Nitrate reductase in Zea mays L. under salinity


  • G. K. Abd-El Baki,

    1. Universität Würzburg, Lehrstuhl für Molekulare Pflanzenphysiologie und Biophysik, Universität Würzburg, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany
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  • F. Siefritz,

    1. Universität Würzburg, Lehrstuhl für Molekulare Pflanzenphysiologie und Biophysik, Universität Würzburg, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany
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  • H.-M. Man,

    1. Universität Würzburg, Lehrstuhl für Molekulare Pflanzenphysiologie und Biophysik, Universität Würzburg, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany
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  • H. Weiner,

    1. Universität Würzburg, Lehrstuhl für Molekulare Pflanzenphysiologie und Biophysik, Universität Würzburg, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany
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  • R. Kaldenhoff,

    1. Universität Würzburg, Lehrstuhl für Molekulare Pflanzenphysiologie und Biophysik, Universität Würzburg, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany
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  • W. M. Kaiser

    1. Universität Würzburg, Lehrstuhl für Molekulare Pflanzenphysiologie und Biophysik, Universität Würzburg, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany
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Correspondence: Werner M.Kaiser. Fax: 0931 8886158; E-mail:


Young maize seedlings (Zea mays L. cv.Giza 2) were exposed to moderate salinity in hydroponic culture. NADH-nitrate reductase (NR) activity (E.C. 1·6.6·1), NR activation state, NR-mRNA-steady state levels and major solute contents in leaves and roots were investigated. With increasing external salt concentration, Na+, Cl, sugars, amino acids and quarternary ammonium compounds accumulated in leaves and roots, with concentrations in leaves exceeding those in roots. The nitrate content of leaves decreased, but increased in roots. The diurnal pattern of NR activity and of NR-mRNA was also changed under salinity, but the NR activation state was not affected. In the first light phase, maximum NR activity increased rapidly in leaves of control plants, but was much slower in leaves from salinized plants. Thus, integrated over the whole day, the NR activity of salt-stressed plants was lower than in control plants. NR transcript levels of control plants were low in the early night phase, started to increase in the second night phase, followed by a distinct peak at 2 h into the light period. This large ‘early morning peak’ of NR-mRNA was hardly affected by salinity, whereas the initial slow increase of m-RNA levels in the early night phase was almost absent in salinized plants. This is considered as one reason for the low NR activity of salinized plants in the first half of the day. It is also suggested that nitrate is a major signal affecting NR expression and activity under salinity. Sugars and amino acids appeared less important.


Crop plants are usually non-halophytes that tolerate only moderate salt concentrations. Under salinity they accumulate salt in their above-ground organs and, to a smaller extent, in roots. Sodium and chloride are largely sequestered in the vacuoles, which prevents salt toxicity. Dehydration of metabolically active subcellular compartments is avoided by accumulation of organic solutes. Many of these ‘compatible solutes’ are N-containing compounds, such as amino acids, amines or betaines. As well as building up low water potentials without encountering salt toxicity, plants must be able to take up and transport cations (K+, Mg2+, Ca2+) and anions (nitrate, phosphate) and to reduce and metabolize nitrate even in the presence of high concentrations of sodium and chloride. In non-halophytes, nitrate uptake and transport appear especially sensitive to salinity. In salt-sensitive pea plants exposed to moderate NaCl concentrations, roots often contained high concentrations of nitrate, whereas nitrate in the shoots became very low. A conclusion was that loading of nitrate into the root xylem is a very salt-sensitive step ( Speer & Kaiser 1994; Peuke et al. 1996 ). This may have severe consequences for whole-plant nitrate assimilation: Nitrate reductase (EC 1·6.6·1) requires nitrate to be induced, and assimilates (sugars, eventually amino acids) appear also involved in the control of NR expression ( Nussaume et al. 1995 ). Further, NR is not only regulated at the transcription level. In higher plants, enzyme activity is rapidly modulated by reversible protein phosphorylation on a serine residue. In the presence of free Mg2+, phospho-NR binds to 14-3-3 proteins which causes a complete inactivation (for a recent review compare Kaiser, Weiner & Huber 1999). Through this process, NR in leaves is rapidly inactivated in the dark and activated in the light. It is also inactivated when stomata close ( Kaiser & Förster 1989). In roots, the NR activation state may also change rapidly, e.g. depending on oxygen supply ( Glaab & Kaiser 1993; Botrel & Kaiser 1997). It was suspected that salinity may have adverse effects on both NR expression and NR activation, which may also affect the NR half life ( Huber & Kaiser 1996). Therefore we investigated the effect of moderate salinity on diurnal variations of nitrate concentrations, of maximum NR activity (NRmax), of NRact (which is measured in the presence of excess Mg2+ and is thought to be the ‘real’ activity of NR in the leaf or root tissue), of the NR activation state (which gives NRact as percent NRmax) and on NR protein content and NR-mRNA levels in maize under salinity. Cation and anion contents, sugars and amino acids were also analysed in order to obtain information on changes of parameters which may be involved in the control of NR expression and activity. In order to distinguish specific salt effects from more general osmotic effects, plants were also exposed to polyethylene glycol (PEG)-solutions iso-osmolal to the other salt solutions applied.


Plant material and growth conditions

Maize (Zea mays L., line G2.) was received from the Seds Agriculture Research Center, Beni-suief, Egypt. The seeds were germinated for 3 d on moistened filter paper and subsequently transferred to hydroponic culture. Plants were grown on pots with perforated plastic tops (six plants per pot), each pot containing 1·6 L of continuously aerated nutrient solution. Growth chambers were temperature controlled (22 °C during day and 18 °C at night) and were illuminated with HQi lamps 400 W (Schreder, Winterbach, Germany), providing at the plant level a photon flux density (PAR) of 320 μmol m−2 s−1, with 11 h of daylight. The standard nutrient solution contained 3 m M KNO3, 1 m M CaCl2, 2 m M MgSO4, 2·7 m M KH2PO4, 1·3 m M K2HPO4, 0·2 m M NaFeEDTA, and trace elements according to Johnson et al. (1957) . The nutrient solution was replaced every third day. If not mentioned otherwise, plants were harvested 2 h into the light, at the times indicated in the legends of figures and tables.

Salt treatment

Salinity was imposed on plants at day 12 after transfer to hydroponics, by a daily increase of 50 m M NaCl, until the desired final salt level (usually 150 m M) was reached. One day later, plants were harvested (day 16 after transfer). Controls were grown for the same time without salt addition.


Shoots and roots were separated. Roots were rinsed with cold water (controls) or with cold glycine betaine solutions equimolar to the salted nutrient solution, in order to prevent osmotic shock. After blotting with tissue paper, fresh weights of roots and shoots were determined separately. Part of the material was dried at 70 °C for 72 h for dry weight determination.

Solute extraction and analysis

About 0·5 g of root or leaf material was homogenized to a fine powder in small mortars in liquid nitrogen. After addition of distilled water (5 mL), samples were allowed to thaw while still being ground. The suspension was boiled briefly (3 min) in a preheated block in order to inactivate the enzymes and precipitate proteins. After centrifugation (15 min, 4°, 16 000 g), the supernatant was kept frozen at –20 °C or used directly after suitable dilution for analysis of inorganic ions, amino acids and sugars. The pellet was washed (resuspended and centrifuged) once again with ice-cold distilled water (5 mL), and was then subjected to treatment with amyloglucosidase (Sigma, Deisenhofen, Germany; 7·5 U/5 mL, 1 h, 37 °C), boiled and centrifuged again and glucose was measured in the supernatant as described below.

Anions (chloride, phosphate, sulfate, nitrate, nitrite, malate, oxalate) were determined by isocratic anion analysis (IC 1002 with automatic sample injector and conductivity detection; Biotronik, Maintal, Germany) and equipped with a self-regenerating suppressor ASRS II 4 mm (Dionex, Idstein, Germany). Sugars (mainly glucose, fructose and sucrose) were determined by isocratic ion chromatography with pulsed amperometric detection (4500 I; Dionex). Amino acid analysis was carried out with an analyser LC 5001 (Biotronik). Cations were determined by ICP-OES (Jobin Ivon, Grasbrunn, Germany). Total soluble protein content was determined in aqueous extracts with BCA reagent (Pierce, Rockford, IL, USA) at 562 nm with bovine serum albumin as standard.

NR activity

Leaves or roots (1 g fresh weight) were ground with mortar and pestle in liquid nitrogen. Two ml extraction buffer (100 m M HEPES-KOH pH 7·6; 20 m M MgCl2, 10 μM flavin adenine dinucleotide (FAD), 5 m M DL-dithiothreitol (DTT), plus the protease inhibitors leupeptin (10 μM), Pefabloc (1 m M) and phenylmethylsulfonyl fluoride (PMSF) (0·2 m M), and in addition 1% polyvinylpyrrolidone (PVP) and 0·05% casein) were added to the still frozen powder and grinding continued until thawing was complete. The suspension was then centrifuged for 12 min (4 °C, 16000 g; Model 24–48R; Hettich Mikro, Tuttlingen, Germany) and the supernatant was removed and kept on ice. The reaction medium consisted (total volume 1 mL) of 50 m M HEPES-KOH pH 7·6, 10 μM FAD, 1 m M DTT, 5 m M KNO3, 0·2 m M NADH and either 20 m M MgCl2 or 20 m M ethylenediaminetetraacetic acid (EDTA). The reaction was started by addition of 200 μL extract and terminated after 5 min by addition of 125 μL zinc acetate solution (0·5 M). After a short centrifugation (4 °C, 5 min, 16000 g), 10 μL phenazine methosulphate (PMS) was added to 950 μL of the supernatant in order to oxidize excess NADH. After 20 min in the dark, the nitrite formed was measured colorimetrically by adding 750 μL of 1% sulfanilamid in 3 M HCl, and 750 mL of 0·02%N-naphtyl-ethylenediamine hydrochloride, and absorption was determined at 546 nm. For each series, blanks and a nitrite standard (20 μM KNO2) were included.

NR activation state

In order to fully activate NR for determination of NRmax, an aliquot of the crude extract was pre-incubated for 15 min at 25 °C in buffer containing EDTA (as above) plus 5 m M 5′-AMP. In these cases, the reaction buffer also contained EDTA (no Mg2+). The actual NR activity (NRact), which is thought to reflect the real NR activity in situ, was determined in the same extract without pre-incubation in the presence of excess (20 m M) free Mg2+. The percentage value of NRact (NRmax = 100%) then gives the activation state of NR.

Western blot analysis

The NR protein level of leaf extracts was analysed by immunoblotting. Ground maize leaves [1 g fresh weight (FW)] were extracted with 2 mL buffer containing 50 mM Hepes-KOH (pH 7·6), 5 m M DTT, 10 μM FAD, 10 μM leupeptin, 1 m M pefablock, 0·2 m M PMSF and 1% PVP. After centrifugation (16 000 g, at 4 °C, 12 min), proteins were precipitated with PEG 8000 (15%) and stirred on ice for 10 min, followed by centrifugation for (20 min, 4 °C, 16 000 g) in a pre-cooled centrifuge. The pellet was dissolved in 200 μL extraction buffer and stored at − 80 °C. After protein determination, an aliquot of each sample was subjected to SDS-PAGE (10% separating gel, 6% stacking gel). The separated proteins were blotted on to nitrocellulose membrane (0·2 μm pore size; Schleicher and Schuell, Dassel, Germany). After blotting, the membranes were blocked with 3% non-fat dry milk powder in TBS (25 m M Tris-HCl, 150 m M NaCl, pH 7·5) containing 0·1% Tween 10 for 1 h at room temperature. Immunodetection of NR was carried out with rabbit serum antibody against purified NR from maize. Prior to use, the antiserum was competed overnight with a leaf extract from nitrate free (ammonium grown) plants. NR was visualized with alkaline phosphatase-labelled goat antirabbit IgG (Southern Biotechnology Associates, Inc., Birmingham, AL, USA) and staining with p-nitro-blue tetrazolium chloride/5-bromo-4-chloro-indolyl-phosphate. Density scanning of NR bands was carried out with an ‘Image Master VDS’ (Pharmacia, Biotech, Uppsala, Sweden). NR bands were identified by co-electrophoresis with purified maize NR (Sigma).

Preparation and analysis of RNA

Total RNA was isolated as described by Kaldenhoff, Kölling & Richter (1993) starting from 0·5 g maize leaf material.

Ribonucleic acids from different preparations were initially standardized by ethidium bromide fluorescence of ribosomal RNA after agarose gel electrophoresis. For Northern hybridization, RNA amounts with equal fluorescence were size-fractionated in a 1% denaturing agarose-formaldehyde gel and transferred to a nylon membrane by capillary-blotting. The nucleic acids were cross-linked by UV-light irradiation for 2 min and then prehybridized in Roti-Hybri-Quick solution (Roth Chemikalien, Karlsruhe, Germany). Hybridization to 32P-labelled probes and washing was performed at 68 °C following the manufactures’ guidelines. The air-dried membrane was exposed to X-ray retina film (XBD) with intensifying screen at − 80 °C and the film was developed after an appropriate time period.

The resulting band-signals were quantified by Image Master VDS software (Pharmacia). The values obtained for the NR-signals were related to those of 28S-rRNA after stripping the filter and hybridizing with a corresponding 32P-labelled probe. The highest value for NR-RNA was set to 100%.

Preparation of cDNA-probes

The NR-cDNA was obtained by reverse transcriptase (RT)-polymerase chain reaction (PCR). From 0·5 μg of poly A-RNA isolated according to Roberts & Paterson (1973), reverse transcription was initiated using primer ZM-NR-AS (5′-TCCCACTTCCACCGCAGATCATCGC-3′). The resulting first-strand cDNA product was amplified by a PCR performed with ZM-NR-AS primer and the primer ZM-NR-S (5′-CGTCGGCAAGCAGTTCACCATGTCC-3′) following the protocol of Titan® RT-PCR-kit (Roche). The PCR-product was cloned into pCR® 2·1-TOPO® (Invitrogen, Groningen, The Netherlands) and sequenced for confirmation of identity to NR. 32P-labelling of the cDNA was performed by following the protocol of Feinberg & Vogelstein (1983).


Growth parameters, water relations and solute contents

Growth of maize plants responded rather sensitively to moderate salinity. Already at low salt concentrations, growth of salinized plants was impaired, although all plants still looked healthy and turgid. Shoot growth was slightly more impaired then root growth. Relative water content was only slightly affected. ( Table 1). Osmotic potentials of tissue sap increased with increasing salinity. It always exceeded the osmotic potential of the nutrient solution.

Table 1.  Growth parameters and relative water content for maize G2. Plants were harvested 21 d after sowing and 6 d after reaching 150 m M (NaCl), 2 h into the light period. Water content is given as percentage of water per FW. Π is the osmotic potential of the cell sap (mosmol/kg). n = (4–6) ± SD
 Salt (m M)
Whole plant FW (g)6·2 ± 0·74·9 ± 0·123·6 ± 0·372·8 ± 0·9
Whole plant DW (g)0·47 ± 0·050·36 ± 0·040·33 ± 0·080·29 ± 0·05
Maximum shoot length (cm)44·9 ± 1·539·2 ± 1·833·3 ± 1·627·5 ± 0·6
Maximum root length (cm)37 ± 3·232·5 ± 4·329·1 ± 1·226·5 ± 3·4
Relative water content92·492·690·889·6
Π of cell sap (leaves)320 ± 11·5370 ± 40·4410 ± 46·1710 ± 130·3
Π of cell sap (roots)360 ± 50·3380 ± 30·5410 ± 60400 ± 109·6

As expected, chloride and sodium were accumulated in the leaves to concentrations exceeding those in the nutrient solution. In contrast, chloride and sodium concentrations in the roots remained at or below those in the external medium, except in control plants, where external chloride was only 10 μM ( Fig. 1).

Figure 1.

Effect of increasing NaCl concentrations on cation and anion concentrations in shoots and roots of maize. Conditions as in Table 1. Each datum point is the mean of four to six separate samples. Bars give SD.

Nitrate concentrations in leaves (harvested 2 h into the light period) were decreased under salt stress, but increased in roots ( Fig. 1), thus confirming previous observations, e.g. with pea plants ( Speer & Kaiser 1994; Speer, Brune & Kaiser 1994). Nitrate concentrations in leaves are often not constant but vary diurnally, at least at limiting nitrate supply ( Man et al. 1999 ). However, in our experiments no clear diurnal time course of the nitrate concentration was found, and salt-stressed plants had lower nitrate concentrations in their leaves throughout the day/night cycle (not shown).

Potassium concentrations in leaves remained almost constant under salinity ( Fig. 1), but Ca2+ and Mg2+concentrations decreased (not shown). In roots, however, potassium concentrations decreased by more than 50% ( Fig. 1), whereas divalent cation concentrations did not change much. Thus, as previously observed for pea ( Speer et al. 1994 ), transport of divalent cations from root to shoot appeared more sensitive to salinity than net potassium transport, just as for nitrate.

Table 2 summarizes concentrations of carbohydrates, amino acids, and total soluble protein content in controls and salt-treated plants. The pool of total amino acids increased in leaves and roots of salinized plants. This was mainly due to accumulation of asparagine (seven-fold), proline (10-fold), GABA (10-fold) and serine (four-fold). Glutamine and glutamate, which are potential signalling compounds for NR expression and regulation, were both slightly higher in leaves under salt stress, but they were not much affected in roots. Quarternary ammonium compounds, which are also thought to be important compatible osmolytes, were only slightly accumulated in salt-stressed maize. Surprisingly, concentrations of total soluble protein were increased under salinity in leaves, but they were hardly affected in roots.

Table 2.  Carbohydrate contents, amino acids, betaines (μmol g−1 FW) and total soluble protein (mg/g FW) in shoots and roots of maize. Same conditions as in Table 1. n = (3–6) ± SD, except for starch and amino acids where n = 2
 ControlNaCl (150 m M)
Hexoses13·4 ± 3·111 ± 3·120·3 ± 3·814·5 ± 4·8
Sucrose 9·3 ± 3·1 3·9 ± 1·226·3 ± 4 4·4 ± 1·1
Starch 2·62 0·2119·2 0·09
Total amino acids16·8 8·7027·510·7
Glutamine 0·44 1·25 1·10 0·77
Glutamate 3·10 1·75 2·90 1·35
Proline 0·17 0·11 2·20 0·36
GABA 0·10 0·15 0·39 0·11
Asparagine 0·12 0·45 1·50 2·20
Serine 1·10 0·77 4·70 1·10
Quarternary ammonium
15·7 ± 3·5 9·2 ± 0·9120·1 ± 9·511·8 ± 2·2
Total soluble protein24·3 ± 10·514·3 ± 0·742·7 ± 7·214·2 ± 2·7

Leaves also accumulated sugars under salinity, especially sucrose, whereas in the roots sugar levels did not change much. Starch levels increased dramatically in leaves of salt-stressed plants ( Table 2). Thus, increased sugar levels were not due to a starch/sugar interconversion, but part of a general increase in carbohydrate contents. In roots, starch levels remained very low.

The salinity-induced changes in leaf and root nitrate and sugar contents might cause changes in NR expression and activity. High sugar levels should have positive effects on NR expression. However, NRmax extracted 2 h into the light period, decreased significantly in leaves under salt stress ( Fig. 2). This was matched by NR protein levels, as indicated by Western blotting ( Fig. 3). In contrast, NRmax increased in the roots ( Fig. 2). Attempts to measure NR protein levels in roots by Western blotting were unsuccessful.

Figure 2.

NRact, NRmax, and activation state in leaves and roots of maize at different concentrations of NaCl. Other conditions as in Table 1. Each datum point is the mean of four to six separate samples. Bars give SD.

Figure 3.

Western blot of NR in leaf extracts from control plants and from plants grown at 150 m M NaCl. Relative optical density of bands is given in percentage. NRmax was measured in the same leaf material, and activity is given in μmol/g FW h.

The activation state of NR was also followed. Stomatal closure has been shown to inactivate NR ( Kaiser & Förster 1989). Under salinity leaves may close stomata, at least transiently. It was therefore surprising that the activation state of leaf-NR was hardly affected by salinity. It should be noted that the rapid kinetics of light-dependent NR activation/inactivation in leaves were not resolved here because the first sample was taken only after 1 h light (morning) or 2 h dark (evening). In summary, the salt-induced changes in NRact resulted in a considerably lower total NR activity per plant (about 50% of control plants), and a higher contribution of the root NR to total NR of the plant.

The above described changes in NR activity and activation state represent an oversimplification, since NR activities vary diurnally, at least in leaves ( Bowsher et al. 1991 ; Man et al. 1999 ). Therefore we also examined NRmax and NRact in leaves and roots during a day/night cycle ( Fig. 4). The NRmax in leaves of control plants was high already in the early light phase and decreased during the day. At the end of the night, NRmax was again high. In contrast, NRmax of salinized plants was low at the end of the night and increased slowly during the day. Accordingly, differences in NRmax and NRact between control plants and salinized plants were not constant during the day, but were most pronounced in the morning. Towards the end of the day these differences became negligible.

Figure 4.

Diurnal time course of NR activity in leaves and roots from control plants and from plants grown at 150 m M NaCl. The shaded part in the graph shows the dark periods. Each datum point is the mean of four separate samples. Bars give SD. Open circles, control plants; closed circles, salt plants.

The changes in the diurnal variation of NRmax in response to salinity could be traced back to differences in the levels of leaf NR-mRNA ( Fig. 5). Consistent with previous results of Huber et al. (1994) , NR-mRNA levels in leaves of control plants went through a distinct maximum during the early morning phase, about 2 h into the light period. However, that increase in NR-mRNA had already started in the late night phase. Interestingly, the pronounced ‘early morning peak’ in the NR-mRNA level was hardly affected by salinity ( Fig. 5b, c), whereas the slow increase in m-RNA during the late night appeared very sensitive. Attempts to measure NR-mRNA also in maize roots were unsuccessful, as signal strength was insufficient.

Figure 5.

Northern blot analysis of NR-RNA steady state levels. Total RNA was extracted from controls (a) or plants grown at 150 m M NaCl (b). The time course of light treatment is symbolized by black (darkness) or white bar (light) in (a). Numbers refer to time of the day. RNA-blots were hybridized with 32P-labelled NR-(NR) and 28S-probes (28S). (c) Quantification of the NR-signal after standardization to the values obtained for 28S-rRNA (grey, controls, black, plants grown at 150 m M NaCl). (a) and (b) give data from a typical experiment out of four identical experiments; (c) gives the mean optical densities. Highest m-RNA-levels at 10 h were set to 100%. Bars indicate SD (n = 4).

Osmotic stress may be part of the salinity syndrome leading to changes in root/shoot NR. Therefore the response of nitrate contents, sugar levels and NR activities was followed in plants that were exposed to nutrient solutions brought to the same osmolality as in the salt treatments, by adding polyethylene glycol ( Table 3). Osmotic stress also decreased nitrate contents in leaves and increased nitrate contents in the roots. However, both changes were not as dramatic as under salt stress. Sugar levels were also elevated by osmotic stress to a similar extent as under salt stress. ( Geigenberger et al. 1997 ; Huber et al. 1999 ). NR activities in the leaf still decreased with increasing osmolality, but not as much as under salt stress. In roots, the situation was less clear, but the usual increase of NR activities in response to stress was absent.

Table 3.  Effect of PEG (600) on NR, nitrate and sugars in leaves and roots of maize. Other conditions as in Table 1. NR is given in μmol g−1 FW h−1), and activation state as (% of NRmax). Sugar and nitrate concentrations are given in μmol g−1 FW, n = (4–6) ± SD
PEG (mM)0·0120175200
LeafNRact3·2 ± 0·62·9 ± 0·82·5 ± 0·72·1 ± 0·4
 NRmax8 ± 1·47 ± 1·04·8 ± 1·64·5 ± 0·7
 Activation state39·9 ± 2·740·2 ± 7·352·6 ± 5·247·5 ± 2·8
 Hexoses10·6 ± 2·811·0 ± 1·529·6 ± 3·025·5 ± 1·4
 Sucrose8·6 ± 2·110·9 ± 2·120·6 ± 7·319·1 ± 4·1
 Nitrate51·8 ± 6·840·6 ± 9·835·5 ± 5·228·5 ± 6·5
Root NRact0·2 ± 0·10·25 ± 0·040·2 ± 0·020·18 ± 0·02
 NRmax0·7 ± 0·20·7 ± 0·20·9 ± 0·10·63 ± 0·2
 Activation state31·8 ± 5·835·7 ± 7·323·8 ± 4·330·9 ± 9·2
 Hexoses7·6 ± 3·29·9 ± 3·212·7 ± 4·19·8 ± 2·5
 Sucrose2·9 ± 0·53·6 ± 1·55·2 ± 1·15·3 ± 0·8
 Nitrate67·7 ± 2·274·8 ± 4·991·5 ± 7·878·8 ± 6·9


Moderate salinity lead to decreased NRmax and NR protein content in leaves of young maize seedlings. In contrast, NR activity increased in roots under salt stress. This confirms and extends previous findings with Ricinus communis L. ( Peuke et al. 1996 ). The increase in root NR activity was too small to compensate for the loss in leaf NR activity. Accordingly, based on whole plant FW, total NR activity decreased under salt stress. But in spite of that, concentrations of free amino acids and of protein in leaf tissues were even higher than in control plants. It is concluded that the salt-dependent decrease in growth was not due to impaired nitrate assimilation. Rather, growth appeared limited by other factors such that nitrate reduction exceeded the demands.

The salt-induced changes in NRmax were largest in the late night- and early morning-phase, and disappeared almost completely during the second light phase, due to a continuous increase of NRmax in salinized plants during the day. Salt stress did not affect NR transcript levels during the day, but decreased them during the night phase. This ‘night-depression’ of NR m-RNA levels is considered as a major reason for the slow increase of NRmax of salinized plants in the initial light phase. As overall sodium and chloride levels in leaves did not change diurnally (not shown), it seems difficult to explain why salinity affected NR-mRNA levels more during the late night than during the day. However, if salt distribution between vacuole and cytsosol would vary diurnally (which has not been measured), this might explain the depression of NR-mRNA levels in the late night phase.

Sugar levels were generally increased under salinity in leaves. This should actually favour NR expression ( Vincentz et al. 1993 ; Nussaume et al. 1995 ), but as shown above, NR activity and NR protein levels (indicated by NRmax and Western blotting) were decreased.

It has been mentioned above that the leaf nitrate content is most probably the decisive factor controlling NR expression and activity in shoots and roots. However, even in 150 m M NaCl, leaves still contained several millimole/L of nitrate which should be sufficient for triggering NR expression. We speculate that nitrate, just like chloride, was sequestered in the vacuole such that concentrations in the cytosol were much lower than bulk nitrate concentrations (compare Schröppel-Meier & Kaiser 1988). This would also explain why nitrate transport from root to shoot was impaired by salt stress, since nitrate loading into the xylem may depend on cytosolic nitrate concentrations in the xylem parenchyma.


This work was supported in part by the DFG, SFB 251. G.K.A.-E.-B. was a recipient of a Ph.D. fellowship of the Egyptian Channel System. We are very grateful to Ann Oaks for providing us with the antimaize NR antiserum, and to Maria Lesch, Eva Wirth, Frieda Reisberg and Astrid Boots for technical assistance with the analysis of inorganic cations, anions, carbohydrates and amino acids. We also thank Beate Otto for substantial help in the isolation of poly A-RNA.