Co-regulation of nitrate reductase and nitrite reductase in cultured spinach cells

• JOURNAL OF • PLANT PHYSIOLOGY

J. Plant Physiol. 157.299-306 (2000) © Urban & Fischer Verlag hl1p:/Iwww.urbanfischer.de/journals/jpp

Co-regulation of nitrate reductase and nitrite reductase in cultured spinach cells Kyoko Ogawa 1 *, Rie Soutome" Keiko Hiroyama" Takashi Hagio 2, Shoji Ida 3, Hiroki Nakagawa 4, Atsushi Komamine s

1 Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, Mejirodai, Bunkyo-ku, Tokyo

112-8681, Japan

2 Cell Manipulation Laboratory, Department of Cell Biology, National Institute of Agrobiological Resources, Ministry of Agriculture, Forestry and

Fisheries, Kannondai, Tsukuba, Ibaraki, 305-8602 Japan 3 Research Institute for Food Science, Kyoto University, Uji, Kyoto, 4

611-0011 Japan

Department of Bioproduction Science, Faculty of Horticulture, Chiba University, Matsudo, Chiba, 271-0092 Japan

5 The Research Institute of Evolutionary Biology, Kamiyoga, Setagaya-ku,Tokyo,

158-0098 Japan

Received June 11, 1999 . Accepted May 20, 2000

Summary The analysis of nitrate reductase(NR)-deficient mutants provides an efficient approach to the study of the regulatory mechanisms of nitrate assimilation. We previously isolated two cell lines , 12F and 1-1 , and suggested that the mutation in the 12F cell line related to translation of NR mRNA which is expressed in the presence of nitrate, and that the mutation in the 1-1 cell line may be in a regulatory gene controlling both genes encoding NR and nitrite reductase (NiR). We investigated transform ants of the two cell lines using particle bombardment with tobacco NR cDNA and analyzed the expression of NR and NiR in the transformants. In the 12F cell line transformants, NR activity and protein was rescued completely and the transformants could grow on N0 3 - medium. This result indicates that the 12F cell line has a mutation that prevents the synthesis of NR protein from NR mRNA. In the 1-1 cell line, the activities of NR and NiR were detected in cells grown on N03 - medium , but at low levels. In the transformants however, activities of NR and NiR and the NR mRNA levels attained levels of observed in the wild-type cells. In addition, the transformants could also grow on N03 medium. These results indicate that the 1-1 cell line has a mutation related to the signal transfer cascade from the N03 - ion to the NR gene . This suggests that NR and NiR genes are co-regulated , and that the presence of NR mRNA may be post-transcriptionally essential for the synthesis of NiR protein .

Key words: Spinacia oleracea - bombardment - nitrate assimilation - nitrate reductase - nitrite reductase

Abbreviations: NR nitrate reductase - NiR nitrite reductase - SOS-PAGE SOS-polyacrylamide gel electrophoresis • E-mail correspondingauthor: [email protected]

0176-1617/00/157/03-299 $15.00/0

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Kyoko Ogawa et al.

Introduction The pathways of nitrate assimilation in higher plants involve two enzymes, nitrate reductase (NR, EC 1.6.6.1),which reduces nitrate to nitrite, and nitrite reductase (NiR, EC 1.7.7.1), which reduces nitrite to ammonium ions. Nitrate assimilation is a highly regulated process (Crawford 1995). Nitrate reduction has long been shown to be regulated by many environmental factors, including the nitrogen source, light, 02/C02, pH, and temperature, as well as endogenous factors, such as metabolites and plant growth regulators (Guerrero et al. 1981). Nitrate reductase is usually considered to be the ratelimiting enzyme in the nitrate assimilation pathway because of its high turnover rate compared with other enzymes in the pathway. It is also thought to playa central role in regulation, a common function of the first enzyme in a pathway (Beevers and Hageman 1969). It has been established that both NR and NiR activities are induced by the presence of nitrate in plants (Guerrero et al. 1981). The isolation of genomic or cDNA clones of NR and NiR has enabled molecular analysis of the regulation of this major metabolic pathway (Rouze and Caboche 1992). Nitrate supply leads to an increase in the steady-state level of mRNA encoding NR in a variety of plant species and tissues (Cheng et al. 1986, Crawford et al. 1988, Gowri and Campbell 1989, Hoff et al. 1991). Nuclear run-off transcription assays (Callaci and Smarrelli 1991) and the study of transgenic plants (Vincentz and Caboche 1991) have provided direct evidence that the regulation of NR expression by nitrate occurs at the transcriptional level. Similarly, it has been demonstrated that the level of NiR mRNA rapidly increases following supply of nitrate in several species (Back et al. 1988, Kramer et al. 1989, Faure et al. 1991). The 5'-promoter sequence of the spinach NiR gene has been shown to confer nitrate-inducibility to a GUS reporter gene (Back et al. 1991), demonstrating with certainty that nitrate controls the transcription of the NiR gene. Faure et al. (1991) reported that nitrate co-regulates the expression levels of NR and NiR transcripts in plants, as in fungi, where NR and NiR genes are controlled by the same positive regulators encoded by nirA and areA in Aspergillus and nit-4 and nit-2 in Neurospora (Scazzocchio and Arst 1989). These regulatory genes were first identified by mutant analysis and have been cloned. Genetic analysis of nitrate assimilation is possible because NR-deficient mutants blocked in the pathway can be rescued by providing ammonium or amino acids as a source of nitrogen. The analysis of NR-deficient mutants provides an effective approach to the study of the regulatory mechanisms of nitrate assimilation (Hoff et al. 1994). Mutants affected in the nitrate assimilation pathway have been isolated in many different plant species (Caboche and Rouze 1990). The NR or NiR activities of most of these mutants have been affected. Unfortunately, until now no regulatory mutants have been obtained in higher plants, and no regulatory genes have been cloned (Hoff et al. 1994).

We previously described the characterization of two NR-deficient mutants, the 12F and 1-1 cell lines, obtained from cultured spinach cells (Ogawa et al. 1994). We suggested that the 12F cell line is a structural gene mutant, a translational mutant of the NR mRNA, and that the 1-1 cell line is a putative regulatory mutant with extremely low levels of production of nitrate-inducible NR and NiR mRNAs. The purpose of this study was to investigate the regulation of the expression of NR and NiR by nitrate in cultured spinach cells. We examined the transformants of NR-deficient mutants, designated the 12F (NR-, NiR+) and the 1-1 (NR-, NiR-) cell lines by particle bombardment with tobacco NR cDNA, and analyzed the expression of NR and NiR in the transformants. Our results support the theory that nitrate coregulates NR and NiR expression. They further suggest that expression of NiR mRNA is regulated by nitrate and that the presence of NR mRNA may be post-transcriptionally essential for the synthesis of NiR protein.

Materials and Methods Plant materials and culture conditions The strain of the wild-type cells in the study used was established in 1980 from the central parts of the hypocotyl-plumule of Spinacia oleracea (cv. Hoyo; Nakagawa et al. 1985 a). These spinach cells (wildtype cells) were grown on Murashige and Skoog's medium (MS medium; Murashige and Skoog 1962) that contained 0.5 mg l -1 naphthaleneacetic acid and 1 mg l -1 6-benzyladenine, as described previously (Nakagawa et al. 1985 a). Two NR-deficient mutants (12F and 1-1 cell lines) were isolated, as described previously (Ogawa et al. 1994). The mutant cells were grown on nitrate-free MS medium, which was prepared by substituting l-glutamine (1 g l -1) and casein hydrolysate (NZ amine type A, Wako, Tokyo) (1 g l -1), plus 20 mmol· l -1 KGI for KN0 3 and NH 4 N03 as the source of nitrogen (without sodium chlorate). Each cell was transferred into fresh medium at eight-day intervals. For particle bombardment, cells were collected by centrifugation. and 0.5 ml of packed cells were spread evenly over the surface of several layers of sterile filter paper on solidified nitrate-free MS medium (0.7 % agar) in a 9 cm diameter Petri dish. This Petri dish was then used for bombardment.

Plasmid DNA and particle bombardment For particle bombardment, the plasm ids pBI221 and pRTNR were used. Plasmid pBI221 has the ~-glucuronidase gene (gus) under the control of the cauliflower mosaic virus (GaMV) 35S promoter. Plasmid pRTNR, kindly provided by Dr. M. Gaboche (INRA, France), has tobacco NRcDNA under the control of the GaMV 35S promoter (Vincentz and Gaboche 1991). The plasmids were purified using a QIAGEN Plasmid Kit (QIAGEN, Hilden, Germany), according to the instruction manual. Plasmid DNA was adhered gold particles (1.0Ilm) as described in the Bio-rad protocol (Munchen, Germany). DNA coating of microparticles was achieved by mixing 50 III of the microparticle suspension (3 mg gold particles in 50 % glycerol), 5 III of plasmid DNA solution

Co-regulation of nitrate reductase and nitrite reductase (11l9Ill-1), 50 III of 2.5mol·l-1 CaCI 2 , and 20 III of 0.1 mol· l-1 spermidine. After spinning, the pellet containing the particles was rinsed with 70 % ethanol, followed with absolute ethanol. and then resuspended in 50 III of absolute ethanol. To load the macrocarriers, 61ll of the coated microparticles were pipetled into the center of a macrocarrier and allowed to dry. A Biolistic PDS-1000/He device (Bio-rad) was employed in these experiments. The distance from the stopping plate to the target cell was approximately 12 cm, and the rupture disc strength was 900 psi. Bombardment was performed only once per Petri dish.

Culture and selection conditions After bombardment, each cell was precultured for 1 week on the solidified nitrate-free MS medium mentioned above . The then cells were transferred to solidified MS medium (nitrate-containing medium). Selection continued on the nitrate-containing medium. After approximately 1 month, the calli were transferred to fresh solidified MS medium, and finally cultured on suspension medium.

DNA isolation and Southern blot analysis Total genomic DNA was isolated from the putative transformants, nontransformants, and Wild-type cells using the DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). Selected cells were initially analyzed by PCR. The primer pairs were designed to amplify an internal 378 bp CaMV 35S promoter fragment; 35S-3: 5'-CAT GCC TGC AGG TCA ACA TG and 35S-4: 5'-CGA GGT CCT CTC CM ATG AA. The PCR conditions applied were 94°C for 5 min, followed by 30 cycles at 94 'c for 1 min , 56 'c for 1 min, 72 °C for 1 min , with a final extension at 72 'c for 5 min using the Perkin-Elmer GeneAmp PCR system 2400 (Norwalk, California, USA). The PCR products were separated on a 1 %agarose gel and transferred to Hybond-N' membranes (Amersham, Buckinghamshire, England). The membranes were hybridized with the Hindlll-8amHI fragment (CaMV 35S promoter) of pB1221.

301

machi et al. 1987), and NiR (Ida 1987) and goat anti-rabbit IgG linked to horseradish peroxidase (E-Y lab., San Mateo, California, USA) as described previously (Maki et al. 1986).

RNA isolation and Northern blot analysis Total RNA was extracted from 1.5 g (fresh weight) of the transformants, nontransformants, and wild-type cells ground in liquid nitrogen using the method of Sam brook et al. (1989). For each sample, 20119 of denatured total RNA was fractionated on 1.0 % agarose gels containing formaldehyde, and transferred to Hybond-N+ membranes (Amersham, Buckinghamshire, England) by Northern blotting. The quantity of RNA loaded onto the gel was routinely checked by ethidium bromide staining of the gel prior to the transfer. The membranes were hybridized with a 32P-labelled DNA probe at 42 'c in 50 % formamide. The probes used were a 3.2 kbp EcoRI cDNA fragment (pIMP4) of spinach NR (Prosser and Lazarus 1990) and a 2.0 kbp EcoRI cDNA fragment (pCIB400) of spinach NiR (Back et al. 1988).

Transient assay for GUS gene After bombardment with pBI221 , each cell was incubated on solidified nitrate-free medium for 2d. Transient expression of the GUS gene was assayed according to the method reported by Klein et al. (1988); the filter paper was transferred to a sterilized plastiC Petri dish containing a substrate mixture for GUS expression. The substrate mixture consisted of 1.9 mmol· L-1 5-bromo-4-chloro-3-indolyl glucuronide (Xgluc; Wako, Tokyo, Japan), 0.5 mmol . L- 1 potassium ferricyanide, 0.5mmol· L-1 potaSSium ferrocyanide, 0.3 % Triton X-100, 20% methanOl , and 100 mmol' L -1 phosphate buffer (pH 7.2). After incubation at 37 °C for 24 h, GUS-expressing cells were detected as blue-coloured spots.

Results Transient assay for GUS gene

Assays of NR and NiR Protein extraction from the transformants, nontransformants, and wildtype cells was performed as described previously (Nakagawa et al. 1985 b). NADH-NR activity was assayed as described previously (Nakagawa et al. 1985 b). One unit of NR activity was defined as the amount required to produce 1 nmol of nitrite per min. NiR activity was assayed in the same extract using methyl viologen reduced by dithionite as the electron donor (Ida and Morita 1973). One unit of NiR activity was defined as the amount required to reduce 1 nmol of nitrite per min . All assays were carried out in triplicate.

The effect of the preculture period on the efficiency of gene delivery and expression was first studied using the GUS gene (pBI221). Each cell was transferred to fresh nitrate-free MS medium and cultured for 1-5 d in a suspension culture. Thereafter, the wild-type cells, the 1-1 cell line, and the 12F cell line were bombarded and assayed for GUS activity. The highest number of blue spots per Petri dish observed was 470 for the wild-type cells at 4 d, 250 for the 1-1 cell line at 3 d, 70 for the 12F cell line at 2 d . However, significant differences in the results of the transient GUS assay were not observed for preculture periods of 1-5 d (data not shown).

Western blot analysis SDS-PAGE was performed according to the procedure of Laemmli (1970) using a 7 .5 %acrylamide gel. Fresh cells were extracted using an extraction buffer as described previously (Nakagawa et al. 1985b), and the extracts were loaded onto the gels. The gel was then transferred to nitrocellulose, as described by Towbin et al. (1979). Immunodetection of the proteins transferred to nitrocellulose was performed using polyclonal antibodies raised against spinach NR (Ka-

Establishment of stable transformants To obtain stable transformants, pRTNR (35S-NR cDNA) was used for bombardment under the same conditions as those standardized for transient gene expression. In 10 transformations (2-4 d preculture periods), a total of 50 Petri dishes per cell line was bombarded. Selection was performed by pick-

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Kyoko Ogawa et al.

Figure 1. Selection and establishment of stable transformants from the NR-deficient mutants after particle bombardment. After preculture for 1 week on solidified nitrate-free MS medium, selection continued for approximately 1 month on solidified MS medium (nitrate-containing medium). The calli were transferred to fresh solidified MS medium and finally cultured on suspension medium. (A) Left, 1 month after the beginning of selection (12F cell line). Right, 1 month after transfer to fresh medium (12F cell line). (B) transformants of the 12F cell line. (e) transform ants of the 1-1 cell line. These cells and the wild-type cells could grow on MS medium, however, the NR-deficient mutants could not. (D) the 12F and 1-1 cell lines on nitrate-free MS medium. WT, the wild-type cells; 12F, the 12F cell line; 12F-T, transformants of the 12F cell line; 1-1, the 1-1 cell line; 1-1-T, transformants of the 1-1 cell line.

ing up calli that had grown on the medium containing nitrate. After bombardment under the conditions described in Materials and Methods, cells were precultured for 1 week on solidified nitrate-free MS medium, and then transferred to solidified MS medium (nitrate-containing medium). As shown in Fig. 1 A-left, calli grown on solidified MS medium were observed after 1 month, and then transferred to fresh solidified MS medium (Fig. 1 A-right). From the 20 calli of the 12F cell line and 35 calli of the 1-1 cell line that were first transferred to fresh solidified MS medium, 10 and 24, respectively, showed the ability to grow on medium containing nitrate after transfer, a result which was repeated four times. Although the 12F and 1-1 cell lines of NR-deficient mutants usually grew on nitrate-free medium (Fig. 1 D), so that could not grow on MS medium, their putative transformants, with introduced NR cDNA, could, like the wild-type cells (Fig. 1 B and e), grow on MS medium. Among the cell lines, six showed good growth in MS liquid medium (the 12F cell line, 6-3.4, 6-3.5 and 6-3.7; the 1-1 cell line, 9-1.3, 9-3.8 and 9-3.12). Figure 2 shows the results of Southern blot analysis of DNA obtained following peR amplification from the six putative transform ants (lanes 4, 5, 6, 8, 9 and 10), nontransformants (lanes 3 and 7), and wild-type cells (lane 2) hybridized with the 378 bp Hindlll-BamHI fragment containing the eaMV 35S promoter of plasmid pB1221. Hybridization bands were observed for all putative transformants. In contrast, no hybridization bands were detected for DNA from the nontransformants and the wild-type cells used as controls. This result confirmed the transfer of NR cDNA to the genomic DNA of the putative transformants obtained by bombardment with plasmid pRTNR. We used these six cell lines for the experiments described here.

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Figure2. Southern blot analysis of the putative transformants. Geno-

mic DNA was isolated from each of the transformants obtained, nontransformants and wild-type cells. peR and Southern blot analysis were performed as described in Materials and Methods. The arrow shows the 378 bp Hindlll-BamHI fragment containing the eaMV 35S promoter of plasmid pB1221. Lane 1, plasmid DNA from pRTNR; lane 2, the wild-type cells; lane 3, the 12F cell line; lanes 4 (6-3.4), 5 (63.5) and 6 (6-3.7), transform ants of the 12F cell line; lane 7, the 1-1 cell line; lanes 8 (9-1.3), 9 (9-3.8) and 10 (9-3.12), transformants of the 1-1 cell line.

Analysis of transformants The transformants obtained and the nontransformants were transferred to fresh MS medium, and the NR and NiR activities were assayed after 3 d of culture; the NR and NiR activities in the Wild-type cells in MS medium increased markedly during this period (Ogawa et al. 1994). As shown in Figure 3, in the case of the nontransformants of the 12F cell line grown in MS medium, no NR activity or protein expression was observed, but the levels of NR activity and protein expression in the transformants were rescued completely to the levels observed in the wild-type cells. The nontransformants of the 12F cell line and the wild-type cells grown in MS medium possessed the same levels of NiR activity, but the level of NiR activity observed in the transformants was the same or higher than that in the nontransformants. In the nontransformants of the 12F cell line, the mRNA encoding NR and NiR was clearly

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Figure3. Comparison of the levels of NR and NiR activities in the transformants, nontransformants and wild·type cells. The top of the figure shows the NR and NiR activities of the transformants obtained , nontransformants and wild-type cells in MS medium (containing 20 mmol . L-1 KN03 ) . The NR and NiR activities were assayed 3d after induction with 20mmol· L -1 KN0 3 . Error bars denote the standard deviation of the meas· urement of three independently processed samples. The bottom of the figure shows immunoblots of NR and NiR protein from the cells as listed in the top of the figure. 12F cells, the NR·deficient 12F cell line; 1-1 cells, the NR-deficient 1- 1 cell line; control, nontransformants; 6-3.4, 6-3.5 and 6-3.7, transformants of the 12F cell line; 9-1.3, 9-3.8 and 9-3.12, transform ants of the 1-1 cell line.

Discussion

expressed , and the expression level was almost the same as that in the wild-type cells (Fig . 4) as determined by Northern blot analysis and previously described (Ogawa et al. 1994). The NR mRNA was overexpressed in the transformants as compared with that in the nontransformants , and the NiR mRNA level in the transformants was observed to be the same as or higher than that in the nontransformants (Fig. 4). In contrast, in the case of the nontransformants of the 1-1 cell line grown in MS medium , the observed levels of NR and NiR activities and proteins were low (Fig. 3). In the transformants of the 1-1 cell line, the NR activity and protein levels were rescued completely to the levels observed in the wildtype cells. The NiR activity and protein levels in the transformants of the 1-1 cell line were also rescued in the same

the sole nitrogen source (MS medium) (Ogawa et al. 1994). Two cell lines were isolated , 1-1 and 12F, which exhibited little or no NR activity, respectively, on N03 - -containing medium. It has been suggested that the mutation in the 12F cell line is related to translation of NR mRNA expressed in the presence of nitrate, and that the mutation in the 1-1 cell line may be in a regulatory gene involved in the regulation of genes encoding NR and NiR. To study the regulation of NR and NiR expression , and particularly to identify whether the 1-1 cell line

manner as the NR activity and protein levels. In the case of

is a regulatory gene mutant, we investigated the transform-

the accumulation of NR mRNA, the nontransformants were distinct from 12F cells and the levels were extremely low in the 1-1 cell line, as previously described (Fig . 4; Ogawa et al.

bacco NR cDNA Particle bombardment is an efficient technique for the

1994). However, the NR mRNA levels of the transformants were rescued to the levels in the wild-type cells or overexpressed as compared to that in the wild-type cells. The NiR mRNA level detected was the same as that in the nontransformants (Fig . 4) .

We previously reported that NR-deficient mutants derived from cultured spinach cells were selected by their chlorate resistance and lack of growth on media containing nitrate as

ants of the two cell lines using particle bombardment with to-

transfer of DNA into plant cells (Gray and Finer 1993, Morikawa et al. 1994). First, we investigated the effect of the preculture period on the efficiency of gene delivery and expression using a transient assay for GUS gene activity. No significant influence of the preculture period was observed

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Kyoko Ogawa et al.

Table 1. Activity levels and Northern blot analysis. This table summarizes Figs. 3 and 4. Each cell line was assayed for NR and NiR activities at 3 d after transfer to medium containing nitrate. Total RNA was extracted and Northern blotting performed the same day.

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Figure4. Northern blot analysis of NR and NiR mRNAs from the transformants. Total RNA was isolated 3d after induction with 20mmol· L-1 KN0 3 . Twenty ~g of total RNA was fractionated by agarose gel electrophoresis in the presence of formaldehyde, and ethidium bromide staining of rRNA was used to confirm equivalent loading (lower panel). Northern blot analysis was performed as described in Materials and Methods.

(data not shown). Moreover, compared with the wild-type cells, a low transient expression was obtained for the mutants, especially for the 12F cell line. These different responses towards bombardment could be explained by a variety of factors, for example, various cell physiological factors - differences in cell cycle stages (Iida et al. 1991), and unsuitability of culture conditions (Kodama et al. 1993), in addition to physical factors (Klein et al. 1988). In order to establish stable transformants, we performed bombardment of the two mutants on day 2, 3 or 4 of the preculture period under the same conditions. As the plasmid used for particle bombardment contained tobacco NR cDNA under the control of the CaMV 35S promoter without the ~-glucuronidase gene (gus) or the nptll gene, we utilized nitrate for the selection of transformants. Finally, from their ability to grow on medium containing nitrate (Fig. 1), and from the results of Southern blot analysis (Fig. 2), we obtained six transformant cell lines. The 12F cell lines were designated 6-3.4, 6-3.5, and 6-3.7, and the 1-1 cell lines were designated 9-1.3, 9-3.8, and 9-3. 12. Table 1 summarizes the results on the levels of NR and NiR activities (Fig. 3) and those of Northern blot analysis (Fig. 4). The finding that the 35S-NR cDNA (pRTNR) rescues both mutants is significant, and demonstrates that active NR protein (and not MoCo or something else) is missing from these cells. In the transformants of the 12F cell line grown on medium containing nitrate, NR mRNA could produce normal levels of NR activity and protein. This result strongly indicates

+, Induction by nitrate. ±, Dedected activity but no induction by nitrate.

that the 12F cell line has a mutation that either prevents NR protein synthesis (e.g. a frameshift) or makes NR so unstable that it does not accumulate (Fig. 5). In contrast, when the 1-1 cell line was transferred to medium containing nitrate from nitrate-free MS medium with 25 mmol . L -1 sodium chlorate, the activities of NR and NiR and expressions of mRNAs of NR and NiR were observed at extremely low levels, as previously described (Ogawa et al. 1994). However, in this experiment, although NiR mRNA was expressed in the nontransformants of the 1-1 cell line, NiR activity was Observed only at low levels and was not nitrate-inducible (Figs. 3 and 4). This difference was because the 1-1 cell line had been grown on nitratefree MS medium without sodium chlorate. We do not yet know how chlorate is involved. However, after the introduction of NR cDNA, NiR activity was rescued in the same way as NR activity. The 1-1 cell line could have cis-acting mutations (a deletion in NR or a mutation that blocks transcription or makes NR mRNA unstable) or trans-acting regulatory mutations. We have also observed that between the wild-type cells and the 1-1 cell line, there was no difference in the nucleotide sequence of the 1.6 kbp 5'-untranslated region (the promoter region) of the NR gene (Takahashi, unpublished results). This suggests that the 1-1 cell line could not have cis-acting mutations, and that the 1-1 cell line has mutation in trans-acting factors (data not shown) from the N03 - ion to the NR gene. It is possible that the 1-1 cell line is a regulatory mutant. In fungi, some NR-deficient mutants showed. constitutive expression of NR protein and NiR activity in the absence of nitrate. This observation has led to the autogenous regulation hypothesis (Cove and Pateman 1969), which suggests that NR interacts with the positive regulator protein nirA/nit-4 when nitrate is absent, thus suppressing the activation of the promoter (Marzluf 1981). In contrast, Deng et al. (1989) reported that in Nicotiana plumbaginifolia, nitrate induction of the transcription of the NR and NiR genes relies neither on NR activity nor on the NR protein since expression of mRNA for NiR, as well as NR, is still induced by nitrate in NR-deficient mutants, even in those in which no NR protein is de-

Co-regulation of nitrate reductase and nitrite reductase

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tected (Pouteau et al. 1989). Moreover, our results also indi-

Acknowledgements. The authors thank Professor Emeritus A. Oaks,

cate that expression of both NR mRNA and NiR activity by ni-

University of Guelph, Canada, for her helpful comments and critical reading of the manuscript. We would like to thank Dr. M. Caboche, INRA, France, for his kind gift of the tobacco NR cDNA construct. Thanks also to Y. Yanagawa, Chiba University, for RI work, and Dr. K. Sueyoshi, Niigata University, for his kind gift of antisera against NR. This work was supported in part by a grant from the «Research for the Future» (JSPS-RFTF96L00604) program of the Japan Society for the Promotion of Science.

trate was observed in the 12F cell line, regardless of the presence of the NR protein. Thus, although NR autoregulation is a valid model in fungi, a similar model is unlikely to apply to higher plants. Faure et al. (1991) suggested that nitrate co-regulated the levels of NR and NiR transcripts in plants. Co-regulation of NR and NiR activities may be required to prevent the deleterious accumulation of nitrite under conditions in which nitrate is assimilated. Our results support the suggestion of Faure et al. (1991) that concerted metabolic regulation of NR and NiR would prevent the accumulation of toxic metabolic intermediates and save energy for plant growth. Crete et al. (1997) reported that NiR was post-transcriptionally regulated by nitrate in Nicotiana plumbaginifolia and Arabidopsis thaliana so that NiR mRNA, which is less abundant and present only at low levels in the absence of nitrate, could be rapidly translated upon nitrate availability. In our experiment, the 12F cell line showed expression of NR and NiR mRNAs by nitrate and normal NiR activity; moreover, the 1-1 cell line showed expression of NiR mRNA by nitrate but no NiR activity when NR mRNA could not be detected. Introduction of NR cDNA into the 1-1 cell line rescued not only NR activity and mRNA expression but also the activity of NiR. Consequently, it is concluded that expression of NiR mRNA is regulated by nitrate, and that the presence of NR mRNA may be post-transcriptionally essential for the synthesis of the NiR protein. We suggest the possibility of a signal from nitrate being conveyed to NiR mRNA, as shown in Figure5. Therefore, analysis of these mutants contributes to the understanding of the complex regulation of nitrate assimilation. We are currently trying to identify the region of the coding sequence necessary for this regulation. Furthermore, it would be necessary to clarify the sequence of the NR alleles in these mutant cell lines. It would be of great interest to clarify these issues.

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