Interaction of sulfur and nitrogen nutrition in tobacco (Nicotiana tabacum) plants: significance of nitrogen source and root nitrate reductase

Abstract: The significance of root nitrate reductase for sulfur assimilation was studied in tobacco (Nicotiana tabacum) plants. For this purpose, uptake, assimilation, and long-distance transport of sulfur were compared between wild-type tobacco and transformants lacking root nitrate reductase, cultivated either with nitrate or with ammonium nitrate. A recently developed empirical model of plant internal nitrogen cycling was adapted to sulfur and applied to characterise whole plant sulfur relations in wild-type tobacco and the transformant. Both transformation and nitrogen nutrition strongly affected sulfur pools and sulfur fluxes. Transformation decreased the rate of sulfate uptake in nitrate-grown plants and root sulfate and total sulfur contents in root biomass, irrespective of N nutrition. Nevertheless, glutathione levels were enhanced in the roots of transformed plants. This may be a consequence of enhanced APR activity in the leaves that also resulted in enhanced organic sulfur content in the leaves of the tranformants. The lack of nitrate reductase in the roots in the transformants caused regulatory changes in sulfur metabolism that resembled those observed under nitrogen deficiency. Nitrate nutrition reduced total sulfur content and all the major fractions analysed in the leaves, but not in the roots, compared to ammonium nitrate supply. The enhanced organic sulfur and glutathione levels in ammonium nitrate-fed plants corresponded well to elevated APR activity. But foliar sulfate contents also increased due to decreased re-allocation of sulfate into the phloem of ammonium nitrate-fed plants. Further studies will elucidate whether this decrease is achieved by downregulation of a specific sulfate transporter in vascular tissues.     

Screening for Arabidopsis mutants affected in the Nii gene expression using the Gus reporter gene

A mutant screen was developed to isolate Arabidopsis thaliana mutants affected in the regulation of the nitrate assimilation pathway. A fusion between the tobacco Nii1 gene (that encodes a foliar nitrite reductase involved in nitrate assimilation) and the Gus reporter gene was introduced into A. thaliana , and shown to be properly regulated by nitrate. Moreover, β -glucuronidase (GUS) activity in the transgenic plants was essentially detected in the cotyledons and leaves, showing that the organ-specific expression of the tobacco Nii1 gene was retained in Arabidopsis . M2 plantlets derived from mutagenized seeds homozygous for the Nii-Gus fusion were screened by histochemical staining of whole plates for GUS activity after growth on nitrate or glutamine. About 250 progenies were screened, leading to the isolation of plants showing an enhanced or reduced staining compared to the control non-mutagenized plants. Several mutants were analyzed for the transmission of the phenotype to the M3 generation, as well as for levels of GUS or nitrite reductase activities or mRNA levels. A major problem encountered during the screening was the high background of false positives that reproducibly showed altered GUS histochemical staining compared to control plants and did not, however, display any changes in GUS activity levels. One interesting family of mutants was isolated that overexpressed GUS activity and Nii mRNA in the absence of nitrate. These mutants turned out to be cnx mutants impaired in the molybdenum cofactor biosynthesis that is necessary for nitrate reductase activity. These results may indicate that active nitrate reductase is necessary for a correct regulation of nitrate assimilation genes by nitrate.     

Changes in the activities of nitrogen assimilation enzymes ofLolium perenne L. during regrowth after cutting

The activities of key enzymes involved in N assimilation were investigated after defoliation of 6-week-old ryegrass plants grown in water culture conditions. In a first experiment, nitrate reductase, glutamine synthetase and glutamate dehydrogenase activities were measured in roots, stubble and leaves on the day of cutting and at 7-day intervals over the following 5-week period of regrowth. Ammonia assimilation enzymes showed little change whereas the nitrate reductase activity sharply decreased 2 weeks after clipping. In a second experiment, the nitrate reductase activity was measured at 2- or 3-day intervals 1 week before and 3 weeks after clipping.In vivo andin vitro assays both showed an increasing activity in leaves up to 8 days after cutting while root activity decreased. The opposite changes then occurred and both organs recovered their initial nitrate reductase activity levels after 12–14 days of regrowth. These fluctuations in nitrate reductase activity were considered to be related to the capacity for C assimilation and the nitrate availability.     

Partitioning of inorganic nitrogen assimilation between the roots and shoots of cerrado and forest trees of contrasting plant communities of South East Brasil

Summary Woody plants growing in cerrado and forest communities of south-east Brasil were found to have low levels of nitrate reductase activity in their leaves suggesting that nitrate ions are not an important nitrogen source in these communities. Only in the leaves of species growing in areas of disturbance, such as gaps and forest margins, were high levels of nitrate reductase present. When pot-grown plants were supplied with nitrate, leaves and roots of almost all species responded by inducing increased levels of nitrate reductase. Pioneer or colonizing species exhibited highest levels of nitrate reductase and high shoot: root nitrate reductase activities. Glutamine synthetase, glutamate synthase and glutamate dehydrogenase were present in leaves and roots of the species examined.¹⁵N-labelled nitrate and ammonium were used to compare the assimilatory characteristics of two species:Enterolobium contortisiliquum, with a high capacity to reduce nitrate, andCalophyllum brasiliense, of low capacity. The rate of nitrate assimilation in the former was five times that of the latter. Both species had similar rates of ammonium assimilation. Results for eight species of contrasting habitats showed that leaf nitrogen content increased in parallel with xylem sap nitrogen concentrations, suggesting that the ability of the root system to acquire, assimilate or export nitrate determines shoot nitrogen status. These results emphasise the importance of nitrogen transport and metabolism in roots as determinants of whole plant nitrogen status.     

Nitrate reductase,nitrite reductase and glutamate dehydrogenase levels in roots and leaves of maize seedlings

Total activities of nitrate and nitrite reductases were higher in 4 to 20 day old maize plants in the leaves than in the roots. The ratio of activities found in the leaves and in the roots respectively was much higher in the case of nitrate reductase than in the case of nitrite reductase. On the other hand higher glutamate dehydrogenase activity in the roots than in the leaves clearly indicates that the roots play a more important role in the assimilation of ammonium than in the assimilation of nitrate. When comparing the distribution of seminal and nodal adventitious roots of maize seedlings with the assimilation of inorganic nitrogen on the basis of enzyme levels, it could be deduced that during the first 20 days of seedling growth seminal roots were more involved in the assimilation of nitrate whereas nodal adventitious roots were more active in ammonium assimilation.     

Diurnal changes in nitrogen assimilation of tobacco roots.

To gain an insight into the diurnal changes of nitrogen assimilation in roots the in vitro activities of cytosolic and plasma membrane-bound nitrate reductase (EC 1.6.6.1), nitrite reductase (EC 1.7.7.1) and cytosolic and plastidic glutamine synthetase (EC 6.3.1.2) were studied. Simultaneously, changes in the contents of total protein, nitrate, nitrite, and ammonium were followed. Roots of intact tobacco plants (Nicotiana tabacum cv. Samsun) were extracted every 3 h during a diurnal cycle. Nitrate reductase, nitrite reductase and glutamine synthetase were active throughout the day-night cycle. Two temporarily distinct peaks of nitrate reductase were detected: during the day a peak of soluble nitrate reductase in the cytosol, in the dark phase a peak of plasma membrane-bound nitrate reductase in the apoplast. The total activities of nitrate reduction were similar by day and night. High activities of nitrite reductase prevented the accumulation of toxic amounts of nitrite throughout the entire diurnal cycle. The resulting ammonium was assimilated by cytosolic glutamine synthetase whose two activity peaks, one in the light period and one in the dark, closely followed those of nitrate reductase. The contribution of plastidic glutamine synthetase was negligible. These results strongly indicate that nitrate assimilation in roots takes place at similar rates day and night and is thus differently regulated from that in leaves.     

Expression of a constitutively active nitrate reductase variant in tobacco reduces tobacco‐specific nitrosamine accumulation in cured leaves and cigarette smoke

Burley tobaccos (Nicotiana tabacum) display a nitrogen‐use‐deficiency phenotype that is associated with the accumulation of high levels of nitrate within the leaf, a trait correlated with production of a class of compounds referred to as tobacco‐specific nitrosamines (TSNAs). Two TSNA species, 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) and N‐nitrosonornicotine (NNN), have been shown to be strong carcinogens in numerous animal studies. We investigated the potential of molecular genetic strategies to lower nitrate levels in burley tobaccos by overexpressing genes encoding key enzymes of the nitrogen‐assimilation pathway. Of the various constructs tested, only the expression of a constitutively active nitrate reductase (NR) dramatically decreased free nitrate levels in the leaves. Field‐grown tobacco plants expressing this NR variant exhibited greatly reduced levels of TSNAs in both cured leaves and mainstream smoke of cigarettes made from these materials. Decreasing leaf nitrate levels via expression of a constitutively active NR enzyme represents an exceptionally promising means for reducing the production of NNN and NNK, two of the most well‐documented animal carcinogens found in tobacco products.     

Influence of Glyphosate on Nitrate Reductase Activity in Soybean (Glycine max)

Experiments were conducted to determine the influence of glyphosate[N-(phosphonomethyl)glycine] on extractable nitrate reductaseactivity during light and dark growth of soybean (Glycine max)seedlings. Glyphosate (5?10^–4 M), applied via root-feedingto three-day-old etiolated seedling, significantly reduced enzymeactivity in roots (48 to 96 h) and leaves (96 h) of seedlingsplaced in the light, but had little effect on enzyme activityin cotyledons compared to enzyme levels in tissues of untreatedseedlings. During dark-growth, nitrate reductase activity increasedwith time in cotyledons of untreated seedlings (activity about85-fold less than in cotyledons of light-grown plants) but muchlower enzyme levels were found in cotyledons of glyphosate-treatedseedlings after 72 and 96 h. In leaves of dark-grown seedlings,glyphosate reduced nitrate reductase levels by 95%. Most inhibitionof extractable enzyme activity occurred in newly developingorgans (leaves and roots) which correlates well with reportsthat glyphosate is rapidly translocated to these sites. However,the fact that glyphosate inhibits growth prior to lowering enzymeactivity levels indicates a secondary effect on nitrate reductase. (Received May 18, 1984; Accepted February 12, 1985)     

Lateral root frequency decreases when nitrate accumulates in tobacco transformants with low nitrate reductase activity: consequences for the regulation of biomass partitioning between shoots and root1

Accumulation of nitrate in the shoot of low-nitrate reductase tobacco transformants leads to an increase of the shoot:root ratio to higher values than in nitrogen-sufficient wild-type plants, even though the transformants are severely deficient in organic nitrogen. In the present paper, wild-type plants and low- nitrate reductase transformants were grown on vertical agar plates to investigate whether this inhibition of root growth by internal nitrate (i) can be reversed by adding sugars to the roots and (ii) is due to slower growth of the main roots or to a decreased number of lateral roots. When grown with a low nitrate supply, the transformants resembled wild-type plants with respect to amino acid and protein levels, shoot-root allocation, lateral root frequency, and rates of growth. When the transformants were grown with a high nitrate supply in the absence of sucrose they grew more slowly and had lower levels of amino acids and protein than wild-type plants, but accumulated more nitrate and developed a high shoot:root ratio. Root length was not affected, but the number of lateral roots per plant decreased. The slower root growth was accompanied by an increase of the concentration of sugars in the roots. Addition of 2% sucrose to the medium partially reversed the high shoot:root ratio in the transformants, but did not increase the frequency of lateral roots. It is concluded that nitrate accumulation in the plant leads to decreased root growth via (i) changes in carbon allocation leading to decreased allocation of sugars to root growth, and (ii) a decrease in the number of lateral roots and a shift in the sensitivity with which root growth responds to the sugar supply. This revised version was published online in June 2006 with corrections to the Cover Date.     

Light and dark assimilation of nitrate in plants

Abstract. Heterotrophic assimilation of nitrate in roots and leaves in darkness is closely linked with the oxidative pentose phosphate pathway. The supply of glucose-6-phosphate to roots and chloroplasts in leaves in darkness is essential for assimilation of nitrite into amino acids. When green leaves are exposed to light, the key enzyme, glucoses-phosphate dehydrogenase, is inhibited by reduction with thioredoxin. Hence the dark nitrate assimilatory pathway is inhibited under photoautotrophic conditions and replaced by regulatory reactions functioning in light. On account of direct photo-synthetic reduction of nitrite in chloroplasts and availability of excess NADH for nitrate reduclase, the rate of nitrate assimilation is extremely rapid in light. Under dark anaerobic conditions also nitrate is equally rapidly reduced to nitrite on account of abolition of competition for NADH between nitrate reductase and mitochondrial oxidation.     

Growth and reproduction of Arabidopsis thaliana in relation to storage of starch and nitrate in the wild-type and in starch-deficient and nitrate-uptake-deficient mutants

We have investigated the interactions between resource assimilation and storage in rosette leaves, and their impact on the growth and reproduction of the annual species Arabidopsis thaliana. The resource balance was experimentally perturbed by changing (i) the external nutrition, by varying the nitrogen supply; (ii) the assimilation and reallocation of resources from rosette leaves to reproductive organs, by cutting or covering rosette leaves at the time of early flower bud formation, and (iii) the internal carbon and nitrogen balance of the plants, by using isogenic mutants either lacking starch formation (PGM mutant) or with reduced nitrate uptake (NU mutant). When plants were grown on high nitrogen, they had higher concentrations of carbohydrates and nitrate in their leaves during the rosette phase than during flowering. However, these storage pools did not significantly contribute to the bulk flow of resources to seeds. The pool size of stored resources in rosette leaves at the onset of seed filling was very low compared to the total amount of carbon and nitrogen needed for seed formation. Instead, the rosette leaves had an important function in the continued assimilation of resources during seed ripening, as shown by the low seed yield of plants whose leaves were covered or cut off. When a key resource became limiting, such as nitrogen in the NU mutants and in plants grown on a low nitrogen supply, stored resources in the rosette leaves (e.g. nitrogen) were remobilized, and made a larger contribution to seed biomass. A change in nutrition resulted in a complete reversal of the plant response: plants shifted from high to low nutrition exhibited a seed yield similar to that of plants grown continuously on a low nitrogen supply, and vice versa. This demonstrates that resource assimilation during the reproductive phase determines seed production. The PGM mutant had a reduced growth rate and a smaller biomass during the rosette phase as a result of changes in respiration caused by a high turnover of soluble sugars ( Caspar et al. 1986 ; W. Schulze et al. 1991 ). During flowering, however, the vegetative growth rate in the PGM mutant increased, and exceeded that of the wild-type. By the end of the flowering stage, the biomass of the PGM mutant did not differ from that of the wild-type. However, in contrast to the wild-type, the PGM mutant maintained a high vegetative growth rate during seed formation, but had a low rate of seed production. These differences in allocation in the PGM mutant result in a significantly lower seed yield in the starchless mutants. This indicates that starch formation is not only an important factor during growth in the rosette phase, but is also important for whole plant allocation during seed formation. The NU mutant resembled the wild-type grown on a low nitrogen supply, except that it unexpectedly showed symptoms of carbohydrate shortage as well as nitrogen deficiency. In all genotypes and treatments, there was a striking correlation between the concentrations of nitrate and organic nitrogen and shoot growth on the one hand, and sucrose concentration and root growth on the other. In addition, nitrate reductase activity (NRA) was correlated with the total carbohydrate concentration: low carbohydrate levels in starchless mutants led to low NRA even at high nitrate supply. Thus the concentrations of stored carbohydrates and nitrate are directly or indirectly involved in regulating allocation.     

The Relationship of Abscisic Acid to Nitrate Reductase Activity in the Potato Plant

Abscisic acid (ABA) at 3.8 µM suppressed both in vivoand in vitro nitrate reductase activity in roots, stems andleaves of potato plants grown in solution culture. Suppressionwas maximal between 24 and 48 h, followed by recovery of activityat 72 h in roots and leaves and at 96 h in stems. Removal from ABA after 24 h resulted in complete recovery ofnitrate reductase activity in roots by 24 h and partial recoveryin leaves. ABA treatment enhanced nitrate accumulation in roots,decreased that of leaves, but had no effect on stem nitratecontent. ABA enhanced decay of the enzyme following nitrate removal;by 7 h activity in roots was 22.5% of the initial value comparedto 55% in the control. ABA showed a less drastic effect on lossof activity in leaves and stems. These results indicate thatABA suppression of nitrate reductase activity is not dependenton nitrate uptake, and although it reduced leaf nitrate contentthere was no clear relationship between tissue nitrate levelsand the ABA response. (Received September 13, 1984; Accepted July 1, 1985)