Changes in growth and activity of enzymes involved in nitrate reduction and ammonium assimilation in tomato seedlings in response to NaCl stress

BACKGROUND AND AIMS: In Tunisia, salt water is largely used for tomato irrigation. In this work, a study was made of the changes in the nitrate reduction and ammonium assimilation into amino acids in tomato seedlings under salinity in order to providee further insight into the salt effects on plant growth. Methods Ten-day-old tomatoes (Solanum lycopersicum) were subjected to 100 mm NaCl stress, and nitrogen metabolism in leaves and roots was studied. KEY RESULTS: The concentrations of Na+ and Cl- rapidly increased in the leaves and in the roots following exposure of tomato seedlings to NaCl stress. In contrast, the NO3- concentrations were lowered first in the roots and later in the leaves. From 5 to 10 d of treatment, salt ions provoked a decrease in the dry weight and an increase in the NH4+ concentrations in the leaves. Inhibition was observed in the leaves for the activities of nitrate reductase (NR, EC 1.6.6.1), ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) and deaminating glutamate dehydrogenase (NAD-GDH, EC 1.4.1.2). NaCl affected these enzyme activities less in the roots than in leaves. This was in accordance with the pronounced decrease of dry weight by salt in leaves compared with that in the roots. CONCLUSIONS: NaCl stress effects on growth, metabolite concentrations and enzyme activities depended on the duration of salt treatment and the plant tissue.     

Glutamate synthase isoforms in rice: immunological studies of enzymes in green leaf, etiolated leaf, and root tissues

Rabbit antiserum was raised against ferredoxin-dependent glutamate synthase (EC 1.4.7.1) purified from green leaves of Oryza sativa L. cv Delta. Ferredoxin-dependent glutamate synthase, detected in green leaf, etiolated leaf, and root tissues cross-reacted completely with the antiferredoxin glutamate synthase immunoglobulin G. In contrast, the immunoglobulin G did not cross-react with NADH-dependent (EC 1.4.1.14) and NADPH-dependent (EC 1.4.1.13) glutamate synthases found in nonphotosynthetic etiolated leaf and root tissues. In addition, ferredoxin-dependent glutamate synthase was separated and distinguished by its affinity to ferredoxin from NAD(P)H-dependent glutamate synthase on ferredoxin-Sepharose affinity chromatography. Based on the immunological studies, it is suggested that ferredoxin-dependent glutamate synthases in green leaf and etiolated leaf tissues are closely related proteins; in contrast, ferredoxin-dependent glutamate synthase in root tissue is a distinct protein from the leaf enzymes.     

Occurrence of ferredoxin-dependent glutamate synthase in plant cell fraction of soybean root nodules (Glycine max)

Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) and NADH-dependent glutamate synthase (EC 1.4.1.14) have been identified in the plant cells of soybean nodules. Ferredoxin-dependent glutamate synthase is 2-fold more active than NADH-dependent enzyme in vitro. Ferredoxin-dependent glutamate synthase cross-reacts with IgG against ferredoxin-dependent glutamate synthase of rice green leaves, whereas NADH-dependent glutamate synthase does not recognize the IgG, indicating that there are two distinct enzyme proteins. Ferredoxin-dependent glutamate synthase is composed of polypeptide chain(s) of 165 kDa and has a high affinity to spinach leaf ferredoxin as an electron carrier.     

NaCl effects on proline metabolism in rice (Oryza sativa) seedlings

Salt-stress effects on osmotic adjustment, ion and proline concentrations as well as proline metabolizing enzyme activities were studied in two rice ( Oryza sativa L.) cultivars differing in salinity resistance: I Kong Pao (IKP; salt-sensitive) and Nona Bokra (salt-resistant). The salt-sensitive cultivar exposed to 50 and 100 m M NaCl in nutritive solution for 3 and 10 days accumulated higher levels of sodium and proline than the salt-resistant cultivar and displayed lower levels of osmotic adjustment. Proline accumulation was not related to proteolysis and could not be explained by stress-induced modifications in Δ¹-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2) or proline dehydrogenase (PDH; EC 1.5.1.2) activities recorded in vitro. The extracted ornithine Δ -aminotransferase (OAT; EC 2.6.1.13) activity was increased by salt stress in the salt-sensitive cultivar only. In both genotypes, salt stress induced an increase in the aminating activity of root glutamate dehydrogenase (GDH; EC 1.4.1.2) while deaminating activity was reduced in the leaves of the salt-sensitive cultivar. The total extracted glutamine synthetase activity (GS; EC 6.3.1.2) was reduced in response to salinity but NaCl had contrasting effects on GS1 and GS2 isoforms in salt-sensitive IKP. Salinity increased the activity of ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) extracted from leaves of both genotypes and increased the activity of NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in the salt-sensitive cultivar. It is suggested that proline accumulation is a symptom of salt-stress injury in rice and that its accumulation in salt-sensitive plants results from an increase in OAT activity and an increase in the endogenous pool of its precursor glutamate. The physiological significance of the recorded changes are analyzed in relation to the functions of these enzymes in plant metabolism.     

The pathway of nitrogen assimilation in plants

The major route of nitrogen assimilation has been considered for many years to occur via the reductive amination of α-oxoglutarate, catalysed by glutamate dehydrogenase. However, recent work has shown that in most bacteria an alternative route via glutamine synthetase and glutamine: 2-oxoglutarate aminotransferase (glutamate synthase) operates under conditions of ammonia limitation. Subsequently the presence of a ferredoxin-dependent glutamate synthase in green leaves and green and blue-green algae, and a NAD(P)H and ferredoxin-dependent enzyme in roots and other non-green plant tissues, has suggested that this route may also function in most members of the plant kingdom. The only exceptions are probably the majority of the fungi, where so far most organisms studied do not appear to contain glutamate synthase. Besides the presence of the necessary enzymes there is other evidence to support the contention that the assimilation of ammonia into amino acids occurs via glutamine synthetase and glutamate synthase, and that it is unlikely that glutamate dehydrogenase plays a major role in nitrogen assimilation in bacteria or higher plants except in circumstances of ammonia excess.     

Glutamate synthase in rice roots. Studies on the electron donor specificity

Rice root glutamate synthase activity was assayed with various reducing systems. Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) and pyridine nucleotide-dependent glutamate synthase (NADH, EC 1.4.1.14; or NADPH, EC 1.4.1.13) exhibited a strict specificity for the electron donor. The ferredoxin-dependent glutamate synthase from rice roots could accept electrons from photoreduced ferredoxin in an illuminated reconstituted spinach chloroplast system. Thioredoxin, a potent electron carrier, was not able to provide either ferredoxin-dependent or pyridine nucleotide-dependent glutamate synthase with electrons as no glutamate formation was detected in the presence of reduced thioredoxin f or m.     

Properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803. Comparison with the Azospirillum brasilense NADPH-dependent enzyme and its isolated alpha subunit

The properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803 were determined by means of kinetic and spectroscopic approaches in comparison to those exhibited by the bacterial NADPH-dependent enzyme form. The ferredoxin-dependent enzyme was found to be similar to the bacterial glutamate synthase alpha subunit with respect to cofactor content (one FMN cofactor and one [3Fe-4S] cluster per enzyme subunit), overall absorbance properties, and reactivity of the FMN N(5) position with sulfite, as expected from the similar primary structure of ferredoxin-dependent glutamate synthase and of the bacterial NADPH-dependent glutamate synthase alpha subunit. The ferredoxin- and NADPH-dependent enzymes were found to differ with respect to the apparent midpoint potential values of the FMN cofactor and of the [3Fe-4S] cluster, which are less negative in the ferredoxin-dependent enzyme form. This feature is, at least in part, responsible for the efficient oxidation of L-glutamate catalyzed by this enzyme form, but not by the bacterial NADPH-dependent counterpart. At variance with earlier reports on ferredoxin-dependent glutamate synthase, in the Synechocystis enzyme the [3Fe-4S] cluster is not equipotential with the flavin cofactor. The present studies also demonstrated that binding of reduced ferredoxin to ferredoxin-dependent glutamate synthase is essential in order to activate reaction steps such as glutamine binding, hydrolysis, or ammonia transfer from the glutamine amidotransferase site to the glutamate synthase site of the enzyme. Thus, ferredoxin-dependent glutamate synthase seems to control and coordinate catalytic activities taking place at its subsites by regulating the reactions of the glutamine amidotransferase site. Association with reduced ferredoxin appears to be necessary, but not sufficient, to trigger the required activating conformational changes.     

A novel ferredoxin-dependent glutamate synthase from the hydrogen-oxidizing chemoautotrophic bacterium Hydrogenobacter thermophilus TK-6

Glutamate synthases are classified according to their specificities for electron donors. Ferredoxin-dependent glutamate synthases had been found only in plants and cyanobacteria, whereas many bacteria have NADPH-dependent glutamate synthases. In this study, Hydrogenobacter thermophilus, a hydrogen-oxidizing chemoautotrophic bacterium, was shown to possess a ferredoxin-dependent glutamate synthase like those of phototrophs. This is the first observation, to our knowledge, of a ferredoxin-dependent glutamate synthase in a nonphotosynthetic organism. The purified enzyme from H. thermophilus was shown to be a monomer of a 168-kDa polypeptide homologous to ferredoxin-dependent glutamate synthases from phototrophs. In contrast to known ferredoxin-dependent glutamate synthases, the H. thermophilus glutamate synthase exhibited glutaminase activity. Furthermore, this glutamate synthase did not react with a plant-type ferredoxin (Fd3 from this bacterium) containing a [2Fe-2S] cluster but did react with bacterial ferredoxins (Fd1 and Fd2 from this bacterium) containing [4Fe-4S] clusters. Interestingly, the H. thermophilus glutamate synthase was activated by some of the organic acids in the reductive tricarboxylic acid cycle, the central carbon metabolic pathway of this organism. This type of activation has not been reported for any other glutamate synthases, and this property may enable the control of nitrogen assimilation by carbon metabolism.     

The evolution of glutamate synthase

DNA coding for the ferredoxin-dependent glutamate synthase (EC1.4.7.1) of spinach chloroplasts has been cloned and sequenced. It consists of 5015 bp and starts with the codon for the N-terminal cysteine of the mature protein. Ferredoxin-dependent glutamate synthase is one of the key enzymes in the early stages of ammonia assimilation in plants, algae and cyanobacteria. In addition to the ferredoxin-dependent enzyme, there are two other forms of glutamate synthase, one of which uses NADH as the electron donor and a second that uses NADPH. Although all three forms catalyze the reductive transamidation of the amido nitrogen from glutamine to 2-oxoglutarate to form two molecules of glutamate, ferredoxin-dependent glutamate synthases differ from the NADH and NADPH-dependent forms in subunit composition and amino acid sequence. The recent availability of sequence data for glutamate synthases from spinach and from two archael species has produced a clearer and more detailed picture of the evolution of this key enzyme in nitrogen metabolism and the origins of the two subunit/domain structure of the enzyme.     

A new assay for ferredoxin-dependent glutamine oxoglutarate amino transferase (glutamate synthase) using electrolytically reduced methyl viologen

A new, quick assay is described for ferredoxin-dependent glutamate synthase from leaves, using electrolytically reduced methyl viologen. Activity, which is stimulated fivefold by ferredoxin, is measured spectrophotometrically as oxidation of methyl viologen. The assay does not require routine estimation of glutamate. Two molecules of glutamate were shown to be formed during the oxidation of two molecules of methyl viologen. Activities correspond to those previously reported for leaf homogenates.     

The development of NAD(P)H-dependent and ferredoxin-dependent glutamate synthase in greening barley and pea leaves

The activity of NAD(P)H-dependent glutamate synthase (E.C. 1.4.1.14) has been demonstrated in extracts from etiolated shoots of pea (Pisum sativum L.) and barley (Hordeum vulgare L.). This activity does not significantly alter upon greening of the etiolated shoots, and is at a similar level in light-grown material. Ferredoxin-dependent glutamate synthase (E.C. 1.4.7.1) has low activity in etiolated shoots but increases rapidly on greening. In light grown leaves ferredoxin-dependent activity is 30–40-fold higher than NAD(P)H-dependent activity. It is not considered that the NAD(P)H-dependent glutamate synthase plays an important role in ammonia assimilation in the photosynthetic tissue of higher plants.     

Different Characteristics of the Two Glutamate Synthases in the Green Leaves of Lycopersicon esculentum

The two glutamate synthases, NAD(P)H- and ferredoxin-dependent, from the green leaves of tomato plants (Lycopersicon esculentum L. cv Hellfrucht frühstamm) differed in their chemical properties and catalytic behavior. Gel filtration of NAD(P)H enzyme gave an apparent molecular size of 158 kilodalton, whereas the ferredoxin enzyme molecular size was 141 kilodalton. Arrhenius plots of the activities of the two enzymes showed that the NAD(P)H enzyme had two activation energies; 109.6 and 70.5 kilojoule per mole; the transition temperature was 22°C. The ferredoxin enzyme however, had only one activation energy; 56.1 kilojoule per mole. The respective catalytic activity pH optima for the NAD(P)H- dependent and the ferredoxin dependent enzymes were around 7.3 and 7.8. In experiments to evaluate the effects of modulators aspartate enhanced the NAD(P)H-linked activity, with a K_a value of 0.25 millimolar, but strongly inhibited that of the ferredoxin-dependent glutamate synthase with a K_i of 0.1 millimolar. 3-Phosphoserine was another inhibitor of the ferredoxin dependent enzyme with a K_i value of 4.9 millimolar. 3-Phosphoglyceric acid was a potent inhibitor of the ferredoxin-dependent form, but hardly affected the NAD(P)H-dependent enzyme. The results are discussed and interpreted to propose different specific functions that these activities may have within the leaf tissue cell.     

The interaction of ferredoxin and glutamate synthase: cross-linking and immunological studies

The water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) serves as an effective reagent for cross-linking spinach leaf ferredoxin and the ferredoxin-dependent spinach leaf enzyme, glutamate synthase. The cross-linked complex was functional in the absence of added ferredoxin, suggesting that ferredoxin is cross-linked to glutamate synthase at the physiological binding site on the enzyme for this iron-sulfur protein electron donor. The ferredoxin:glutamate synthase stoichiometry of the cross-linked complex was estimated to be 2:1. The absorbance spectrum of the oxidized, cross-linked complex was very similar to that of an electrostatically stabilized, noncovalent, 2:1 complex of the two proteins. An antibody raised against spinach NADP+ reductase, which recognizes a ferredoxin-binding site on glutamate synthase, does not recognize the cross-linked ferredoxin-glutamate synthase complex. This implies that the ferredoxin-binding sites on the two enzymes are structurally similar enough so that an antibody raised against one of these ferredoxin-dependent enzymes recognizes an epitope at the ferredoxin-binding site of the second enzyme. Cross-linking of ferredoxin to its binding site on glutamate synthase renders this epitope inaccessible to the antibody.     

Enzymes of Ammonia Assimilation, Photosynthesis, and Respiration in Alfalfa Leaves of Different Ages

The activities of enzymes involved in ammonia metabolism ferredoxin-dependent glutamate synthase (Fd-GOGAT), glutamine synthetase (GS) and glutamate dehydrogenase (GDH), the rates of photosynthetic oxygen evolution, dark respiration, and the activity of RuBP carboxylase (RuBPC) were determined in alfalfa (Medicago sativa L.) leaves taken from the apex (apical leaves), from the second to the fourth internode (mature leaves) and from the bottom of the canopy (basal leaves). Photosynthetic rate and the activities of RuBPC, GS and Fd-GOGAT showed their maximum in the mature leaves. The respiration rate together with amino acid and ammonium contents decreased with leaf age, whereas the opposite was true for GDH activity. Basal leaves still maintained substantial levels of chlorophylls, GS and Fd-GOGAT activities and oxygen evolution rate, thus suggesting that photosynthesis has some role in the reassimilation of the nitrogen liberated during protein degradation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Salinity-induced tissue-specific diurnal changes in nitrogen assimilatory enzymes in tomato seedlings grown under high or low nitrate medium.

We studied the salt stress (100 mM NaCl) effects on the diurnal changes in N metabolism enzymes in tomato seedlings (Lycopersicon esculentum Mill. cv. Chibli F1) that were grown under high nitrogen (HN, 5 mM NO(3)(-)) or low nitrogen (LN, 0.1 mM NO(3)(-)). NaCl stress led to a decrease in plant DW production and leaf surface to higher extent in HN than in LN plants. Total leaf chlorophyll (Chl) content was decreased by salinity in HN plants, but unchanged in LN plants. Soluble protein content was decreased by salt in the leaves from HN and LN plants, but increased in the stems-petioles from LN plants. Nitrate reductase (NR, EC 1.6.1.6) showed an activity peak during first part of the light period, but no diurnal changes were observed for the nitrite reductase (NiR, EC 1.7.7.1) activity. Glutamine synthetase (GS, EC 6.3.1.2) and glutamate synthase (Fd-GOGAT, EC 1.4.7.1) activities increased in HN plant leaves during the second part of the light period, probably when enough ammonium is produced by nitrate reduction. NR and NiR activities in the leaves were more decreased by NaCl in LN than in HN plants, whereas the opposite response was obtained for the GS activity. Fd-GOGAT activity was inhibited by NaCl in HN plant leaves, while salinity did not shift the peak of the NR and Fd-GOGAT activities during a diurnal cycle. The induction by NaCl stress occurred for the NR and GS activities in the roots of both HN and LN plants. Glutamate dehydrogenase (GDH, EC 1.4.1.2) activity shifted from the deaminating activity to the aminating activity in all tissues of HN plants. In LN plants, both aminating and deaminating activities were increased by salinity in the leaves and roots. The differences in the sensitivity to NaCl between HN and LN plants are discussed in relation to the N metabolism status brought on by salt stress.     

Senescence and protein degradation in leaf segments of young winter wheat: influence of leaf age

Leaf senescence in intact wheat plants can be strongly influencedby altered source/sink relations. Interactions with other plantparts are no longer possible in detached leaves and thereforedifferences in their senescence behaviour reflect the physiologicalstatus of the leaf before cutting. The net degradation of chlorophyllsand of selected enzyme proteins (detected by SDS-PAGE and immunoblotting)was delayed in detached young leaves as compared to senescingor mature leaves excised from the same field-grown wheat plants.The physiological leaf age was therefore decisive for the velocityof artificial senescence. Net degradation rates of the enzymesinvestigated varied in detached leaves. The protein quantitiesof plastidial glutamine synthetase, phosphoribulokinase andphosphoglycolate phosphatase decreased more rapidly than thoseof ferredoxin-dependent glutamate synthase and nitrite reductase.Differences were also detected between two enzymes involvedin the same metabolic pathway (photorespiratory carbon cycle)but located in different subcellular compartments: the plastidialenzyme phosphoglycolate phosphatase was lost more rapidly thanglycolate oxidase (peroxisomal enzyme). Key words: Detached leaves, senescence, proteolysis, leaf age, wheat     

Antigenic similarities between ferredoxin-dependent nitrite reductase and glutamate synthase fromChlamydomonas reinhardtii

Polyclonal antisera were prepared against ferredoxin-nitrite reductase (EC 1.7.7.1) and ferredoxin-glutamate synthase (glutamate synthase (ferredoxin); EC 1.4.7.1) from the green algaChlamydomonas reinhardtii. The anti-glutamate synthase antibodies recognized both glutamate synthase and nitrite reductase, but inhibited only the ferredoxin-linked activity of the latter enzyme and not the activity dependent on methyl viologen. Analogously, the anti-nitrite reductase antibodies recognized glutamate synthase and nitrite reductase but the first enzyme was only poorly inhibited. Free ferredoxin protected the nitrite reductase against its inactivation by anti-glutamate synthase antibodies. These results indicate that the ferredoxin-dependent glutamate synthase and nitrite reductase from this alga share common antigenic determinants, and that these are located at the ferrodoxin-binding domains.     

Ammonia Assimilation in Canavalia ensiformis Plants under Water and Salt Stress

Nitrogen fixation and ammonia assimilation in nodules have beenthoroughly studied under stress conditions, but the behaviorof enzymes involved in ammonia assimilation to organic compoundsin plants of the Leguminosae family subjected to stress stillremains to be conclusively established. We found that understress conditions, C. ensiformis plants can switch from theirusual pathway of assimilation to an alternative one dependingon the nature of the stress and the tissue in which the processtakes place. In roots, it switches from the glutamate dehydrogenase(GDH) pathway to the glutamine synthetase (GS)/glutamate synthase(GOGAT) cycle under water stress but not under salt stress.However, in leaves under salt stress, GDH activity is maintainedbut GS activity markedly decreases (Received March 24, 1987; Accepted March 4, 1988)