The chloroplastic glutamine synthetase (GS-2) of tobacco is phosphorylated and associated with 14-3-3 proteins inside the chloroplast.

The chloroplastic isoform of glutamine synthetase (GS-2, EC 6.3.1.2) from Nicotiana tabacum L. is phosphorylated at the serine residues. At least three of the six GS-2 subunits separated by two-dimensional polyacrylamide gel electrophoresis cross-reacted with an antibody raised against phosphoserine. This provoked the question as to whether 14-3-3 proteins might be present in the chloroplast and bind to chloroplastic GS-2. Although two different 14-3-3 proteins of 32 and 30 kDa were present in total leaf extracts, in the soluble fraction of chloroplasts, only the 32-kDa 14-3-3 protein was immunodetected with an antibody raised against a conserved region of 14-3-3 protein from corn. This demonstrates the presence of a chloroplast-located isoform of 14-3-3 proteins in tobacco. To examine a putative binding of GS-2 to these 14-3-3 proteins in vivo, the native GS-2 holoenzyme was probed with a 14-3-3 antibody. The strong cross-reaction between GS-2 and the 14-3-3 antibody clearly points to a binding of GS-2 and 14-3-3 in tobacco chloroplasts. Only those oligomers of GS-2 that were strongly associated with 14-3-3 proteins were catalytically active.

Isolation and characterization of nitrite-reductase-deficient mutants of Chlorella sorokiniana (strain 211-8k).

A method is presented to isolate mutants of Chlorella sorokiniana with defects in NO3- metabolism. Three nitrite-reductase (NIR; E.C.1.7.7.1)-deficient mutants were obtained from 500 pinpoint-colony-forming clones. The final screening was performed using NO3-, NO2- or NH4+ as N-source. The mutants isolated absorb NO3- with rates close to those measured for the wild type and they excrete NO2- into the medium. The ratio between NO3- uptake and NO2- excretion was 1:1. The sensitivity of NO3- uptake to NH4+ was reduced in the mutant strains as it was in the N-starved wild type of Chlorella. Nitrate reductase (NR; EC 1.6.6.1) expression and NR activity were slightly reduced compared to the wild type due to feedback regulation in the mutant strains. No NIR protein was found in the three mutants. However, NIR activity was obtained (50% of the wild-type) for one mutant strain. The NIR-deficient mutants and the already available NR-deficient mutants will be promising tools for investigations of the nitrate assimilation pathway on the molecular level and for studies searching for signaling of C and N metabolism by inorganic N-compounds.

Reduction of the nitrogen and carbon content in swine waste with algae and bacteria.

Animal waste causes environmental problems like eutrophication of ground and surface water or the pollution of the atmosphere because of its high NH4+ content. The aim of our study was to fix the nitrogen of swine waste as biomass. Therefore, an isolated alga, Chlorella sp., and bacteria naturally living in liquid manure were grown in batch cultures (containing diluted swine waste supplied with a nutrient solution) and continuous cultures (undiluted liquid manure) to achieve reduction of NH4+ and total organic carbon (TOC) contents. For continuous cultivation, a photobioreactor of our own design was used. The batch cultivation of Chlorella sp. and bacteria in swine waste resulted in good growth of both groups of organisms and in a reduction of 25% NH4+ and 80% TOC. In the continuous cultivation a steady state was not achieved owing to a change in the composition of the bacterial population. NH4+ was totally removed, but NO2- (up to 100 mM) was transiently released. NO3- was not detected. These effects might be explained by the presence of heterotrophic nitrifiers, which are able to oxidize NH4+ to NO2- and to reduce NO2- to gaseous compounds.

The effects of SO2 fumigation on the nitrogen metabolism of aseptically grown spruce seedlings.

Aseptically grown spruce seedlings were cultivated in a hydroponic system, where the roots were separated from the shoots by a gastight, silicone material. The plants were fumigated with four SO(2) concentrations (93, 190, 270 and 530 microg m(-3)) for nine weeks. Up to 270 microg m(-3) of SO(2), an inhibition of nitrogen metabolism (enzyme activities of nitrate reductase (NR) and glutamine sythetase (GS) and nitrate content) in the shoot was compensated by a stimulation in the root, while nitrogen uptake was unaffected. Only the treatment with 530 microg m(-3) of SO(3) decreased enzyme activities, nitrate content in both roots and shoots as well as nitrate uptake, and inhibited the growth of plants. Increases in the content of thiols and superoxidismutase activity are discussed in terms of SO(2) detoxification.

Influence of a high Mn supply on Norway spruce (Picea abies (L.) Karst.) seedlings in relation to the nitrogen source.

Effects of 3, 25, 100, 200 and 800 microM Mn on biomass and pigment, starch and nitrate concentrations were studied in Norway spruce (Picea abies (L.) Karst.) seedlings grown with either NO(3) (-) or NH(4) (+) as the sole nitrogen source. After 77 days of exposure to 800 microM Mn, shoot growth had ceased in about 50% of the seedlings independently of the N source. Despite high Mn concentrations in roots and shoots of the Mn-treated seedlings, no visible symptoms of Mn toxicity were evident. The rate of root elongation was decreased by treatment with >/= 200 microM Mn when N was supplied as NO(3) (-), but not when it was supplied as NH(4) (+). This difference could be attributed to the higher Mn concentrations in root tips of the NO(3) (-)-grown seedlings compared with the NH(4) (+)-grown seedlings. In Mn-treated seedlings, the concentration of Mg, and to a lesser extent that of Ca, decreased. Depletion of these elements might account for the observed growth depression. Potassium concentrations were similar in the control and Mn-treated seedlings. Treatment of seedlings with 800 microM Mn for 50 days led to several physiological changes: starch accumulated, the concentrations of nitrate and phenolic compounds increased, pigment concentrations decreased, and in vivo nitrate reductase activity in roots was reduced.

Vegetative storage proteins in poplar : induction and characterization of a 32- and a 36-kilodalton polypeptide.

Bark, wood, and root tissues of several Populus species contain a 32- and a 36-kilodalton polypeptide which undergo seasonal fluctuations and are considered to be storage proteins. These two proteins are abundant in winter and not detectable in summer as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection. An antibody raised against the 32-kilodalton storage protein of Populus trichocarpa (T. & G.) cross-reacts with the 36-kilodalton protein of this species. The synthesis of the 32- and 36-kilodalton proteins can be induced in micropropagated plants by short-day conditions in the growth chamber. These proteins are highly abundant in structural roots, bark, and wood and combined represent >25% of the total soluble proteins in these tissues. Nitrate concentration in the leaves and nitrate uptake rate decreased dramatically when LD plants were transferred to short-day conditions; the protein content in leaves was unaffected. A decrease of the 32- and 36-kilodalton polypeptides occurs after transferring induced plants back to LD conditions. Both polypeptides are glycosylated and can be efficiently purified by affinity chromatography using concanavalin A-Sepharose 4B. The 32- and the 36-kilodalton polypeptides have identical basic isoelectric points and both consist of at least three isoforms. The storage proteins show a loss in apparent molecular mass after deglycosylation with trifluoromethanesulfonic acid. It is concluded that the 32- and 36-kilodalton polypeptides are glycoforms differing only in the extent of glycosylation. The relative molecular mass of the native storage protein was estimated to be 58 kilodalton, using gel filtration. From the molecular mass and the elution pattern it is supposed that the storage protein occurs as a heterodimer composed of one 32- and one 36-kilodalton subunit. Preliminary data suggest the involvement of the phytochrome system in the induction process of the 32- and 36-kilodalton polypeptides.

Glutamine synthetase oligomers and isoforms in sugarbeet (Beta vulgaris L.).

The α-amino-N compounds that accumulate in the thickening storage root of sugarbeet (Beta vulgaris L.) were synthesized in the leaves (NO 3 (-) nutrition) and also in the lateral roots (NH 4 (+) nutrition). Ammonium stimulated glutamine synthetase (GS, EC 6.3.1.2) activity, especially in the lateral roots. With non-denaturing polyacrylamide-gel isoelectric focussing, simultaneously active charge-isomers of GS were separated in both leaves and roots. The leaf isoforms were active in an octameric and also in a tetrameric form. In the root only octameric isoforms were found. The tetramer was more active than the octamer in the leaf blade and vice versa in the leaf stem. Only the tetramer needed ß-mercaptoethanol for activity stabilization in vitro. A reactivation, however, of an inactive tetramer by the addition of thiol/thioredoxin was not possible. The same isoforms of GS were separated in different organs of sugarbeet but with different patterns of relative activity. The activity pattern depended also on the N-source of the plant. With increasing age of the plant the number of active GS isoforms declined in both leaves and roots although the in-vitro activity remained unchanged (NO 3 (-) -fed plants) or even increased (NH 4 (+) -fed plants).

The effect of endogenous and externally supplied nitrate on nitrate uptake and reduction in sugarbeet seedlings.

The pericarp of the dormant sugarbeet fruit acts as a storage reservoir for nitrate, ammonium and α-amino-N. These N-reserves enable an autonomous development of the seedling for 8-10 d after imbibition. The nitrate content of the seed (1% of the whole fruit) probably induces nitrate-reductase activity in the embryo enclosed in the pericarp. Nitrate that leaks out of the pericarp is reabsorbed by the emerging radicle. Seedlings germinated from seeds (pericarp was removed) without external N-supply are able to take up nitrate immediately upon exposure via a low-capacity uptake system (vmax = 0.8 µmol NO 3 (-) ·(g root FW)(-1)·h(-1); Ks = 0.12 mM). We assume that this uptake system is induced by the seed nitrate (10 nmol/seed) during germination. Induction of a high-capacity nitrate-uptake system (vmax = 3.4 µmol NO 3 (-) ·(g root FW)(-1)·h(-1); Ks = 0.08 mM) by externally supplied nitrate occurs after a 20-min lag and requires protein synthesis. Seedlings germinated from whole fruits absorb nitrate via a highcapacity uptake mechanism induced by the pericarp nitrate (748 nmol/pericarp) during germination. The uptake rates of the high-capacity system depend only on the actual nitrate concentration of the uptake medium and not on prior nitrate pretreatments. Nitrate deprivation results in a decline of the nitrate-uptake capacity (t1/2 of vmax = 5 d) probably caused by the decay of carrier molecules. Small differences in Ks but significant differences in vmax indicate that the low- and high-capacity nitrate-uptake systems differ only in the number of identical carrier molecules.

Characterization of Nitrate Reductase Deficient Mutants of Chlorella sorokiniana.

After x-ray irradiation, 13 mutants of Chlorella sorokiniana incapable of using NO(3) (-) as N source were isolated using a pinpoint method. Using immunoprecipitation and Western blot assays, no nitrate reductase was found in five strains while in eight mutants the enzyme was detected. The latter strains contained different patterns of nitrate reductase partial reactions. All isolates were of the nia-type as indicated by the inducibility of purine hydroxylase I and by complementation of nitrate reductase activity in the Neurospora crassa mutant Nit-1. A restoration of NADP-nitrate reductase in Nit-1 was also obtained with NH(4) (+)-grown cells indicating that Mo-cofactor is constitutive in Chlorella. Complementation experiments among the Chlorella mutants resulted in restoration of NADH-nitrate reductase activity. The characteristics of some of the Chlorella mutants are discussed in view of an improper orientation of Mo-cofactor in the residual nitrate reductase protein.

Synthesis and degradation of nitrate reductase during the cell cycle of Chlorella sorokiniana.

Studies on the diurnal variations of nitrate reductase (NR) activity during the life cycle of synchronized Chlorella sorokiniana cells grown with a 7:5 light-dark cycle showed that the NADH:NR activity, as well as the NR partial activities NADH:cytochrome c reductase and reduced methyl viologen:NR, closely paralleled the appearance and disappearance of NR protein as shown by sodium dodecyl sulfate gel electrophoresis and immunoblots. Results of pulse-labeling experiments with [35S]methionine further confirmed that diurnal variations of the enzyme activities can be entirely accounted for by the concomitant synthesis and degradation of the NR protein.

Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana.

Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrate uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.

Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana.

Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrite uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.

Inhibition of nitrate transport by anti-nitrate reductase IgG fragments and the identification of plasma membrane associated nitrate reductase in roots of barley seedlings.

Membrane associated nitrate reductase (NR) was detected in plasma membrane (PM) fractions isolated by aqueous two-phase partitioning from barley (Hordeum vulgare L. var CM 72) roots. The PM associated NR was not removed by washing vesicles with 500 millimolar NaCl and 1 millimolar EDTA and represented up to 4% of the total root NR activity. PM associated NR was stimulated up to 20-fold by Triton X-100 whereas soluble NR was only increased 1.7-fold. The latency was a function of the solubilization of NR from the membrane. NR, solubilized from the PM fraction by Triton X-100 was inactivated by antiserum to Chlorella sorokiniana NR. Anti-NR immunoglobulin G fragments purified from the anti-NR serum inhibited NO3- uptake by more than 90% but had no effect on NO2- uptake. The inhibitory effect was only partially reversible; uptake recovered to 50% of the control after thorough rinsing of roots. Preimmune serum immunoglobulin G fragments inhibited NO3- uptake 36% but the effect was completely reversible by rinsing. Intact NR antiserum had no effect on NO3- uptake. The results present the possibility that NO3- uptake and NO3- reduction in the PM of barley roots may be related.

Regulation of assimilatory nitrate reduction at the level of nitrite in Chlorella fusca.

Batch cultures of Chlorella fusca excreted nitrite into the medium if gassed with air (0.03% CO2), but they did not if supplied with air containing 5% CO2. After a change from high to low CO2 concentration in the gas stream, nitrite excretion started immediately. After an increase in CO2 concentration to 5%, nitrite uptake started within only 30 min. Changes of in-vitro activities of nitrate reductase, nitrite reductase and glutamine synthetase did not correspond to changes of nitrite concentration in the medium and therefore could not explain these observations. A nitrite-binding site, whose activity corresponded with both nitrite excretion and uptake, was detected at the chloroplast envelope. From these data an additional regulatory step in the assimilatory nitrate-reduction sequence is suggested. This includes an envelopeprotein fraction probably regulating the availability of nitrite within the chloroplast.

Induction of a high-capacity nitrate-uptake mechanism in barley roots prompted by nitrate uptake through a constitutive low-capacity mechanism.

Roots of nitrate-starved and nitrate-pretreated seedlings of Hordeum vulgare were used to investigate the induction of a high-capacity uptake mechanism for nitrate. When exposed to 0.2 mmol·l(-1)KNO3, nitrate-starved roots took up nitrate at a rate of approx. 1 µmol·(g FW)(-1)·h(-1); K(+) was absorbed at a rate ten-times higher. Nitrate uptake accelerated after a lag of about 1 h, until it matched the rate of K(+) uptake about 4 h later. p-Fluorophenylalanine (FPA), which prevents the synthesis of functioning proteins, suppressed the development of the high-capacity mechanism. Pretreatment of the roots with 0.2 mmol·l(-1) Ca(NO3)2 for 24 h established the high-capacity mechanism. Pretreated roots were able to absorb nitrate at high rates immediately upon exposure to 0.2 mmol·l(-1)KNO3, in the absence or presence of FPA. The high-capacity mechanism, once established, appeared to have a protein turnover as slow as that of the low-capacity mechanism or that of the mechanism involved in the uptake of K(+). In contrast, the mechanisms for the transport of nitrate and K(+) into the xylem vessels were completely blocked by FPA within 1 h of application, confirming earlier evidence for a rapid turnover of the transport proteins in the xylem parenchyma.Nitrate reduction proceeded at rates which were roughly one-tenth as large as the rates of the respective nitrate-uptake processes, indicating that nitrate-reductase activity was determined by the rate of nitrate uptake and not vice versa.We conclude that the formation of a high-capacity nitrate-uptake mechanism in barley roots occurs in response to nitrate uptake through a constitutive mechanism of low capacity which appears to function as a sensing mechanism for nitrate in the environment of the roots.

A Circadian Rhythm in the Number of Daughter Cells in Synchronous Chlorella fusca var vacuolata.

Chlorella fusca var vacuolata cells were transferred to continuous darkness or weak light (0.07 watts per square meter) (both were called waiting time, WT) after a 12-hour light and 12-hour dark schedule. A daily dilution is performed at the end of the light/dark schedule, resulting in always the same average production of 18 autospores per mother cell. After 12 and 24 hours of WT in darkness, the production of autospores in a subsequent light/dark schedule was 50 and 100%, respectively. If the WT was performed in weak light (0.07 watts per square meter) the lowest production was obtained, independently of the length of WT. However, an interruption of this weak light by dark pulses (3 hours) increased the autospore production by an amount that depends upon the phase of the circadian rhythm, and varied up to 70% of the control (WT in permanent darkness). If the WT (total darkness) was interrupted by light pulses of 0.5 hour (white, same as used for growth), a phase response curve of productivity resulted. Pulses between the 12th and 18th hour of WT in darkness gave a 3-hour delay of maximum; later on pulses shifted the maximum autospore production 3 hours ahead.

An L-cysteine Dependent Nitrate Reductase Inactivating Factor in Synchronous Chlorella sorokiniana.

Nitrate reductasee activity of cell-free Chlorella systems is inactivated in the presence of specific thiols. Out of 14 different thiols tested only L-cysteine and to a certain extent D-cysteine catalyzed an inactivation of the Chlorella nitrate reductase. This systeme was active only with reduced L-cysteine, since oxidized cystine had no effect, demonstrating that the reduced thiol group is necessary. A factor not identical with the nitrate reductase itself seems involved in this inactivation system. Evidence for this is that purified nitrate reductase is not inactivated by L-cysteine; however, when purified nitrate reductase was added to extracts inactivated by L-cysteine it was inactivated as well. The highest rate of degradation was found at a pH around 8. The data suggest that the inactivation factor from Chlorella is a proteinase, which has to be activated by L-cysteine. These results are discussed in relation to the regulation of assimilatory nitrate reduction.

A comparison of the high-active and low-active form of nitrate reductase in synchronous Chlorella sorokiniana.

Chlorella sorokiniana possesses two forms of nitrate reductase (EC 1.6.6.1.). One with low activity is present in cells at the end of the light-dark cycle, the other with high activity is present after 1 h of illumination. The two forms can be distinguished by gel electrophoresis, isopycnic centrifugation, assay of the partial reactions and their sensitivity to antibodies, respectively. These differences are discussed with respect to an effect of intracellular nitrate on the activation of nitrate reductase.

A Thioredoxin-Mediated Activation of Glutamine Synthetase and Glutamate Synthase in Synchronous Chlorella sorokiniana.

The effects of thioredoxin, dithioerythrol, and mixtures of both on enzymes involved in N metabolism of Chlorella sorokiniana have been studied. Glutamine synthetase, inactivated in vivo, was activated 8-fold by thioredoxin and dithioerythrol. By the same treatment, the activity of glutamate synthase was stimulated nearly 4-fold. Thus, two key enzymes of N metabolism were shown to be regulated via thioredoxin. The enzymes of the nitrate reducing system, i.e. nitrate reductase and nitrite reductase, were not affected by thiols. From these results, a model of NO(3) (-) metabolism is put forward which considers the regulating effect of light.

Regulation of Glutamine Synthetase by Light and during Nitrogen Deficiency in Synchronous Chlorella sorokiniana.

A method is described to achieve density labeling of proteins in unicellular algae by using (13)CO(2). This is a satisfactory procedure especially for work on nitrogen metabolism. The increase in activity of glutamine synthetase (EC 6.3.1.2.) and glutamate synthase (EC 1.4.7.1.) in Chlorella sorokiniana mediated by a dark/light shift and by nitrogen starvation were investigated. Using the method of density labeling and isopycnic centrifugation, we demonstrated that the increase in enzyme activity after a dark/light shift is based on activation rather than de novo synthesis. The increase in enzyme activity after transfer to nitrogen-deficient medium is based both on activation and de novo synthesis.