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Shuichi Akaba, Marie-Therese Leydecker, Therese Moureaux, Takayuki Oritani and Tomokazu Koshiba 4 1 Department of Biology, Tokyo Metropolitan University, Hachioji-shi, Tokyo, 192-0397 Japan 2 Laboratoire de Biologie Cellulaire, Institut National de la Recherche Agronomique, Route de Saint-Cyr, F-78026, Versailles Cedex, France 3 Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, Aoba-ku, Sendai, 981 Japan

Cytokinin (CK) and low-intensity light effects in modulating nitrate reductase (NR) activity, NR protein and NR encoding mRNAs were studied in tobacco cell suspension cultures. NR activity was strikingly enhanced by CK in dark- as well as in light-grown cells whereas it was less affected by light alone. NR protein accumulation was stimulated by the hormone in the light only; then a CK light-dependent regulation of NR activity was suggested. Light enhanced the steady-state levels of hybridisable total NR mRNA and a light-inductive effect was also observed after transfer from dark to light; this effect was dependent on sucrose supply and was enhanced in CK-supplied cells. NR poly(A) mRNA were assayed in cell poly(A) RNA, purified by oligo(dT)-cellulose chromatography. In growing cells CK enhanced (i) the steady state levels of hybridisable NR poly(A) mRNA and (ii) the ratio of hybridisable NR poly(A) mRNA to total hybridisable NR mRNA. CK-induced accumulation of hybridisable NR poly(A) mRNA resulted in a correlative enhancement of the accumulation of NR protein, provided cells were grown in the light. Overall stimulatory effects of CK on the amounts of bulk poly(A) mRNA and on the mean-size of mRNA poly(A) tails were also observed. It is suggested that CK effect on gene expression involves a modulation of mRNA polyadenylation.

An antisense nitrite reductase (NiR, EC 1.7.7.1) tobacco ( Nicotiana tabacum L.) transformant (clone 271) was used to gain insight into a possible correlation between nitrate reductase (NR, EC 1.6.6.1)-dependent nitrite accumulation and nitric oxide (NO(.)) production, and to assess the regulation of signal transduction in response to stress conditions. Nitrite concentrations of clone 271 leaves were 10-fold, and NO(.) emission rates were 100-fold higher than in wild type leaves. Increased protein tyrosine nitration in clone 271 suggests that high NO(.) production resulted in increased peroxynitrite (ONOO(-)) formation. Tyrosine nitration was also observed in vitro by adding peroxynitrite to leaf extracts. As in mammalian cells, NO(.) and derivatives also increased synthesis of proteins like 14-3-3 and cyclophilins, which are both involved in regulation of activity and stability of enzymes.

The molybdenum cofactor is shared by nitrate reductase (NR), xanthine dehydrogenase (XDH), and abscisic acid (ABA) aldehyde oxidase in higher plants (M. Walker-Simmons, D.A. Kudrna, R.L. Warner [1989] Plant Physiol 90:728-733). In agreement with this, cnx mutants are simultaneously deficient for these three enzyme activities and have physiological characteristics of ABA-deficient plants. In this report we show that aba1 mutants, initially characterized as ABA-deficient mutants, are impaired in both ABA aldehyde oxidase and XDH activity but overexpress NR. These characteristics suggest that aba1 is in fact involved in the last step of molybdenum cofactor biosynthesis specific to XDH and ABA aldehyde oxidase; aba1 probably has the same function as hxB in Aspergillus. The significance of NR overexpression in aba1 mutants is discussed.

The nitrate assimilatory pathway has been the matter of intensive molecular and cellular analysis in solanaceous species. In this report we will present data on the function of the N-terminal part of the enzyme nitrate reductase, on the cloning of genes of the molybdenum cofactor biosythesis, and on the characterization of a transcription factor presumed to be involved in the expression of genes of the nitrate assimilatory pathway.

Nitrate (NR) and nitrite reductase (NiR) catalyse the reduction of nitrate to ammonium. The regulation of NR and NiR gene expression by carbohydrates (C) and nitrogen (N) metabolites was studied using detached leaves. In the dark, glucose fructose and sucrose supplied to detached green leaves of dark-adapted Nicotiana plumbaginifolia plants resulted in NR mRNA and protein accumulation and the loss of circadian rhythmicity in the size of the transcript pool. The characterization of transgenic plants expressing either a NR cDNA controlled by the 35S CaMV promoter or a transcriptional fusion between the tobacco nia1 (NR structural gene) promoter and the beta-glucuronidase reporter gene, led us to conclude that C metabolite control is taking place at the transcriptional level. Under low light conditions (limiting photosynthetic conditions), the supply of glutamine or glutamate resulted in a drop in the level of NR mRNA. Exogenously supplied carbohydrates partially antagonized this inhibitory effect suggesting that the availability of N and C metabolites affects the expression of the NR gene. The effects of carbohydrates and glutamine on NiR expression were also studied. NiR mRNA levels in the dark were relatively insensitive to feeding with glucose. Glutamate and glutamine were less efficient at decreasing NiR mRNA than NR mRNA levels. In contrast to NR, NiR mRNA levels were significantly increased by light treatments, indicating that NiR display regulatory characteristics reminiscent of photosynthetic genes such as the small subunit of ribulose bisphosphate carboxylase than to NR.

The nitrate assimilation pathway is known to be tightly controlled in plants by many endogenous and environmental factors (Beevers and Hageman 1983), the most important being the nature and availability of the nitrogen source, and light. Nitrate compartmentalization and flux between plant tissues and inside plant cells are clearly the first critical steps in that control, but are, as yet, refractory to molecular analysis. The second step, nitrate reduction into ammonium, is catalysed by two enzymes, a likely cytosolic nitrate reductase (NR), and a plastidial nitrite reductase, both induced by nitrate. Interestingly, each factor known to control the overall pathway appears to control NR, which is thus thought to play a central role in regulation of the nitrate assimilation pathway.

Protein breakdown during germination of maize at 28° is closely correlated with the appearance of protease activity. In the first 2 days of germination, a slight disaggregation of only G 3 glutelins into more simple elements (albumin-globulins) can be observed. Between 2 and 2.5 days, there is extensive breakdown of all protein fractions, the rate of which coincides with the rate of appearance of proteolytic activity. After 2.5 days these phenomena slow down and the bulk of the endosperm proteins disappears. Three acid proteases in endosperm extracts of germinated grain (P 11 , P 21 and P 22 ) have been isolated by affinity chromatography and gel filtration, and partially characterized. P 11 (MW 40 000) which is present in the ungerminated grain, cannot hydrolyse prolamins and is insensitive to reducing agents. P 21 (MW 36 000) and P 22 (MW 12 000), which appear on day 3 of germination, can degrade prolamins in vitro . Reducing agents enhance their activity and prevent their aggregation or denaturation. Comparative assays with different substrates suggest our enzyme preparations are principally endotype proteases with little contaminating carboxypeptidase activity.

Quantitative and qualitative (amino acid composition) changes of albumins and globulins in developing grain of normal and opaque-2 (o2) maizes were examined in proteins extracted sequentially with water and 0.5 M naCl, and isolated by salting out with trichloroacetic acid. The amount of albumins per grain reached a maximum at mid-development then declined. Globulins, virtually absent in very young grain, increased in level until maturity. The variations were more marked for o2 than normal maize. Amino acid compositions changed little with development. The selectivity and exhaustiveness of sequential extraction, and the physiological role of albumins and globulins in grain are discussed.

Nitrate reductase was purified from leaves of Nicotiana plumbaginifolia using either 5'AMP-Sepharose chromatography or two steps of immunoaffinity chromatography involving monoclonal antibodies directed against nitrate reductase from maize and against ribulose-1,5-bisphosphate carboxylase from N. plumbaginifolia. Nitrate reductase obtained by the first method was purified 1000-fold to a specific activity of 9 units/mg protein. The second method produced an homogenous enzyme, purified 21,000-fold to a specific activity of 80 units/mg protein. SDS/PAGE of nitrate reductase always resulted in two bands of 107 and 99.5 kDa. The 107-kDa band was the nitrate reductase subunit of N. plumbaginifolia; the smaller one of 99.5 kDa is thought, as commonly reported, to result from proteolysis of the larger protein. The molecular mass of 107 kDa is close to the values calculated from the coding sequences of the two nitrate reductase genes recently cloned from tobacco (Nicotiana tabacum cv Xanthi).

1. Introduction The enzymes octopine and nopaline dehydrogenase are responsible for the synthesis of the unusual amino acid derivatives octopine and nopaline, specific for crown-gall tumors [l-4]. The nature of the enzyme synthesized is determined by the Ti-plasmid har- boured in the

Nitrate reductase (NR, EC 1.6.6.1) from higher plants is a homodimeric enzyme carrying a molybdenum cofactor at the catalytic site. Tungsten can be substituted for molybdenum in the cofactor structure, resulting in an inactive enzyme. When nitratefed Nicotiana tabacum plants were grown on a nutrient solution in which tungstate was substituted for molybdate, NR activity in the leaves decreased to a very low level within 24 hours while NR protein accumulated progressively to a level severalfold higher than the control after 6 days. NR mRNA level in molybdate-grown plants exhibited a considerable day-night fluctuation. However, when plants were treated with tungstate, NR mRNA level remained very high. NR activity and protein increased over a 24-hour period when nitrate was added back to N-starved molybdate-grown plants. NR mRNA level increased markedly during the first 2 hours and then decreased. In the presence of tungstate, however, the induction of NR activity by nitrate was totally abolished while high levels of NR protein and mRNA were both induced, and the high level of NR mRNA was maintained over a 10-hour period. These results suggest that the substitution of tungsten for molybdenum in NR complex leads to an overexpression of the NR structural gene. Possible mechanisms involved in this deregulation are discussed.

In the leaves of nitrate-fed tobacco plants, the malate level increased 3-fold during the day and decreased during the night. These 24-h oscillations continued to occur in plants placed in continuous light conditions. On the other hand, the malate content declined markedly and showed no rhytmic variations when plants were transferred to continuous darkness. The fluctuation pattern of malate level were similar to those of in vitro nitrate reductase (NR, EC 1.6.6.1) activity although they were not parallel. Moreover, when NR activity, and presumably nitrate-reduction, were strongly decreased either in plants grown with tungstate (an inhibitor of NR activity) or in plants starved of nitrogen, leaf malate accumulation during the day was completely suppressed. The supply of nitrate to N-starved plants induced NR activity and leaf malate accumulation unless tungstate was present in the nutrient solution. These suggest that the malate content in leaves is dependent upon the level of nitrate reduction.

From previous observations concerned with the qualitative and quantitative evolution of protein groups in developing maize (Zea mays L.) grain, it may be deduced that the amount per grain of any amino acid incorporated into the proteins is linearly related to the amount of true protein in the grain. In this study, such a linear relationship is characterised quantitatively from experimental data by regressing the individual amino acid against the total protein accumulated in the developing grain of both normal (+) and opaque-2 (o2) maize. The linear least squares regression on total nitrogen content in the grain is also used for specifying the quantitative variations of any free and protein-incorporated amino acid in the grain. The slope Bj of the regression line represents the relative rate of accumulation of amino acid (j) in grain protein (or nitrogen) and its limiting content in protein (or nitrogen) of a grain which accumulates a large amount of it. Bj, which is compared to the amount of amino acid (j) present in the protein of immature grains, or of mature endosperm, or germ, is close to the amount of amino acid (j) present in the protein of mature endosperm. The same holds for the Bj value determined from opaque-2 maize and which differs from the amount of Bj determined using the normal variety for many amino acids. Using experimental data reported in the literature, a linear relationship has also been found in describing quantitative variations of amino acid in the developing grain of normal and Hiproly barley.

NADH: nitrate reductase (EC 1.6.6.1) was purified from Nicotiana plumbaginifolia leaves. As recently observed with nitrate reductase from other sources, this enzyme is able to reduce nitrate using reduced bromphenol blue (rBPB) as the electron donor. In contrast to the physiological NADH-dependent activity, the rBPB-dependent activity is stable in vitro. The latter activity is non-competitively inhibited by NADH. The monoclonal antibody ZM.96(9)25, which inhibits the NADH: nitrate reductase total activity as well as the NADH: cytochrome c reductase and reduced methyl viologen (rMV): nitrate reductase partial activities, has no inhibitory effect on the rBPB: nitrate reductase activity. Conversely, the monoclonal antibody NP.17-7(6) inhibits nitrate reduction with all three electron donors: NADH, MV or BPB. Among various nitrate reductase-deficient mutants, an apoprotein gene mutant (nia. E56) shows reduced terminal activities but a highly increased rBPB:nitrate reductase activity. rBPB:nitrate reductase thus appears to be a new terminal activity of higher plant nitrate reductase and involves specific sites which are not shared by the other activities.

The influence of light-dark cycles and nitrate supply on nitrate reductase (NR) mRNA levels was studied in two plant species, tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum) using specific NR DNA probes. In the same series of experiments, changes in the levels of NR protein (NRP) by enzyme-linked immunosorbent assay and changes in the level of NADH-nitrate reductase activity (NRA) were also followed. During a light-dark cycle, it was found that in both tomato and tobacco, NR mRNA accumulation increased rapidly during the dark period and reached a maximum at the beginning of the day, while NRP reached a peak 2 and 4 hours after mRNA peaked, for tomato and tobacco, respectively. At the end of the day, the amount of mRNA was decreased by a factor of at least 100 compared to sunrise in both species. These results demonstrate that light is involved, although probably not directly, in the regulation of the NR gene expression at the mRNA level. The peak of NRA in tobacco coincided with the peak in NR mRNA accumulation (i.e. sunrise), whereas in tomato the peak of NRA was approximately 5 to 6 hours after sunrise. There is no obvious correlation between NRP and NRA levels during the day. In nitrogen starvation experiments, a rapid decrease of NRP and NRA was detected, while NR mRNA levels were not significantly altered. Upon nitrate replenishment, nitrogen-starved plants accumulated NR mRNA rapidly. These results suggest that the availability of nitrogen affects the expression of NR activity at the transcriptional as well as at the post-transcriptional levels.

Over a 24-h light-dark cycle, the level of mRNA coding for nitrate reductase (NR; EC 1.6.6.1) in the leaves of nitrate-fed Nicotiana tabacum L. plants increased throughout the night and then decreased until it was undetectable during the day. The amount of NR protein and NR activity were two-fold higher during the day than at night. When plants were transferred to continuous light conditions for 32 h, similar variations in NR gene expression, as judged by the above three parameters, still took place in leaf tissues. On the other hand, when plants were transferred to continuous dark conditions for 32 h, the NR-mRNA level continued to display the rhythmic fluctuations, while the amount of NR protein and NR activity decreased constantly, becoming very low, and showed no rhythmic variations. After 56 h of continuous darkness, the levels of NR mRNA, protein and activity in leaves all became negligible, and light reinduced them rapidly. These results indicate the circadian rhythmicity and light dependence of NR expression.