Overexpression of cytosolic glutamine synthetase. Relation to nitrogen,light, and photorespiration

In plants, ammonium released during photorespiration exceeds primary nitrogen assimilation by as much as 10-fold. Analysis of photorespiratory mutants indicates that photorespiratory ammonium released in mitochondria is reassimilated in the chloroplast by a chloroplastic isoenzyme of glutamine synthetase (GS2), the predominant GS isoform in leaves of Solanaceous species including tobacco (Nicotiana tabacum). By contrast, cytosolic GS1 is expressed in the vasculature of several species including tobacco. Here, we report the effects on growth and photorespiration of overexpressing a cytosolic GS1 isoenzyme in leaf mesophyll cells of tobacco. The plants, which ectopically overexpress cytosolic GS1 in leaves, display a light-dependent improved growth phenotype under nitrogen-limiting and nitrogen-non-limiting conditions. Improved growth was evidenced by increases in fresh weight, dry weight, and leaf soluble protein. Because the improved growth phenotype was dependent on light, this suggested that the ectopic expression of cytosolic GS1 in leaves may act via photosynthetic/photorespiratory process. The ectopic overexpression of cytosolic GS1 in tobacco leaves resulted in a 6- to 7-fold decrease in levels of free ammonium in leaves. Thus, the overexpression of cytosolic GS1 in leaf mesophyll cells seems to provide an alternate route to chloroplastic GS2 for the assimilation of photorespiratory ammonium. The cytosolic GS1 transgenic plants also exhibit an increase in the CO(2) photorespiratory burst and an increase in levels of photorespiratory intermediates, suggesting changes in photorespiration. Because the GS1 transgenic plants have an unaltered CO(2) compensation point, this may reflect an accompanying increase in photosynthetic capacity. Together, these results provide new insights into the possible mechanisms responsible for the improved growth phenotype of cytosolic GS1 overexpressing plants. Our studies provide further support for the notion that the ectopic overexpression of genes for cytosolic GS1 can potentially be used to affect increases in nitrogen use efficiency in transgenic crop plants.     

Involvement of photorespiration and glycolate pathway in carbonic anhydrase induction and inorganic carbon concentration in Chlamydomonas reinhardtii

Interaction between induction of carbonic anhydrase (CA) activity, induction of inorganic carbon (C_i) concentrating mechanisms and the photorespiratory glycolate pathway has been studied in wild type 6145c and photorespiratory mutant 18–7F (low in phosphoglycolate phosphatase activity) cells of C. reinhardtii . Cell transfer from high CO₂ (5%, v/v) to low CO₂ (0.03%) provoked an increase of extracellular and total (extracellular plus intracellular) CA in both wild type and mutant cells. During adaptation to low CO₂ conditions, both strains excreted ammonium to the medium at a similar rate in the presence of l -methionine- d-l -sulfoximine (MSX), an inhibitor of glutamine synthetase (GS). MSX also provoked ammonium excretion by air adapted wild type and mutant cells, even though both strains had high levels of CA activity and of C_i concentrating activities.
GS increased in both strains after transfer from high to low CO₂ conditions. However, this increase was abolished by aminooxyacetate, an inhibitor of the glyoxylate-serine aminotransferase, and by glycolaldehyde, an inhibitor of triose phosphate to ribulose 1,5-bisphosphate conversion. CA synthesis did not occur in the presence of either aminooxyacetate or glycolaldehyde. Algae grown in high CO₂ in the presence of aminooxyacetate did not induce C_i concentrating mechanisms. Integration of these three processes, i.e., CA synthesis, C_i-concentration, and photorespiratory glycolate pathway is proposed in the framework of carbon metabolism of the alga.     

Photorespiratory ammonia assimilation in the leaves of barley, sorghum and Moricandia arvensis

Effects of methionine sulphoximine (MSX) on glutamine synthetase activity and ammonia accumulation in the leaves of barley, sorghum and Moricandia arvensis were studied. Glutamine synthetase activity of sorghum leaves was relatively resistant to MSX treatment during initial stages. All the three plant species exhibited considerable accumulation of ammonia arising from non-photorespiratory sources, in response to MSX treatment. Minimal estimates of the rate of glycine decarboxylation were made employing MSX and isonicotinyl hydrazide together in the treatment solution. Changes in the pool sizes of amino acids involved in photorespiratory nitrogen metabolism as affected by MSX have also been investigated.     

The role of transamination in the synthesis of homoserine in peas

Incubation of intact pea plants (Pisum sativum), or detached shoots, in continuous light caused a substantial increase (up to 4-fold in 2 days) in levels of homoserine. Amino acids supplied to leaves in the transpiration stream enhanced the accumulation, with glutamate, aspartate, and asparagine causing similar enhancement. Aminooxyacetate (AOA), a transamination inhibitor, at 1 millimolar prevented the accumulation. ¹⁴C-labeling experiments showed that succinate was a good source of carbon for homoserine synthesis; carbon from aspartate or asparagine was also incorporated into homoserine. For each precursor, the transfer of label was prevented by AOA. The keto acid analog of homoserine was rapidly transaminated in leaves to give homoserine. The results suggest that accumulating homoserine is synthesised by transamination rather than being derived from aspartate via the aspartate kinase/homoserine dehydrogenase pathway. The latter pathway was shown to be operating in the chloroplasts, and was sensitive to threonine (but was not inhibited by AOA), suggesting that this path has a role in synthesis of aspartate-derived amino acids but is not involved in the accumulation of excess homoserine in the pea.     

Diurnal variation of asparaginase in developing pea leaves

Levels of asparaginase activity from developing pea leaves (Pisum sativum) were found to change on a daily basis, increasing during the light period and decreasing in the dark. During extended periods of light, high levels of activity were maintained, while prolonged dark reduced activity to a low value. Half-expanded leaves exhibited the greatest change in activity over the photoperiod. Very young or mature leaves displayed little or no diurnal variation in asparaginase activity.     

Ammonium and amino acid metabolism in excised leaves of wheat (Triticum aestivum) senescing in the dark

Several parameters of amino acid metabolism were studied in detached primary leaves of wheat (Triticum aestivum L. cv. Castell) during a 14 day incubation period in the dark. Protein loss was accompanied by a 5-fold increase in the total amount of free amino acids during the first 4 days of the incubation period with asparagine being the most important. Beyond this stage a pronounced intracellular accumulation of ammonium occured. A gradual decrease in the levels of free amino acids and ammonium at the later stages of senescence could in part be accounted for by leakage from the leaves. Additionally, some nitrogen was lost due to ammonia volatilization. The rapid decay of the glutamine synthetase (GS; EC 6.3.1.2)-glutamate synthase (Fd-GOGAT; EC 1.4.7.1) system and the fast decline of glutamate-pyruvate transaminase (GPT; EC 2.6.1.2) activity appear to be predominant features of senescence in the dark. Decreasing Fd-GOGAT activity was slightly compensated by a small and temporary increase in the activity of NADH-GOGAT (EC 1.4.1.14). Glutamateoxalocetate transaminase (GOT: EC 2.6.1.1) activity, although declining continuously, proved to be much more persistent. Changes in glutamate dehydrogenase (GDH; EC 1.4.1.3) activity closely resembled the profile of ammonium evolution in the leaves and NADP-isocitrate dehydrogenase (IDH; EC 1.1.1.42) activity revealed a temporary maximum during the period of rapid increase in GDH activity. Increased activity of GDH could also be induced by exogenous ammonium. Ammonium accumulation could, at least partly, be caused by increased asparaginase (EC 3.5.1.1) activity which accompanied the rapid conversion of asparagine to aspartic acid. Asparagine aminotransferase (EC 2.6.1.14) activity declined sharply from the beginning of the senescence period. Although the activity profile of glutaminase (EC 3.5.1.2) was similar to that of asparaginase, glutamine was of little importance quantitatively and an analogous relationship between glutamine and glutamic acid could not be detected.     

Photorespiratory ammonium assimilation in chloroplasts of Chlamydomonas reinhardtii

The effect of CO₂ concentration on the rate of photorespiratory ammonium excretion and on glutamine synthetase (GS) and carbonic anhydrase (CA) isoenzymes activities has been studied in Chlamydomonas reinhardtii cw-15 mutant (lacking cell wall) and in the high CO₂-requiring double mutant cia-3/cw-15 (lacking cell wall and chloroplastic carbonic anhydrase). In cw-15 cells, both the extracellular (CA_ext) and chloroplastic (CA_chl) CA activities increased after transferring cells from media bubbled with 5% CO₂ in air (v/v, high-C_i cells) to 0.03% CO₂ (low-C_i cells), whereas in cia-3/cw-15 cells only the CA_ext was induced after adaptation to low-C_i conditions and the CA_chl activity was negligible. During adaptation to low-C_i conditions in the presence of 1 mM of l-methionine-D,L-sulfoximine (MSX), a specific inhibitor of GS activity, both mutant strains excreted photorespiratory ammonium into nitrogen free medium. In addition, the ammonium excretion rate by cw-15 in the presence of MSX was lower in cells grown and kept at 5% CO₂ than in high-C_i cells adapted to 0.03% CO₂. The double mutant cia-3/cw-15 excreted photorespiratory ammonium at a higher rate than did cw-15. Total GS activity (GS-1 plus GS-2) increased during adaptation to 0.03% CO₂ in both strains of C. reinhardtii. However, only the activity GS-2, which is located in the chloroplast, increased during the adaptation to low CO₂, whereas the cytosolic GS-1 levels remained similar in high and low-C_i cells. We conclude that: (1) cia-3/cw-15 cells lack chloroplastic CA activity; (2) in C. reinhardtii photorespiratory ammonium is refixed in the chloroplasts through the GS-2/GOGAT cycle; and (3) chloroplastic GS-2 concentration changes in response to the variation of environmental CO₂ concentration.     

Effect of glutamine on glutamine-synthetase regulation in the green alga Monoraphidium braunii

Glutamine-synthetase (GS; EC 6.3.1.2) activity and protein levels were measured in crude extracts from Monoraphidium braunii Näegeli, strain 202-7d, cultures grown under different nitrogen sources. Only ammonium and l-glutamine promoted a partial enzyme inactivation, which, in the case of l-glutamine, was accompanied by a significant repression of GS. Methionine sulfoximine (MSX), a strong inhibitor of GS, produced a drastic inactivation of GS which was concomitant with a marked increase in GS protein as measured by rocket immunoelectrophoresis. Such an increase was prevented in the presence of cycloheximide. The effect of the l-glutamine analog on GS activity and protein was partially inhibited if l-glutamine was also added to cell cultures, possibly indicating competition in the transport of these two substances. In addition, the effects of MSX were reversed after longer times when cultures were treated with smaller concentrations of inhibitor. Treatment of cell cultures with azaserine, a specific inhibitor of glutamate synthase, the second enzyme acting in the ammonium assimilation pathway, promoted a strong GS inactivation and a partial repression of this enzyme, which paralleled a specific increase in the intracellular pools of glutamine High-performance liquid chromatography measurements of intracellular amino-acid concentrations showed that glutamine levels correlated negatively with GS concentration. A role for glutamine as a negative effector of GS synthesis is proposed.Abbreviations GS l-glutamine synthetase - GOGAT l-glu-tamine:2-oxoglutarate amidotransferase - MSX methionine sulfoximine During the course of this work, J.A. was supported by a fellowship from Junta de Andalucía, and J.M. G-F. by a fellowship from the Spanish Ministerio de Educatión y Ciencia. This work was supported by the Junta de Andalucía.     

Antisense reduction of serine hydroxymethyltransferase results in diurnal displacement of NH4+ assimilation in leaves of Solanum tuberosum

Serine hydroxymethyltransferase (SHMT) is part of the mitochondrial enzyme complex catalysing the photorespiratory production of serine, ammonium and CO(2) from glycine. Potato plants (Solanum tuberosum cv. Solara) with antisensed SHMT were generated to investigate whether photorespiratory intermediates accumulated during light lead to nocturnal activation of the nitrogen-assimilating enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT). The transformant lines contained 70-90% less SHMT protein, and exhibited a corresponding decrease in mitochondrial SHMT activity. SHMT antisense plants displayed lower photosynthetic capacity and accumulated glycine in light. Glycine was converted to serine in the second half of the light period, while serine, ammonium and glutamine showed an inverse diurnal rhythm and reached highest values in darkness. GS/GOGAT protein levels and activities in the transgenics also reached maximum levels in darkness. The diurnal displacement of NH(4)(+) assimilation was accompanied by a change in the subunit composition of GS(2), but not GS(1). It is concluded that internal accumulation of post-photorespiratory ammonium is leading to nocturnal activation of GS/GOGAT, and that the time shift in ammonia assimilation can constitute part of a strategy to survive photorespiratory impairment.     

Involvement of the Accumulation of Glutamine in the Initiation of Adventitious Buds in Stem Segments of Torenia

Active meristematic divisions in stem segments of Torenia culturedin vitro can be induced in the epidermis by application of cytokininor the calcium ionophore A23187 [GenBank] , resulting in the differentiationof adventitious buds. Endogenous free glutamine accumulatedat a high concentration in the epidermal tissues during theearly stages of such cultures. The accumulation of glutaminewas caused by an increase in glutamine synthetase (GS) activity,and the increase of GS activity was suppressed by the applicationof some inhibitors of GS activity, mRNA synthesis, protein synthesis,or calmodulin. Incorporation of these inhibitors into the culturemedium also inhibited initiation of adventitious buds. The inhibitoryeffect of an inhibitor of GS, methionine sulfoximine (MSX),was apparent only at the very begining of the culture, and theeffect could be overcome by the simultaneous addition of glutamine.The inhibitory action of MSX on initiation of buds seemed tobe caused by an accumulation of ammonium ions. Reduction inlevels of NH₄NO₃ in or its elimination from the culture mediumstimulated the initiation of adventitious buds. Therefore, boththe accumulation of glutamine and the reduction in levels ofammonium ions seem to play a role in the initiation of adventitiousbuds in stem., segments of Torenia. ¹Present address: Faculty of Agriculture, University of Saga,Honjo-cho, Saga, Saga, 840 Japan. (Received October 3, 1988; Accepted March 9, 1989)     

酰胺态氮瞬间调节荚膜红假单孢菌光合固氮活性的GS传感机制

天门冬酰胺(Asn)和谷氨酰胺(Gln)对荚膜红假单孢菌固氮酶活性抑制,在表观上类似于氨关闭效应,这种抑制效应由GS参与,相似于氨抑的传感机制。中断Gln代谢的6-diazo-5-oxo-L-norleucine(DON)存在时,氨抑的持续时间延长,与此相类似,Gln抑制加剧,这可能归之于Gln的积累。但是,Gln抑制被methionine sulfoximine(MSX,GS的抑制剂)消除,消除时MSX对Gln的浓度比值约为0.2,与氨抑消除所需的MSX对氨的浓度比值相当。此外,MSX消除氨抑不为DON拮抗,表明Gln抑制固氮酶活性由GS传感。然而,不能抑制GS转谷酰基活性的methionine suffone(MSF,谷氨酸的类似物)却与MSX相同,能消除Gln和氨对固氮活性的抑制。上述观察结果也可延伸至Asn的关闭固氮酶活性效应。     

Effect of Methionine Sulfoximine on the Accumulation of Ammonia in C(3) and C(4) Leaves : The Relationship between NH(3) Accumulation and Photorespiratory Activity

Additions of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase (GS), result in an increase in NH₃ in seedling leaves of C₃ (wheat [Triticum aestivum cv. Kolibri] and barley [Hordeum vulgare var Perth]) and C₄ (corn [Zea mays W6A × W182E] and sorghum [Sorghum Vulgare var MK300]) plants. NH₃ accumulation is higher in C₃ (about 17.8 micromoles per gram fresh weight per hour) than in C₄ (about 4.7 micromoles) leaves. Under ideal conditions, when photosynthesis is not yet inhibited by the accumulation of NH₃, the rate of NH₃ accumulation is about 16% of the apparent rate of photosynthesis. A maximum accumulation of NH₃ was elicited by 2.5 millimolar MSX and was essentially independent of the addition of NO₃⁻ during either the growth or experimental period. When O₂ levels in the air were reduced to 2%, MSX resulted in some accumulation of NH₃ (6.0 micromoles per gram fresh weight per hour). At these levels of NH₃, there was no significant inhibition of rates of CO₂ fixation. There was also a minor, but significant, accumulation of NH₃ in corn roots treated with MSX. Inhibitors of photorespiration (isonicotinic hydrazide, 70 millimolar; 2-pyridylhydroxymethanesulfonic acid, 20 millimolar) or transaminase reactions (aminooxyacetate, 1 millimolar) inhibited the accumulation of NH₃ in both C₃ and C₄ leaves. These results support the hypothesis that GS is important in the assimilation of NH₃ in leaves and that the glycine-serine conversion is a major source of that NH₃.     

Low assimilation efficiency of photorespiratory ammonia in conifer leaves

Glutamine synthetase (GS) localized in the chloroplasts, GS2, is a key enzyme in the assimilation of ammonia (NH₃) produced from the photorespiration pathway in angiosperms, but it is absent from some coniferous species belonging to Pinaceae such as Pinus. We examined whether the absence of GS2 is common in conifers (Pinidae) and also addressed the question of whether assimilation efficiency of photorespiratory NH₃ differs between conifers that may potentially lack GS2 and angiosperms. Search of the expressed sequence tag database of Cryptomeria japonica, a conifer in Cupressaceae, and immunoblotting analyses of leaf GS proteins of 13 species from all family members in Pinidae revealed that all tested conifers exhibited only GS1 isoforms. We compared leaf NH₃ compensation point (γ_NH3) and the increments in leaf ammonium content per unit photorespiratory activity (NH₃ leakiness), i.e. inverse measures of the assimilation efficiency, between conifers (C. japonica and Pinus densiflora) and angiosperms (Phaseolus vulgaris and two Populus species). Both γ_NH3 and NH₃ leakiness were higher in the two conifers than in the three angiosperms tested. Thus, we concluded that the absence of GS2 is common in conifers, and assimilation efficiency of photorespiratory NH₃ is intrinsically lower in conifer leaves than in angiosperm leaves. These results imply that acquisition of GS2 in land plants is an adaptive mechanism for efficient NH₃ assimilation under photorespiratory environments.     

Photorespiratory ammonium release by the cyanobacterium Anabaena cylindrica in the presence of methionine sulfoximine

A release of ammonium by non-nitrogen-fixing Anabaena cylindrica (grown on NH₄Cl) in the presence of MSX (methionine sulfoximine) and absence of any external nitrogen source was found. In the light the release was maximal at 0.2 mM MSX, a concentration which did not affect net CO₂ fixation nor the glycollate excretion, but inhibited the glutamine synthetase activity and the reassimilation of ammonium. It is suggested that the major source of the ammonium released is the photorespiratory conversion of glycine to serine as (1) the release was stimulated by increase in light intensity, (2) high CO₂ (3%) lowered the release, if not given as a longer pretreatment (as CO₂ or HCO ₃ ^- ) when a stimulation was observed, (3) glyoxylate and glutamate stimulated the release, the latter compound particularly under nitrogen-deficient conditions and (4) isonicotinic acid hydrazide caused a reduced release of ammonium. Furthermore, a substantial part of the ammonium released by N₂-fixing A. cylindrica in presence of MSX may thus originate from the glycollate pathway. The data show that in the light the glycine to serine conversion is active in cyanobacteria with a concomitant production of ammonium which is assimilated by glutamine synthetase.Abbreviations MSX L-methionine-Dl-sulfoximine - INH isonicotinic acid hydrazide - RuDP ribulose 1,5-diphosphate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - GS glutamine synthetase - GOGAT glutamate synthase - DTT Dl-dithiothreitol     

Glutamine synthetase and glutamate synthase from Sclerotinia sclerotiorum

Glutamine synthetase (GS, EC 6.3.1.2) and glutamate synthase (GOGAT, EC 1.4.1.13) were purified from Sclerotinia sclerotiorum and some of their properties studied. The GS transferase and biosynthetic activities, as well as GOGAT activity, were sensitive to feedback inhibition by amino acids and other metabolites. GS showed a marked dependence on ADP in the transferase reaction and on ATP in the Mg²⁺-dependent biosynthetic reaction. Regulation of GS activity by adenylylation/deadenylylation was demonstrated by snake venom phosphodiesterase treatment of the purified enzyme. GOGAT required NADPH as an electron donor; NADH was inactive. GOGAT was strongly inhibited by p-chloromercuribenzoate and the inhibition was reversed by cysteine. The enzyme was also markedly inhibited by o-phenanthroline, 2,2′-bipyridyl and azaserine. l-Methionine-dl-sulphoximine (MSX) and azaserine inhibited the incorporation of ¹⁵N-labelled ammonium sulphate into washed cells of S. sclerotiorum. MSX and azaserine respectively also inhibited purified GS and GOGAT activities. GDH activity was not detected in cell-extracts. Thus the GS/GOGAT pathway is the main route for the assimilation of ammonium compounds in this fungus.     

Changes in ammonium ion content and glutamine synthetase activity in rice leaves caused by excess cadmium are a consequence of oxidative damage

Ammonium ion accumulation and the decrease in glutamine synthetase (GS)activity induced by CdCl₂ were investigated in relation to lipidperoxidation in detached rice leaves. CdCl₂ was effective inincreasing ammonium ion content, decreasing GS activity and increasing lipidperoxidation. Free radical scavengers (glutathione, thiourea, sodium benzoate)and an iron chelator (2,2-bipyridine) were able to inhibit the decreasein GS activity and ammonium ion accumulation caused by CdCl₂ and atthe same time inhibit CdCl₂-induced lipid peroxidation. Paraquat,which is known to produce oxygen radicals, decreased GS activity, increasedammonium ion content, and increased lipid peroxidation. GS1 appears to be thepredominant isoform present. Excess Cd caused a decrease in GS1 but not in GS2in detached rice leaves. An increase in lipid peroxidation preceded ammoniumionaccumulation and the decrease in GS1 activity. These results suggest that thedecrease in GS activity and the accumulation of ammonium ions in detached riceleaves are a consequence of oxidative damage caused by excess Cd.     

CONTRASTING EFFECTS OF METHIONINE SULFOXIMINE ON UPTAKE AND ASSIMILATION OF AMMONIUM IN ULVA INTESTINALIS (CHLOROPHYCEAE)1

Ammonium is assimilated in algae by the glutamine synthetase (GS)–glutamine:2‐oxoglutarate aminotransferase pathway. In addition to the assimilation of external ammonium taken up across the cell membrane, an alga may have to reassimilate ammonium derived from endogenous sources (i.e. nitrate reduction, photorespiration, and amino acid degradation). Methionine sulfoximine (MSX), an irreversible inhibitor of GS, completely inhibited GS activity in Ulva intestinalis L. after 12 h. However, assimilation of externally derived ammonium was completely inhibited after only 1–2 h in the presence of MSX and was followed by production of endogenous ammonium. However, endogenous ammonium production in U. intestinalis represented only a mean of 4% of total assimilation attributable to GS. The internally controlled rate of ammonium uptake (V_i) was almost completely inhibited in the presence of MSX, suggesting that V_i is a measure of the maximum rate of ammonium assimilation. After complete inhibition of ammonium assimilation in the presence of MSX, the initial or surge (V_s) rate of ammonium uptake in the presence of 400 μM ammonium chloride decreased by only 17%. However, the amount that the rate of ammonium uptake decreased by was very similar to the uninhibited rate of ammonium assimilation. In addition, the decrease in the rate of ammonium uptake in darkness (in the absence of MSX) in the presence of 400 μM ammonium chloride matched the decrease in the rate of ammonium assimilation. However, in the presence of 10 μM ammonium chloride, MSX completely inhibited ammonium assimilation but had no effect on the rate of uptake.     

NITROGEN METABOLISM AND AMINO ACID NUTRITION IN THE SOIL ALGA STICHOCOCCUS BACILLARIS (CHLOROPHYCEAE)1

Nitrate-grown cells of Stichococcus bacillaris Naeg. (UTEX 314) contained much higher activities of glutamine synthetase (GS) and NADPH-glutamate dehydrogenase (GDH) than ammonium-grown cells. Methylamine, a non-metabolizable ammonium analog, caused a decrease in GS activity in nitrate-grown cells suggesting that GS is regulated by the size of the endogenous ammonium pool. The decrease in GS observed in methylammonium-loaded nitrate-grown cells was accompanied by an increase in NADPH-GDH activity. Stichococcus bacillaris can be grown in the presence of methionine sulfoximine (MSX), a potent inhibitor of GS. However, only a fraction of a control cell population showed a requirement for glutamine or arginine for growth following MSX addition. Fully adapted MSX-grown cells were indistinguishable from control cells in their ability to photosynthesize and utilize amino acids as nitrogen sources. Alanine, arginine, asparagine, glutamine, glycine and proline were good nitrogen sources, and maximum capacity for amino acid transport was developed in cells grown on these amino acids. Compared to nitrate-grown cells the activity of GS in ammo acid-grown cells was low, whereas NADPH-GDH was very active. The activity of NADH-GDH in amino acid-grown cells was highest under heterotrophic conditions.     

Tobacco isoenzyme 1 of NAD(H)-dependent glutamate dehydrogenase catabolizes glutamate in vivo

Glutamate (Glu) dehydrogenase (GDH, EC 1.4.1.2-1.4.1.4) catalyzes in vitro the reversible amination of 2-oxoglutarate to Glu. The in vivo direction(s) of the GDH reaction in higher plants and hence the role(s) of this enzyme is unclear, a situation confounded by the existence of isoenzymes comprised totally of either GDH beta- (isoenzyme 1) or alpha- (isoenzyme 7) subunits, as well as another five alpha-beta isoenzyme permutations. To clarify the in vivo direction of the reaction catalyzed by GDH isoenzyme 1, [(15)N]Glu was supplied to roots of two independent transgenic tobacco (Nicotiana tabacum) lines with increased isoenzyme 1 levels (S4-H and S49-H). The [(15)N]ammonium (NH(4)(+)) accumulation rate in these lines was elevated approximately 65% compared with a null segregant control line, indicating that isoenzyme 1 catabolizes Glu in roots. Leaf glutamine synthetase (GS) was inhibited with a GS-specific herbicide to quantify any contribution by GDH toward photorespiratory NH(4)(+) reassimilation. Transgenic line S49-H did not show enhanced resistance to the herbicide, indicating that the large pool of isoenzyme 1 in S49-H leaves was unable to compensate for GS and suggesting that isoenzyme 1 does not assimilate NH(4)(+) in vivo.     

Photorespiratory NH(4)(+) production in leaves of wild-type and glutamine synthetase 2 antisense oilseed rape

Exposure of oilseed rape (Brassica napus) plants to increasing leaf temperatures between 15 degrees C and 25 degrees C increased photorespiratory NH(4)(+) production from 0.7 to 3.5 micromol m(-2) s(-1). Despite the 5-fold increase in the rate of NH(4)(+) production, the NH(4)(+) concentration in root and leaf tissue water and xylem sap dropped significantly, whereas that in the leaf apoplastic fluid remained constant. The in vitro activity of glutamine synthetase (GS) in both leaves and roots also increased with temperature and in all cases substantially exceeded the observed rates of photorespiratory NH(4)(+) production. The surplus of GS in oilseed rape plants was confirmed using GS2 antisense plants with 50% to 75% lower in vitro leaf GS activity than in the wild type. Despite the substantial reduction in GS activity, there was no tendency for antisense plants to have higher tissue NH(4)(+) concentrations than wild-type plants and no overall correlation between GS activity and tissue NH(4)(+) concentration was observed. Antisense plants exposed to leaf temperatures increasing from 14 degrees C to 27 degrees C or to a trifold increase in the O(2) to CO(2) ratio did not show any change in steady-state leaf tissue NH(4)(+) concentration or in NH(3) emission to the atmosphere. The antisense plants also had similar leaf tissue concentrations of glutamine, glycine, and serine as the wild type, whereas glutamate increased by 38%. It is concluded that photorespiration does not control tissue or apoplastic levels of NH(4)(+) in oilseed rape leaves and, as a consequence, that photorespiration does not exert a direct control on leaf atmosphere NH(3) fluxes.