Detection of Nitrate Reductase Cross-Reacting Material in Wild-Type and Mutant Cells of Nicotiana tabacum

Biochem. Physiol. Pflanzen 180, 63-74 (1985)

Detection of Nitrate Reductase Cross-Reacting Material in Wild-Type and Mutant Cells of Nicotiana tabacum J. SCHIEMANN and A. J. MULLER Institute of Genetics and Crop Plants, Academy of Sciences of the GDR, Gatersleben Key Term Index: nitrate reductase, antibody, mutant, enzyme synthesis, cell culture; Nicolillna tabacum

Summary Nitrate reductase (NR) cross-reacting material in extracts of Nicotiana tabacum cell cultures was quantitatively estimated by its ability to protect the active enzyme from inhibition by antiserum. In wild-type cells, the nitrate·induced increase of NR activity was accompanied by a parallel increase of antigenicity, suggesting that the induction of ~R activity is due to de novo synthesis of the "poprotein. Mutant cells that lack NADH-NR activity were found to possess NR cross-reacting material. This inactive NR protein was inducible by nitrate in case of the structural gene mutants NiaG3 and Nia28 and constitutive at high level in case of the cofactor mutant CnxG8. The relative ,wtigenicity of the structural gene mutant extracts was dependent on the antiserum used, indicating alterations in enzyme structure that lowered its affinity to certain NR-specific antibodies.

Introduction

Induction of NR activity by nitrate as its natural substrate has been found in higher plants, algae, fungi, and bacteria and seems to be a general phenomenon (see reviews HEWITT 1975; SRIVASTAVA 1980; GCERRERO et al. 1981; MARzLUF 1981; STOCTHAl\IER 1976). In many organisms, the presence of nitrate is an absolute requirement for the formation of NR activity. By contrast, tobacco (Nicotiana tabacum) exhibits partially constitutive expression of NR. Upon growth on reduced nitrogen such as ammonium succinate or amino acids as sole nitrogen source and in the absence of nitrate, both cultured cells (MULLER and GRAFE 1978; ME:\lDEL and MULLER 1979; HEIMER and RIKLIS 1979; SAALBACH 1980) and various tissues of whole tobacco plants (MULLER 1983) develop appreciable NADH-NR activity (up to 20 % of the fully induced level). The constitutive and the nitrate-induced NR activity have, by genetic evidence, been shown to be due to the same enzyme (MULLER 1983). Nitrate-induced de novo synthesis of the NADH-NR has been demonstrated in cultured tobacco cells by boyant density separations (ZIELKE and FILNER 1971). This paper describes the application of an immunological procedure to determine the levels of KR-related proteins irrespective of their retention of any catalytic function. Abbreviations: NR, nitrate reductase; anti-NR I and 11, rabbit antisera directed against nitrate reductase of tobacco and squash, re~pectively; CRM, cross-reacting material; BSA, bovine serum albumin; )[o-co, molybdenum- cofactor; BVH, reduced benzyl viologen; CR, cytochrome c reductase

64

J. SCHIEMANN and A. J. MULLER

One objective of the present study was to see whether the nitrate-induced increase of NR activity in cultured tobacco cells is fully accounted for by de novo synthesis of the enzyme or in addition does involve activation of a precursor protein. Using immunological methods, FUNKHOUSER and RAMADOSS (1980) demonstrated that ammonia-grown Chlorella vulgaris cells, which do not possess NR activity, contain an inactive form of NR which is activated upon addition of nitrate. On the other hand, NR-CRM was lacking in uninduced mycelia of Neurospora crassa (AMY and GARRETT 1979, 1980), barley seedlings (Kuo et al. 1981; SOMERS et al. 1983 b), and cucumber cotyledons (ULITZSCH and SCHIEMANN 1984). In addition to wild type cells, we also examined cell cultures of three NR-deficient tobacco mutants (MULLER and GRAFE 1978; MENDEL and MULLER 1979; MULLER 1983). The mutants Nia63 and Nia28 lack NADH-NR activity due to a mutation in the structural gene for this enzyme. Mutant Cnx68 has lost NADH-NR activity as a consequence of a mutation that affects formation of active Mo-co. We used a protection from inhibition assay to quantitatively estimate the amount of NR CRM in the mutant cells under inducing and noninducing conditions with the aid of different antisera. The objective of these studies was to establish whether this assay is well suited for the detection of mutant forms of NR and whether the synthesis of the defective NR molecules is regulated in the same way as the wild-type enzyme. Material and Methods Extraction of leaf nitrate reductase Young leaves from greenhouse-grown tobacco plants (Nicotiana tabacum cv. Gatersleben) were washed with distilled water. The middle vein was removed, and the leaf material was ground in a mortar with liquid nitrogen. The dry powder was mixed in a ratio of 1: 4 with cold extraction buffer containing 100 mM KINa-phosphate, pH 7.5, 1 mM EDTA, 12.5 mM 2-mercaptoethanol, lOIlM leupeptine, and lOIlM FAD. The homogenate was centrifuged at 3°C for 30 min at 22,000 . g and the supernatant was passed through filter paper. The crude extract was mixed with 1/4 vol. glycerol and stored at -70°C. Cell lines and culture conditions Log phase cells of the wild-type cell line S21-1 and 3 NR-deficient mutant cell lines of Nicotiana tabacum cv. Gatersleben, Cnx68/2, Nia63 (MULLER and GRAFE 1978), and Nia28 (MULLER 1983) were used. Mutant selection, culture conditions, and media have been described elsewhere (MULLER and GRAFE 1978). The cells were grown as surface callus cultures on agar medium containing ammonium succinate (20 mM) as sole nitrogen source and subcultured every 7 d. Callus was harvested at the end of the exponential growth phase (7 d after subculture). For induction, KN0 3 up to a final concentration of 50 mM was added to the culture 15 h before harvesting. Preparation of cell-free callus extract Cell-free callus extract was prepared according to MENDEL and MULLER (1979) with slight modifications. 1.5 ml of ice-cold extraction buffer (100 mM KINa-phosphate, pH 7.5, 1 mM EDTA, 2 mM 2-mercaptoethanol, 3 % BSA, lOIlM FAD) were added to each gram of cells. Reducing agents as 2-mercaptoethanol, cysteine, glutathione, or Cleland's reagent which are used to protect SH-groups of NR disturb the nitrite determination by diazocoupling colorimetric assay (WELANDER 1978; SCHIEMANN, unpublished). Therefore, the concentration of 2-mercaptoethanol was reduced in the

Detection of Nitrate Reductase

65

callus extraction buffer. In contrast to tobacco leaf material where 12.5 mM 2-mercaptoethanol are essential to isolate active NR, 2 mM 2-mercaptoethanol is sufficient for NR extraction from callus. The cells were sonicated 1 min under efficient cooling with the microtip of a Branson B-12 Sonifier (Branson Co., Danbury, USA) at maximal output. The homogenate was centrifuged at 3°C for 30 min at 22,000 . g and the supernatant was passed through filter paper. For the anti-NR inhibition protection assay, wild type cells from the same passage as mutant cells were used as control. The extracts were used immediately for assay. NADH-NR assay

NADH-NR assay was performed according to MENDEL and MULLER (1978) with slight modifications. The reaction mixture contained (in 0.65 ml total volume): 0.1 ml enzyme extrad, 0.4 ml 100 mM KINa-phosphate, pH 7.5, 0.05 ml100 mM KNO a. The reaction was started by the addition of 0.1 ml 2 mM NADH. After incubation at 25°C for 30 min, the reaction was terminated by incubating for 5 min in boiling water and cooling in an ice bath. The nitrite formed was determined by the diazo coupling colorimetric assay as described by NICHOLAS and NASON (1957) by addition of 0.3 ml 1 % sulphanilamide in 3 N HCI and 0.3 ml 0.02 % N-(l-naphthyl)ethylenediamine dihydrochloride. To remove the turbidity, samples were centrifuged for 10 min at 5,000· g. After 30 min the colour intensity was measured at 540 nm. 1 enzyme unit of NADH-NR activity is defined as the amount of enzyme forming 1 nanomole of nitrite per hour. Anti-NR inhibition assay

For the anti-NR inhibition assay, enzyme extract was preincubated with increasing amounts of anti-NR in a final volume of 200,tt1. The preincubation was carried out at 25°C for 30 min. Phosphate buffer and nitrate were added and the NR activity assay was started with 100,tt1 NADH solution as described above. Anti-NR I was prepared as described by ULITZSCH and SCHIEMANN (1984). Anti-NR II (Anti-NR Serum 12(79 J. SMARRELLI) was a kind gift of Dr. W. H. CAMPBELL, Syracuse (USA). The antiserum was precipitated two times in 50 % saturated ammonium sulphate at pH 7.0, dissolved in water and dialyzed against 100 mM phosphate buffer, pH 7.5.

Results

Antibodies raised against tobacco NR (anti-NR I) as well as those raised against squash NR (anti-NR II) inhibited the NADH-NR activity of tobacco extracts. The reaction was completed within 30 min. Longer preincubation periods did not result in a higher degree of inhibition (data not shown). The degree of inhibition was also independent of whether the preincubation was performed at 4 °C for 60 min or at 25°C for 30 min. Centrifugation did not influence the degree of inhibition, indicating that the inhibition was not due to precipitation of the enzyme-antibody complex. The relationship between enzyme inhibition and amount of antiserum added approximately followed a hyperbolic function (Fig. 1, 2) and thus met the expectation for a reversible reaction. However, very low amounts of antiserum had no inhibitory effect, so that the inhibition curves took the form of shouldered (sigmoid) curves. The anti-NR I repeatedly gave more extended shoulders than the anti-NR II (see also Fig. 4, 5). The presence of a shoulder might indicate that more than one antibody are needed to inactivate one enzyme molecule. The antigenicity of extracts was measured by the amount of antiserum required for 50% inhibition of NR activity (AS 5o). This value was found to be linearly related to the amount of enzyme, when different dilutions of the same extract were tested for inhibition 4a

J.

66

SCHIEMANN

and A. J.

MULLER

70

60

~ I

I

o

« z

40 30

20 10

o

2

3

4

567

antiserum added CjJ1J

8

9

10

Fig. 1. Anti-NR I inhibition assay of different amounts of tobacco leaf NR. 45ftl (e---e), 30ftl (0---0), and 151'1 (0---0) of crude leaf extract (specific activity

1,625 units/mg protein) were inhibited by increasing amounts of anti-NR I. Enzyme extraction, preincubation, and NADH-NR assay as described in Material and Methods.

by increasing amounts of antiserum (Fig. 1, 2). Thus, the AS50 value per unit of NADHNR activity (0.05,tt1 in case of serum I; 0.0012,tt1 in case of serum II) was independent of the extract concentration during preincubation.

Antigenicity in induced and uninduced wild-type cells Tobacco callus cultures grown on reduced nitrogen have a low level of NR activity, which upon addition of nitrate increases 5- to 10-fold. To find out whether or not the nitrate-induced increase of NR activity is accompanied by an increase of antigenicity, we tested NR extracted at different stages of induction process (0, 3 and 16 h) for inhibition by anti-NR 1. The results presented in Table 1 show that the 3 extracts gave about the same AS 50 value per unit of NR activity. Thus, the NR CRM level increased parallel to the NR activity. The obvious conclusion that the nitrate-induced increase of NR activity cannot be attributed to the activation of an immunologically detectable inactive form of the enzyme, was confirmed by additional experiments,in which the antigenicity of mixtures of induced and uninduced estracts was determined, using anti-NR I as well as anti-NR II. It was found that the mixed extracts had no higher AS 50 value per unit of NR activity than the induced extract alone (data not shown).

67

Detection of Nitrate Reductase 100

70

g'80 'c

•

E

f60

60

."

~

'iij

~

'S

:p

---~.------------

~40

a:::

z

,,~

o>R. 20

~40

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I

~ <{30 z

0.2

20 10

o

0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 antiserum added 1)

Cf

0.18

0.2

Fig. 2. Anit-NR II inhibition assay of different amounts of tobacco leaf NR. Experimental conditions as in Fig. 1. e---e, 45,u1 extract; 0 - - -0 , 15,u1 extract.

~

Table 1. Anti-NR I inhibition assay at different stages of NR induction by nitrate. NR was extracted at different times after addition of nitrate to tobacco callus cultures grown on ammonium succinate medium. Extracts of callus induced for 3 and 16 h were diluted 4.5-fold and 6-fold, respectively Time after addition of nitrate (h)

°

3 16

(units/g FW)

NADH-NR activity (units/assay)

Antiserum required for 50 % inhibition of NR activity (,ul)

(Ill)

220 1,060 1,300

16.6 17.5 16.3

1.25 1.07 1.13

0.075 0.061 0.069

ASool unit

Antigenicity in mutants GRAF et al. (1975) used the protection of inhibition assay of a standardized NR preparation from spinach leaves to detect the amount of NR CRM in an inactivated enzyme preparation. They found a quantitative relationship between the original activity of the inactivated NR preparation and the quantity of anti-NR (spinach) to produce 50 % inhibition of the standard NR activity. Similarly, we estimated the amount of NR CRM

68

J.

Cl t:

SCHIEMANN

and A. J.

MULLER

80

·c

·iii 70 E III

~60

+'

.s;

~ 50 - - - - - - - - - til

a:

z

~

40 30 20

10

o

2

3

4 5 6 7 antiserum added (fl)

8

9

10

Fig. 3. Anti-NR inhibition protection assay with extracts from induced and non-induced Cnx 68 callus with and without temperature treatment. 50 ttl of crude NR extract (volume activity 770 units/ml) were inhibited by increasing amounts of anti-NR I. Before the addition of anti-NR, lOOpl of uninduced (0-- - 0), induced (0-- - 0). or induced temperature treated ( . -- -. ) Cnx 68 extract was added. e - - -e, control with 100 ttl extraction buffer.

present in NR-deficient mutants by the ability of the mutant extract to compete with the active enzyme for antibody binding. For this protection of inhibition assay, the mutant extract was mixed with a NR extract of induced wild-type cells. The amount of antiserum required to inhibit the NR activity of the mixture to 50 %(AS 50) was determined and compared to the AS 50 of the control (wild-type) extract. The antigenicity of the mutant extract was calculated in the following way: .. . . relatIve antIgenICIty

AS (mixture) - AS (control)

so 50 = -...::..:-'-----'---.::.:...-'-----'-

AS50 (control)

X

volume of control extract volume of mutant extract

.

The NR activity of the control extract served as a basis for expressing antigenicity in absolute terms (in NR unit equivalents per ml or per g fresh weight). This permitted a direct comparison of the results of experiments that were performed with control extracts of different NR activity. (A prerequisite to such comparisons is that all experiments were carried out with the same antiserum and gave the same AS 50 value per unit of NR activity.)

69

Detection of Nitrate Reductase A

0.25 0.5 0.75 antiserum (squash) added 1)

Cf

B

2.5 5 7.5 10 12.5 15 17.5 antiserum (tobacco) added [1"1)

Fig. 4. Anti-N R (squash, A,. tobacco, B) inhibition protection assay with extracts from induced Cnx68 callus.

100,u1 crude NR extract (volume activity 490 units/ml) + 100,u1 extraction buffer (e---e), +50,u1 extraction buffer +50,u1 induced Cnx68 extract (6---6), + 100,u1 induced Cnx68 extract ( .6 ---.6) were incubated as described in Material and Methods.

Results presented in Fig. 3 show that the addition of two volumes of an induced Cnx68 extract to one volume of control extract increased the AS50 from 3.1,tt1 to 9.3,tt1 of anti-NR I. Thus, the antigenicity of the mutant extract was the same as that of the control extract (770 NR unit equivalents per ml). Uninduced Cnx68 cells had only a slightly lower antigenicity (660 NR unit equivalents per ml or 85% of the control value). Heating the mutant extract for 5 min at 50° resulted in a nearly complete loss of antigenicity, as shown by the very low ability of the heated extract to protect NR from inhibition by anti-NR I (Fig. 3) or anti-NR II (data not shown). The same heat treatment caused a total loss of NR activity in wild-type extracts. In other experiments (Fig. 4, 5), the influence of extracts from different NR-deficient mutants on the inhibition curve was examined. In addition, the two antisera (anti-NR I and anti-NR II) were compared with respect to their ability to bind the inactive mutant enzyme. With both antisera, it was found that the level of antigenicity was highest in Cnx68 , lower in Nia63, and still lower in Nia28 cells, and that the antigenicity of Nia28 and Nia63 was inducible by nitrate. However, the use of anti-NR II led to con5

Biochem. Physiol. Pflanzen, Bd. 180

70

J. SCHIEMANN and A. J. MULLER A

B

10

o

0.02

0.04

0.06

0.08

0.1

antiserum (squash)added [1'1)

o

1 2 3 4 antiserum (tobacco)C¥:fded

[,..n

Fig. 5. Anti-NR (squash, A; tobacco, B) inhibition protection assay with extracts from induced and uninduced Nia63 and Nia28 callus.

25 III crude NR extract (volume activity 1,360 units/ml) + 100 III extraction buffer ( . - - - . ) , uninduced Nia28 extract (0---0), induced Nia28 extract ( . -- -.), uninduced N ia63 extract ( ~ --- ~ ), or induced Nia63 extract ( • - - - . ) were incubated asdescribed in Material and Methods. The activities (units/25 III wild type extract) after addition of 100 III extraction buffer or mutant extract were: 34, buffer; 34, Nia63 induced and uninduced; 38, Nia28 uninduced; 39, Nia28 induced.

siderably higher values for the antigenicity of Nia28 and Nia63 extracts, as compared to the use of anti-NR I (Fig. 5). In contrast, comparable amounts of NR CRM were detected in the Cnx68 extract by both antisera (Fig. 4). Table 2 summarizes results from different induced and uninduced cell cultures of the Nia28, Nia63 , and Cnx68 mutants. Discussion

The results presented in Table 1 provide further evidence that nitrate-dependent induction of NR in tobacco callus is due to de novo synthesis of the enzyme and not to activation of an immunologically detectable precursor protein. The same conclusion was drawn from immunological studies of NR induction in Neurospora crassa (AMY and GARRETT 1979, 1980), barley (SOMERS et al.1983b), and cucumber (ULITZSOH and SORlE-

Detection of Nitrate Reductase

71

Table 2. Antigenicity of induced and uninduced Nia28, Nia63, and Cnx68 extracts. Values given are from different experiments. The average NR activity of the control was 930 units per mI. For callus extraction, 1.5 ml extraction buffer were added to each gram of cells. a, b, c: values from the same experiment as in Fig. 3, 4, 5, respectively Antigenicity (NR unit equivalents/ml) Mutant line Cnx68/2

Nia63

Nia28

Induced by nitrate

Determined by anti-NR I

Determined by anti-NR II

+ + +

66()a 77()a 1,015 b 2,500 10c

n.d. n.d. 1,150 b n.d. "'" 435 c n.d. >420 c n.d. 160c 130

+ + + +

60 ~420c

255 15 c 30 120c 95

~410c

310

MANN 1984). In contrast to the temporary accumulation of inactive NR apoprotein which was observed during the nitrate-dependent induction process in cucumber cotyledons (ULITZSCH and SCHIEMANN 1984), all stages of NR induction in tobacco callus exhibited the same ratio of NR activity to the amount of NR apoprotein. The analysis of antigenicity in mutant cells reveals that all three mutant lines studied, although completely lacking NADH-NR activity, contain NR CRM. Moreover, it shows that in the two structural gene mutants Nia63 and Nia28 the level of NR CRM is regulated in a way similar to the regulation of NR activity in thc wild-type line: Uninduced cells had a low CRM level, which increased after addition of nitrate. In contrast, the cofactor mutant Cnx68J2 exhibited completely constitutive synthesis of NR CRM. When comparing the antigenicity of the mutants with that of the wild-type, one must take into account that the NR level (and consequently also the CRM level) of cell cultures varies considerably, even under the standardized growth conditions employed in this study. For this reason, the present results do not permit an exact quantitative comparison between the cell lines. It can only be concluded that the CRM level estimated with the aid of the anti-NR II is significantly lower in the Nl:a63 and Nia28 lines as compared to the wild-type line, and that the Cnx68J2 line has a CRM level that is as high as and probably higher than the wild-type level. These results confirm and extend previous conclusions concerning the presence of NR apoprotein in the mutants. Cell line Cnx68J2 is known to constitutively synthesize 7.6 S demolybdo NR, which is detectable by its retained diaphorase (NADH-CR) activity (MENDEL and MULLER 1979, 1980; MENDEL et al. 1981). The higher than wild-type level of NADH-CR activity was interpreted to mean that Cnx68/2 has an enhanced 5*

72

J.

SCHIEMANN

and A. J.

MULLER

level of NR apoprotein. This conclusion is supported by our demonstration of an unusual high level of NR CRM in uninduced and induced Cnx68/2 cells. The CRM may comprise not only dimeric (7.6 S) demolybdo-NR molecules, but also monomers and partially degraded NR apoprotein. The detection of nitrate-inducible NR CRM in Nia28 cells corresponds well to the finding that this mutant, which lacks NADH-NR activity and is unable to utilize nitrate for growth, has retained BVH-NR activity (MULLER and MENDEL 1982). It can therefore be concluded that Nia28 cells synthesize a structurally modified NR that has lost the ability to utilize NADH as electron donor, but can still catalyze nitrate reduction in the presence of artificial electron donors. The presence of NR CRM in Nia63 cells indicates that this mutant also synthesizes a defective form of NR, which however differs from the Nia28 enzyme in its complete inability to catalyze nitrate reduction with any electron donor (MENDEL and MULLER 1979). Recently, a low level of NR-associated NADH-CR activity could be detected in extracts of induced Nia63 cells (MENDEL, personal communication) suggesting that at least part of the immunologically detectable NR apoprotein has retained diaphorase activity. The protection of inhibition assay measures the ability of an extract to bind those antibodies, which are capable of inhibiting the catalytic activity of NR. It is obvious that this assay (like other immunological methods) gives no direct answer to the question of whether a reduced antigenicity is due to a reduced concentration of NR apoprotein in the extract or due to structural modifications of the apoprotein and the resulting reduction of its affinity to the antibodies. However, alterations in antigen structure may influence the reaction of two antisera in different ways. We found that anti-NR II indicated a considerably higher level of NR CRM in the structural gene mutants than anti-NR I did. Thus, the two sera differed from each other not only in titer, but also in their ability to recognize mutant forms of NR. The antibody composition of these sera may have additionally been influenced by the different enzyme preparations used for immunization of the rabbits. For preparing anti-NR II, intact squash cotyledon NR was isolated using blue-Sepharose and polyacrylamide gel electrophoresis (SMARELLI and CAMPBELL 1981). For preparing anti-NR I, NR from tobacco leaves was isolated using blue-Sepharose and chromatography on Ultroge1. The tobacco NR preparations used for immunization had lost their NADH-NR activity and were identified by their NADH-CR activity. In SDS polyacrylamide gel electrophoresis they showed two main bands of approximately 58,000 D and 40,000 D (SCHIEMANN and MANTEUFFEL 1982). Whilst demonstrating the importance of antiserum quality for the detection of mutant forms of NR, the present results do not answer the question of whether the anti-NR II was capable of detecting any modified NR protein. Our observation that this antiserum failed to recognize heat-inactivated wild-type and mutant NR of tobacco contrasts with the finding of AMY and GARRETT (1979) that Neurospora crassa NR inactivated by the same heat-treatment procedure retained its antigenicity. It remains to be established if other NR-specific antisera and/or other immunological techniques will be able to detect heat-inactivated and other highly altered forms of tobacco NR. In this connection it should be noted that the protection of inhibition assay and immunoelectro-

Detection of Nitrate Reductase

73

phoresis gave comparable results in studies of NR-deficient mutants of Neurospora crassa (AMY and GARRETT 1979) and barley (Kuo et al. 1981; SOMERS et al. 1983a). Acknowledgement We wish to express our gratitude to Dr. W. H. CAMPBELL, Syracuse (USA), for kindly providing antiserum against squash nitrate reductase.

References AMY, N. K., and GARRETT, R. H.: Immunoelectrophoretic determination of nitrate reductase in Neurospora crassa. Anal. Biochem. 95, 97-107 (1979). AMY, N. K., and GARRETT, R. H.: Repression of nitrate reductase activity and loss of antigenically detectable protein in Neurospora crassa. J. Bacteriol. 144,232-237 (1980). FUNKHOUSER, E. A., and RAMADOSS, C. S.: Synthesis of nitrate reductase in Chlorella. II. Evidence for synthesis in ammonia-grown cells. Plant Physiol. 65, 944-948 (1980). GRAF, L., NOTTON, B. A., and HEWITT, E. J.: Serological estimation of spinach nitrate reductase. Phytochemistry 14, 1241-1243 (1975). GUERRERO, M. G., VEGA, J. M., and LOSADA, M.: The assimilatory nitrate-reducing system and its regulation. Ann. Rev. Plant Physiol. 32, 169-204 (1981). HEIMER, Y. M., and RIKLIS, E.: On the mechanism of development of nitrate reductase activity in tobacco cells. Plant Sci. Lett. 16, 135-138 (1979). HEWITT, E. J.: Assimilatory nitrate-nitrite reduction. Ann. Rev. Plant Physiol. 26, 73-100 (1975). Kuo, T., KLEINHOFS, A., SOMERS, D., and WARNER, R. L.: Antigenicity of nitrate reductase-deficient mutants in Hordeum vulgare L. Mol. Gen. Genet. 181, 20-23 (1981). MARZLUF, G. A.: Regulation of nitrogen metabolism and gene expression in fungi. Microbiol. Rev. 45, 437-461 (1981). MENDEL, R. R., and MULLER, A. J.: Reconstitution of NADH-nitrate reductase in vitro from nitrate reductase-deficient Nicotiana tabacum mutants. Mol. Gen. Genet. 161, 77-80 (1978). MENDEL, R. R., and MULLER, A. J.: Nitrate reductase-deficient mutant cell lines of Nicotiana tabacum. Further biochemical characterization. Mol. Gen. Genet. 177, 145-153 (1979). MENDEL, R. R., and MULLER, A. J.: Comparative characterization of nitrate reductase from wild-type and molybdenum cofactor-defective cell cultures of Nicotiana tabacum. Plant Sci. Lett. 18, 277-288 (1980). MENDEL, R. R., ALIKULOV, Z. A., Lvov, N. P., and MULLER, A. J.: Presence of the molybdenum cofactor in nitrate reductase-deficient mutant cell lines of Nicotiana tabacum. Mol. Gen. Genet. 181, 395-399 (1981). MULLER, A. J.: Genetic analysis of nitrate reductase-defieient tobacco plants regenerated from mutant cells. Evidence for duplicate structural genes. Mol. Gen. Genet. 192, 275-281 (1983). MULLER, A. J., and GRAFE, R.: Isolation and characterization of cell lines of Nicotiana tabacum lacking nitrate reductase. Mol. Gen. Genet. HI1, 67-76 (1978). MULLER, A. J., and MENDEL, R. R.: Expression of different niaA alleles in cultured cells and whole plants of Nicotiana tabacum. Abstr. Int. Symp. on Nitrate Assimilation - Molecular and Genetic Aspects. Gatersleben, GDR, P10 (1982). NICHOLAS, D. J. D., and NASON, A.: Determination of nitrate and nitrite. In: Methods of Enzymology, Vol. III (S. P. COLOWICK and N. O. KAPLAN, eds.), p.983. Academic Press, New York 1957. SAALBACH, I.: Dissertation, Akademie der Wissenschaften, Berlin, DDR, 1980. SCHIEMANN, J., and MANTEUFFEL, R.: Antibodies against tobacco nitrate reductase. Abstr. Int. Symp. on Nitrate assimilation - Molecular and Genetic Aspects. Gatersleben, GDR, P17 (1982).

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SMARRELLI, J., and CAMPBELL, W. H.: Immunological approach to structural comparisons of assimilatory nitrate reductases. Plant PhysioI. 68, 1226-1230 (1981). SOMERS, D. A., Kuo, T., KLEINHOFS, A., and WARNER, R. L.: Nitrate reductase-deficient mutants in barley. Immunoelectrophoretic characterization. Plant PhysioI. 71, 145-149 (1983 a). SOMERS, D. A., Kuo, T., KLEINHOFS, A., WARNER, R. L., and OAKS, A.: Synthesis and degradation of barley nitrate reductase. Plant Physiol. 72, 949-952 (1983 b). SRIVASTAVA, H. S.: Regulation of nitrate reductase activity in higher plants. Phytochem. 19, 725733 (1980). STOUTHAMER, A. H.: Biochemistry and genetics of nitrate reductase in bacteria. In: Advances in Microbiology (A. H. ROSE and D. W. TEMPEST, eds.) vol. 14, p.315. Academic Press, London 1976. ULITZSCH, M., and SCHIEMANN, J.: Induction of NADH-dependent nitrate reductase activity in cucumber cotyledons by de novo synthesis of the enzyme. Biochem. Physiol. Pflanzen 179, 115-121 (1984). WELANDER, M.: The effect of mercaptoethanol on the activity of enzymes of nitrogen metabolism in leaves from Urtica dioica and Spinacia oleracea. PhysioI. Plant. 43, 242-246 (1978). ZIELKE, H. R., and FILNER, P.: Synthesis and turnover of nitrate reductase induced by nitrate in cultured tobacco cells. J. BioI. Chern. 246, 1772-1779 (1971).

Received June 28, 1984; accepted August 1, 1984 Authors' address: Dr. J. SCHIEMANN, Dr. habil. A. J. MULLER, Zentralinstitut fiir Genetik und Kulturpflanzenforschung, Akademie der Wissenschaften der DDR, DDR - 4325 Gatersleben.