldonado et al. 1980). Wild-type strains, on nitrate or nitrite media, and mu- tants 301, 102, 104 and 307, on any nitrogen source used other than ammonia, exhibit higher NAD(P)I t -CR activi- ties than the basal levels found on ammonia, and show concomitantly a new, faster band of diaphorase activity

when subjected to electrophoresis. This diaphorase has been recently characterized as a true subunit with NAD(P)H-CR activity of the NR complex of C. reinhardii (FernAndez 1981). BVH-NR of mutant 305 has a lower molecular weight (177,000) than the native NAD(P)H-NR (241,000), but shows the same kinetic and enzymatic behaviour as the corresponding terminal activity of the wild-type enzyme complex (FernAndez 1981). Moreover, NAD(P)H-CR from mutants 301, 102, 104, and 307, and BVH-NR from 305 have been shown to have active subunits of the NR of C. reinhardii by in vitro complementation studies (FernAn- dez 1981 ; Fern~indez and CArdenas 1981 a).

Mutant 203 was devoid of all the activities required for reduction of nitrate to nitrite and possibly is affected in a regulatory gene or is a double mutant (Sosa et al. 1978).

Another interesting point that became clear in the pres- ent work is that BVH-NR from mutant 305 induced in any nitrogen source used apart from ammonia was always fully active. In contrast, NR from wild-type strains grown on nitrate or nitrite was always obtained in a partially inac- tive form and needed to be reactivated by ferricyanide be- fore assay. NR from green algae and higher plants are re- versibly inactivated by reduction and reactivated by oxida- tion (Losada et al. 1970; Losada 1974; Palacian et al. 1974). Moreover, nitrate and nitrite have been described in certain cases as protecting agents against this inactivation by reduc- tion (Kaplan et al. 1978; Losada et al. 1981). Addition of ammonia or removal of the nitric nitrogen source produced the same inactivating effect on C. reinhardii NR (Losada et al. 1973; Florencio et aI. 1980; Florencio and Vega 198t). Regulation studies in the nit-A mutant of C. reinhardii, which possesses only BVH-NR insensitive to inactivation by NAD(P)H, suggest that ammonia both stimulates NR proteolytic breakdown and stops NR synthesis. Nitrate would act by binding to the enzyme thus stabilizing it against proteolytic degradation, a phenomen which also ap- pears after prolonged periods (22 h) of nitrogen starvation (Hipkin et al. 1980).

Our results strongly suggest that regulation of NR syn- thesis and degradation may operate at, at least two levels. First, ammonia clearly represses all the activities of the NR complex. This repression seems to be exerted through the glutamine synthetase route in a direct or indirect way (Culli- more and Sims 1980, 1981 ; Flores et al. 1980). Second, the NR biosynthesis does not depend so much on the nitrate presence in the culture media as on the integrity of the whole NR complex. On N-free, aspartate or urea media, the sole difference in the enzymatic machinery responsible for the nitrate reduction between wild-type and mutant strains is that defective NR, incapable of inactivation, are induced in mutants but not in the intact wild-type cells. The lack of activity observed in wild-type cells derepressed on urea, aspartate or N-free media (Figs. 1, 2 and 3) could be explained in this way. A requirement of functional integ- rity for the N R redox inter-conversion has been demon- strated in vitro in Spinacea oleracea (Palacian et al. 1974).

On the basis of these results, we propose that the NR molecule of C. reinhardii plays an autoregulatory role on its own synthesis and degradation, which may take place either via the reduced inactive form of the enzyme or via the N R protein identification by a regulatory gene product. In the first case, N R is maintained in its active form by nitrate or nitrite and the enzyme level remains constant within the cells by steady turnover. In the absence of either

168

nitrate or nitrite, the enzyme becomes inactivated by reduc- tion and this inactive form gives the signal to inhibit its own synthesis or to favor its degradat ion. The defective enzymes f rom mutants cannot be inactivated by reduction and thus their repression can only be exerted by ammonia . In the second case, an autoregula t ion o f the N R protein can be also considered, as p roposed for the enzymes of Aspergillus nidulans (Cove and Pa teman 1969) and Neuro- spora crassa (Coddington 1976; Tomset t and Gar re t t 1981). Defective N R molecules, in the absence of ni trate or nitrite, are not recognized by the regulatory gene product and thus are incapable of impeding the positive act ion of the lat ter on the enzyme synthesis that, however, is still repressible by ammonia . The reversible inact ivat ion by reduction would be a manner of control l ing enzyme activity at short times. In any case, integrity of the N R complex seems to be essential for fine regulation.

N i R induct ion is similar in all mutan t and wild-type strains: it is repressed by ammonia and appears in nitrate, nitrite, or N-free media (Fig. 4) Ni t ra te does not have to be converted into nitrite to induce N i R activity. Differences between the regulat ion systems of N R and N i R indicate that both enzymes have separate regulat ion mechanisms, as has been suggested in N. crassa (Coddington 1976; Gar - rett and Amy 1978). The N i R overinduct ion in mutan t 307 may be due to ple iotropic effects of muta t ions on N R struc- ture like those described in A. nidulans (Cove and Pateman 1969) and N. tabacum (Mendel and Miiller 1979).

Acknowledgements. The authors thank Prof. M. Losada for helpful advice. This work was supported by Grants from Centro de Estu- dios de la Energia (Spain) and Philips Research Laboratories (The Netherlands). One of us (E.F.) thanks the Ministerio de Educaci6n y Ciencia (Spain) for a fellowship.

References

Bailey JL (1967) Techniques in protein chemistry, 2nd. Elsevier, Amsterdam, p 340

Beevers L, Hageman RH (1969) Nitrate reduction in higher plants. Annu Rev Plant Physiol 20: 495-522

Coddington A (1976) Biochemical studies on the nit mutants of Neurospora crassa. Mol Gen Genet 145:195-206

Cove DJ, Pateman JA (1969) Autoregulation of the synthesis of nitrate reductase in Aspergillus nidulans. J Bacteriol 97:1374-1378

Cullimore JV, Sims AP (1980) An association between photorespir- ation and protein catabolism: studies with Chlamydomonas. Planta 150:392-396

Cullimore JV, Sims AP (1981) Glutamine synthetase of Chlamydo- monas: its role in the control of nitrate assimilation. Planta 153:18-24

Fern~ndez E (1981) Caracterizaci6n molecular del complejo enzi- mMico NAD(P)H-nitrato reductasa de algas verdes mediante el estudio de estirpes silvestres y mutantes de Chlamydomonas reinhardii. Doctoral Dissertation, University of Sevilla, Spain

Fernfindez E, Cfirdenas J (1981 a) In vitro complementation of assimilatory NAD(P)H-nitrate reductase from mutants of Chla- mydomonas reinhardii. Biochim Biophys Acta 657:1-12

Fern~mdez E, C/trdenas J (1981 b) Occurrence of xanthine dehy- drogenase in ChIamydomonas reinhardii: A common cofactor shared by xanthine dehydrogenase and nitrate reductase. Planta 153:254-257

Florencio FJ, Vega JM (1981) Regulaci6n de la asimilaci6n de nitrato en Chlamydomonas Chlamydomonas reinhardii. Pro- ceedings of the Second Congress of the Spanish Fed of Exp Biol Soc. FESBE, Madrid, p 96

Florencio FJ, Vega JM, Losada M (1980) Efecto del amonio y

de la obscuridad en la asimilaci6n fotosint~tica del nitrato por el alga verde Chlamydomonas reinhardii. Proceedings of the First Spanish-Portuguese Congress of Biochem. SEB-SPB, Coimbra, p 195

Flores E, Guerrero MG, Losada M (1980) Short-term ammonium inhibition of nitrate utilization by Anacystis nidulans and other cyanobacteria. Arch Microbiol 128:137-144

Garrett RH, Amy NK (1978) Nitrate assimilation in fungi. Adv Microbiol Physiol 18:1-65

Herrera J, Paneque A, Maldonado JM, Barea JL, Losada M (1972) Regulation by ammonia of nitrate reductase synthesis and ac- tivity in Chlamydomonas reinhardii. Biochem Biophys Res Commun 48 : 996-1003

Hipkin CR, A1-Bassam BA, Syrett PJ (1980) The roles of nitrate and ammonium in the regulation of the development of nitrate reductase in Chlamydomonas reinhardii. Planta 150:13-18

Jovin T, Charambach A, Naughton MA (1964) Apparatus for preparative temperature regulated polyacrylamide gel electrophoresis. Anal Biochem 9:351-364

Kaplan D, Mayer AM, Lips SH (1978) Nitrite activation of nitrate reductase in higher plants. Planta 138:205-209

Losada M (1974) Interconversion of nitrate and nitrite reductase of the assimilatory type. In: Fischer EH, Krebs EG, Neurath H, Stadtman ER (eds) Metabolic interconversions of enzymes, III Internat Symp. Springer, Berlin Heidelberg New York, p 257

Losada M, Guerrero MG, Vega JM (1981) Tile assimilatory reduc- tion of nitrate. In: Bothe H, Trebst A (eds) Biology on inorgan- ic nitrogen and sulfur. Springer, Berlin Heidelberg New York, pp 3~63

Losada M, Herrera J, Maldonado JM, Paneque A (1973) Mecha- nism of nitrate reductase reversible inactivation by ammonia in Chlamydomonas. Plant Sci Lett 1:31-37

Losada M, Paneque A, Aparicio PJ, Vega JM, Cfirdenas J, Herrera J (1970) Inactivation and repression by ammonium of the ni- trate reducing system in Chlorella. Biochem Biophys Res Com- mun 38:100%1015

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265-275

MacDonald DW, Cove DJ, Coddington A (1974) Cytochrome c reductases from wild-type and mutant strains of Aspergillus nidulans. Mol Gen Genet 128:187-199

Maldonado JM, James DM, Norton BA, Hewitt EJ (1980) Occur- rence and properties of cytochrome e reductase in cereaIs. Pro- ceedings of the Second Congress of the Fed of Eur Soc of Plant Physiol. FESPP, Santiago, pp 468-469

Mendel RR, Miiller AJ (1979) Nitrate reductase-deficient mutant cell lines of Nicotiana tabaeum. Further biochemical character- ization. Mol Gen Genet 177:145-153

Morris I, Syrett PJ (1963) The development of nitrate reductase in Chlorella and its repression by ammonium. Arch Mikrobiol 47:32-41

Morris GJ, Coulson G, Clarke A (1979) The cryopreservation of Chlamydomonas. Cryobiology 16: 401-410

Nichols GL, Shehata SAM, Syrett PJ (1978) Nitrate reductase defi- cient mutants of Chlamydomonas reinhardii. Biochemical char- acteristics. J Gen Microbiol 108:79-88

Palacian E, De la Rosa FF, Castillo F, Gomez-Moreno C (1974) Nitrate reductase from Spinaeea oleracea. Reversible inactiva- tion by NAD(P)H and by thiols. Arch Biochem Biophys 161:441-447

Paneque A, Losada M (1966) Comparative reduction of nitrate by spinach nitrate reductase with NADHz and NADPH2. Bio- chim Biophys Acta 128:202-204

Paneque A, Del Campo FF, Ramirez JM, Losada M (1965) Flavin nucleotide nitrate reductase from spinach. Biochim Biophys Acta 109 : 79-85

Pistorius EK, Fiinkhouser EA, Voss H (1978) Effect of ammonium and ferricyanide on nitrate utilization by Chlorella vulgaris. Planta 141 : 279-282

169

Ramirez JM, Del Campo FF, Paneque A, Losada M (1966) Ferre- doxin-nitrite reductase from spinach. Biochim Biophys Acta 118 : 58-71

Snell FD, Snell CT (1949) Colorimetric methods of analysis, vol 2. Van Nostrand, New York, p 804

Sosa FM, Ortega T, Barea JL (1978) Mutants from Chlamydomon- as reinhardii affected in their nitrate assimilation capability. Plant Sci Lett 11:51-58

Subramanian KN, Sorger GJ (1972) The role of molybdenum in the synthesis of Neurospora nitrate reductase. Biochim Biophys Acta 256 : 533 543

Thacker A, Syrett PJ (1972) Disappearance of nitrate rednctase activity from Chlamydomonas reinhardii. New Phytol 71:435-441

Tomsett AB, Garrett RH (1981) Biochemical analysis of mutants defective in nitrate assimilation in Neurospora crassa: evidence for autogenous control by nitrate reductase. Mol Gen Genet 184:183-190

Vennesland B, Guerrero MG (1979) Reduction of nitrate and ni- trite. In: Gibbs M, Latzko E (eds) Encyclopedia of plant phys- iol, New Series, vol 6. Springer-Verlag, Berlin, pp 425-444

Wallace W, Johnson CB (1978) Nitrate reductase and soluble cyto- chrome c reductase(s) in higher plants. Plant Physiol 61:748 752

Wang CC, Raper JR (1970) Isozyme patterns and sexual morpho- genesis in Schizophyllum. Proc Natl Acad Sci USA 66 : 882-889

Wray JL, Filner P (1970) Structural and functional relationships of enzyme activities induced by nitrate in barley. Biochem J 119:715 725

Zumft WG, Paneque A, Aparicio PJ, Losada M (1969) Mechanism of nitrate reduction in Chlorella. Biochem Biophys Res Com- mun 36 : 980-986

Communicated by H. B6hme

Received January 12 / March 22, 1982