Evidence for a cytosolic-dependent light induction of chloroplastic glutamine synthetase during greening of etiolated rice leaves

During the greening of etiolated rice leaves, total glutamine synthetase activity increases about twofold, and after 48 h the level of activity usually observed in green leaves is obtained. A density-labeling experiment with deuterium demonstrates that the increase in enzyme activity is due to a synthesis of the enzyme. The enhanced activity obtained upon greening is the result of two different phenomena: there is a fivefold increase of chloroplastic glutamine synthetase content accompanied by a concommitant decrease (twofold) of the cytosolic glutamine synthetase. The increase of chloroplastic glutamine synthetase (GS₂) is only inhibited by cycloheximide and not by lincomycin. This result indicates a cytosolic synthesis of GS₂. The synthesis of GS₂ was confirmed by a quantification of the protein by an immunochemical method. It was demonstrated that GS₂ protein content in green leaves is fivefold higher than in etiolated leaves.Abbreviations AbH heavy chain of antibodies - AbL light chain of antibodies - AP acid phosphatase - CH cycloheximide - G6PDH glucose-6-phosphate dehydrogenase - GS glutamine synthetase - GS₁ cytosolic glutamine synthetase - GS₂ chloroplastic glutamine synthetase - LC lincomycin - NAD-MDH NAD malate dehydrogenase - NADP-G3PDH NADP glyceraldehyde-3-phosphate dehydrogenase     

Expression of glutamine-synthetase genes in cotyledons of germinating Phaseolus vulgaris L.

In the legume Phaseolus vulgaris L., glutamine synthetase (GS; EC.6.3.1.2.) is encoded by four actively transcribed genes, gln-, gln-, gln- and gln-. We have studied the expression of these genes in cotyledons during seed germination and have studied the effect of light and nitrate on this process. An RNase-protection method, used to detect the abundances of GS mRNAs, revealed that the four GS genes are differentially expressed in the germinating cotyledons. The gln-. mRNA was present in dry seeds and was the most abundant GS mRNA during early stages of germination. The gln- and gln- mRNAs were first detectable 2 d after sowing and their abundances differed in light- and dark-grown cotyledons at later stages of germination. The gln- mRNA (which encodes the plastid-located GS) was detectable only in light-grown cotyledons, at a low abundance. A nitrate supply of 2 mM had only a minor effect on the expression of the GS genes. Western immunodetection and ion-exchange high-performance liquid chromatography demonstrated that the polypeptide and isoenzyme were present in extracts of dry seeds and represented the major GS products at 2 d and 4 d. Both the and polypeptides appeared at the 2-d stage. The role of differential GS gene expression in controlling cotyledonary GS activity is discussed.Abbreviations 1D, 2D one-, two-dimensional - GS glutamine synthetase - GS_t GS transferase activity - IEX-HPLC ion-exchange high-performance liquid chromatography - kDa kilodaltons - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We are grateful to the Association of Commonwealth Universities and the Science and Engineering Research Council for financially supporting R.S. and to the S.E.R.C. for a grant to support M.J.B. We would like to thank Dr K.J.F. Farnden (University of Otago, New Zealand) and Dr T.H.N. Ellis (John Innes Institute, Norwich) for scanning the autoradiographs for Fig. 2.     

Activity of the tetramer and octamer of glutamine synthetase isoforms during primary leaf ontogeny of sugar beet (Beta vulgaris L.)

In extracts from the primary leaf blade of sugar beet (Beta vulgaris L.) we separated a chloroplastic isoform (GS 2) of glutamine synthetase (GS, EC 6.3.1.2) and one or two (depending on leaf age) cytosolic isoforms (GS 1a and GS 1b). The latter were prominent in the early (GS 1a) and late stages of leaf ontogeny (GS 1a and GS 1b), whereas during leaf maturation GS 2 was the predominantly active GS isoform. The GS 1 isoforms were active exclusively in the octameric state although tetrameric GS 1 protein was detected immunologically. Their activity stayed at a relatively constant level during leaf ontogeny; an increase was observed only in the senescent leaf. The activity of GS 2, however, changed drastically during primary leaf ontogeny and was modulated by changes in the oligomeric state of the active enzyme. In the early and late stages of leaf ontogeny when GS 2 activity was low (lower than that of the GS 1 isoforms), GS 2 was active only in the octameric state. In the maturing leaf, when GS 2 activity had reached its maximum level (much higher than that of the GS 1 isoforms), 80 of total GS 2 activity was due the activity of the tetrameric form of the enzyme and 20 was due to octameric GS 2. Tetrameric GS 2 was a hetero-tetramer and thus not the unspecific dissociation product of homo-octameric GS 2. In addition, GS 2 activity was modulated by an activation/inactivation of the tetrameric GS 2 protein. Due to an activation of the GS 2 tetramer, the activity of tetrameric GS 2 increased during leaf maturation from zero level 23-fold compared with that of GS 1a and 18-fold compared with that of GS 1b. Possible activators of tetrameric GS 2 are thiol-reactive substances. During leaf senescence, GS 2 activity decreased to zero; this decrease was due to an inactivation of the tetrameric GS 2 protein probably caused by oxidation.Abbreviations FLL final lamina length - FPLC fast protein liquid chromatography - GS glutamine synthetase - GHA -glutamyl hydroxamate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase Dr. Roger Wallsgrove's (Rothamsted Experimental Station, Harpenden, UK) generous gift of GS antiserum is greatly appreciated.     

Physiological characteristics of glutamine synthetases I and II of Frankia sp. strain CpI1

Frankia sp. strain CpI1 has two glutamine synthetases designated GSI and GSII. Biosynthetic activities of both GSI and GSII were strongly inhibited by ADP and AMP. Alanine, aspartate, glycine and serine inhibited both GSI and GSII activities, whereas asparagine and lysine inhibited only slightly. Glutamine inhibited GSII but did not affect GSI. Since GSII is more heat labile than GSI, their relative heat stabilities can be used to determine their contribution to total GS activity. In cells grown on ammonia and on glutamine as sole combined-nitrogen sources most GS activity detected in crude extracts was due to GSI. In cells transferred to glutamate, GSI accounted for all GS activity in the first 15 h and then heat labile GSII was induced and increased to account for 40% of total GS activity within 50 h. Transfer of N₂-fixing cells to ammonia-containing medium led to a rapid decrease of GSII and a slow increase of GSI activity within 24 h. Conversely, when ammonia-grown cells were transferred to combined nitrogen-free medium, GSI activity gradually decreased and GSII increased before total activity leveled off in 50 h. GSII appears to be an ammonia-assimilating enzyme specifically synthesized during perceived N-starvation of Frankia cells.     

Glutamine synthetase of Chlamydomonas: Rapid reversible deactivation

A 70% reduction in glutamine synthetase (GS) activity was observed within 5 min when 5 mM NH₃ and darkness was applied to steady-state cells of Chlamydomonas utilising NO₃. The enzyme was reactivated in vivo by reillumination of the culture and in vitro by treatment with thiol reagents. The activity modulations affected the synthetase and transferase activities similarly and were not influenced by protein synthesis inhibitors. Deactivation of GS was also observed when steady-state cells were treated with an uncoupler of phosphorylation, carbonylcyanide m-chlorophenylhydrazone (CCCP) or inhibitors of the electron transport chain but under these conditions the activity modulation affected over 90% of the activity and was irreversible. The mechanism of the physiological deactivation of GS is discussed in relation to both the in vivo and in vitro findings.Abbreviations GS glutamine synthetase (EC 6.3.1.2.) - GSs glutamine synthetase, synthetase activity - GSt glutamine synthetase, transferase activity - CAP chloramphenicol - CCCP carbonylcyanide m-chlorophenyl hydrazone - CHX cycloheximide - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DSPD disalicylidene propanediamine - DTT dithiothreitol - GSH reduced glutathione     

Large-scale partial purification of phytochrome from green leaves of Avena sativa L.

Phytochrome from 10 or 11-d-old oat (Avena sativa L. cv. Garry) leaves, which were harvested just prior to sunset from plants grown in a greenhouse in the absence of supplemental illumination, was purified an estimated 250-fold by sequential poly(ethylenimine) and ammonium-sulfate fractionations, followed by linear-gradient hydroxyapatite chromatography. Compared to earlier protocols, the one presented here is substantially more rapid, provides improved yield and purity, can be used with larger quantities of tissue, and eliminates an apparently immunodominant contaminant with a molecular mass of about 115 kDa (kilodalton). Phytochrome obtained by this procedure has an apparent monomer size of 123 kDa as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is estimated to be 0.6% pure. This purity permitted spectral analysis at wavelengths below 500 nm, in which region phytochromes from green and etiolated oat shoots do not differ markedly, as they do at longer wavelengths.Abbreviations Da Dalton - HA hydroxyapatite - Pfr, Pr farredand red-absorbing form of phytochrome, respectively - SDS sodium dodecyl sulfate This research was supported by the U.S. Department of Energy (contract DE-AC-09-81SR10925 to L.H.P.). The excellent technical assistance of Mrs. Donna Tucker and Mrs. Danielle Neal is gratefully acknowledged.     

Glutamine synthetase of Chlamydomonas: its role in the control of nitrate assimilation

Work is described which suggests that glutamine synthetase (GS) could play an important and direct regulatory role in the control of NO₃ assimilation by the alga. In both steady-state cells and ones disturbed physiologically by changes in light or nitrogen supply the assimilation of NO₃ appears to be limited by the activity of GS. Moreover although in normal cells NH₃ can completely inhibit NO₃ uptake, promote the deactivation of nitrate reductase (NR) and repress the synthesis of NR and nitrite reductase (NIR), these controls are relaxed in cells in which GS is deactivated by treatment with L-methionine-DL-sulfoximine (MSO). It is proposed that the reversible deactivation of GS may play an important part in the regulation of NO₃ assimilation although it is still not clear whether the enzyme itself or products of its metabolism are responsible.Abbreviations GS glutamine synthetase - GS_s glutamine synthetase, synthetase activity - GS_t glutamine synthetase, transferase activity - NR nitrate reductase - NIR nitrite reductase - GDH glutamate dehydrogenase - CHX cycloheximide - MSO L-methionine-DL-sulfoximine - FAD flavine adenine dinucleotide     

Phytochrome-mediated swelling of etiolated leaf protoplasts and its possible biological significance

Summary Red light, mediated by the photoreceptor phytochrome, induces maize leaf unrolling as well as leaf expansion. Protoplasts prepared from maize leaves still in the rolled condition swell in a red-far red photoreversible manner indicating that phytochrome mediates this phenomenon. To determine if protoplast swelling is related to leaf unrolling, leaf expansion, or both, we compared red-light induced swelling of protoplasts from rolled maize leaves to protoplasts prepared from tissues that are known to grow in response to light but do not unroll. We also compared the swelling response of protoplasts from rolled vs. unrolled leaves. In all cases, we found that swelling correlated with the unrolling response and not leaf expansion.     

Factors involved in the coordinate appearance of nitrite reductase and glutamine synthetase in the mustard (Sinapis alba L.) seedling

The extent to which the appearances of nitrite reductase (NIR; EC 1.7.7.1) and glutamine synthetase (GS; EC 6.3.1.2) are coordinated was studied in mustard (Sinapis alba L.) seedlings. It was established by immunotitration that the increased activities of NIR and GS in the presence of light and nitrate can be attributed to the de-novo synthesis of enzyme protein. The bulk of the NIR and GS was found in the developing cotyledons. In the absence of nitrate in the growth medium there was no coordinate appearance of NIR and GS. While light strongly stimulated the appearance of GS, the level of NIR was hardly affected and remained low. On the other hand, in the presence of nitrate in the medium the appearances of NIR and GS were strictly coordinated, the GS level being considerably above that of NIR. It is argued that phytochrome-controlled synthesis of GS in the absence of nitrate is part of the mechanism to reassimilate ammonium liberated during proteolysis of storage protein and metabolism of the resulting amino acids, whereas the strictly coordinated synthesis in the presence of light and nitrate indicates the dominance of nitrate assimilation under these circumstances. The fact that the level of GS was always considerably above that of NIR appears to be a safety measure to prevent ammonium accumulation.Abbreviations FR standardized far-red light (3.5 W·m^–2), to drive the high-irradiance reaction of phytochrome - GS glutamine synthetase, EC 6.3.1.2 - NIR nitrite reductase, EC 1.7.7.1 This work was supported by Heidelberger Akademie der Wissenschaften (Forschungsstelle Nitratassimilation).     

Water droplets in intercellular spaces of barley leaves examined by low-temperature scanning electron microscopy

Low-temperature scanning electron microscopy was used to examine fracture faces in leaf blades taken from well-watered or drought-stressed barley (Hordeum vulgare L. cv. Mazurka) seedlings. The leaf blades were freeze-fixed while hydrated and were examined with or without gold-coating. There were droplets (with a smooth surface at the resolution achieved) on the surface of cell walls in leaf blades (0.91 g^-1 water content) from well-watered seedlings grown in an environment of 67% relative humidity. These were mainly on the vascular bundle sheath, the guard and subsidiary cells, and on some mesophyll cells around the substomatal cavity and between the stoma and vascular bundle. The droplets occurred, more abundantly, in the same places in seedlings from 100% relative humidity. They occurred on a few guard cells from wilting leaf blades (0.81 g·g^-1 water content) and were absent from severely drought-stressed leaf blades (0.15 g·g^-1 water content). The droplets sublimed at the same moment as both water which was in leaf cells and water which was allowed to condense (after freeze-fixation) on the wall surface. It is suggested that the droplets are aqueous. Their possible origin and importance is discussed.     

Phloem unloading following reactivation in predarkened mature maize leaves

Mature leaf blades of 48-h predarkened maize plants (Zea mays L. cv. Prior) were excised, and treated apically as the source (light, normal air) and basally as the sink (light or dark, air without CO₂). After providing the source portion with ¹⁴CO₂, the sink portions were harvested after 2, 7 or 14 h by freezing with liquid nitrogen, grinding, and freeze-drying. Extracts, fractionated by ionexchange resins into neutral, basic and acid fractions, were chromatographed on thin cellulose layers, and autoradiographed. Identification of labeled compounds was carried out by co-chromatography with authentic labeled substances. Activities of enzymes pertaining to the metabolism of sucrose were checked. Results show that the source supplies sucrose to the sink, where it is unloaded and metabolized by acid invertase (EC 3.2.1.26) in both the light and the dark. Starch appearing in the sink only in the light, after 7 h of re-illumination, yields labeled glucose upon hydrolysis. Although sucrose-phosphate synthetase (EC 2.4.1.14) is active in sinks and in isolated vascular-bundle fragments, it remains questionable whether sucrose unloaded from sieve tubes is metabolized by a method other than inversion. Sucrose synthetase (EC 2.4.1.13) was found to be inactive. Obviously, the main metabolite of unloaded sucrose is glucose-6-phosphate, giving access to the glycolytic pathway. The main difference between the sinks in the light and the dark is the lack of labeled glycine and serine in the dark. This indicates that in the light decarboxylation of glycine yields CO₂, which is recycled photosynthetically.Abbrevations Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - TLC thin-layer chromatography - UDPGlc uridine 5-diphosphate glucose     

Purification and characterization of phosphoribulokinase from wheat leaves

Homogeneous phosphoribulokinase (PRK; ATP: d-ribulose-5-phosphate 1-phosphotransferase, EC 2.7.1.19) was isolated from wheat leaves with a specific activity of 15 kat mg^-1 protein. The purification included ammonium sulfate cuts, isoelectric precipitation, and hydrophobic and affinity chromatography on pentylagarose and Blue Sepharose CL 6B, respectively. Gel filtration of the purified enzyme yielded a 83000 Da protein. Subunits of about 42000 Da were estimated from sodium dodecyl sulfate-polyacrylamide gels. Wheat leaf PRK was stable for at least four weeks when stored at 4°C. Saturation curves for ribulose 5-phosphate (Ru5P) and ATP followed Michaelis-Menten kinetics (K _m values: K _{m Ru5P}=50–80 M; K _{m ATP}=70 M). The saturation curve for MgCl₂ was sigmoidal (half-maximal velocity <0.5 mM). The affinity for Ru5P, ATP and Mg²⁺ was not affected by pH changes comparable to pH shifts in the stroma. In contrast to chloroplast fructose-bisphosphatase (Zimmermann et al. 1976, Eur. J. Biochem. 70, 361–367) the affinity for ligands remained unchanged in the dithiothreitol-activated and in the non-activated state. The activity of PRK was increasingly sensitive to inhibition by 3-phosphoglyceric acid with decreasing pH below pH 8.0.Abbreviations DTT dithiothreitol - EDTA ethylenediamine-tetraacetic acid - PRK phosphoribulokinase - Ru5P ribulose-5-phosphate - SDS-PAGE sodium dodecyl sulfate-polyacryl-amide gel electrophoresis     

Isoforms of Glutamine Synthetase in the Marine Coccolithophorid Emiliania huxleyi (Prymnesiophyceae)

Two isoenzymes of glutamine synthetase (EC 6.3.1.2), GS₁ and GS₂, have been purified from cells of Emiliania huxleyi using Cibacron blue dye ligand chromatography and gel filtration, separated by ion-exchange chromatography on Mono-Q and partly characterized. Each enzyme is a homohexamer with a molecular mass of 402 kDa for GS₁ and 501 kDa for GS₂. The molecular mass of the subunits of GS₁ and GS₂ was estimated to be 61 and 78 kDa, respectively. As in higher plants, GS₁ is slightly more thermostable than GS₂ and much less stimulated by thiols than GS₂. For these reasons, GS₁ was designated as the cytosolic enzyme and GS₂ as the chloroplastic one. Although the K_ms for NH₂OH are about the same, GS₂ possesses a much higher affinity for glutamine than GS₁. As in bacteria, ATP appears to play an important role in the allosteric regulation of GS₂. l-Ala and CTP are potent inhibitors of GS₁ activity. CTP, carbamoyl-phosphate and l-Ala exert a cumulative inhibitory effect on GS₁ activity. GS₂ is also inhibited to some extent by l-Ala and l-His. NH₂-terminal sequence analysis of GS₂ did not show any homology with bacteria, cyanobacteria or higher plants.     

Temperature and oxygen regulated expression of a glutamine synthetase gene fromVibrio alginolyticus cloned inEscherichia coli

Glutamine synthetase (GS) synthesis inVibrio alginolyticus was regulated by temperature, oxygen and nitrogen levels. A GS gene,glnA fromV. alginolyticus was cloned on a 5.67 kb insert in the recombinant plasmid pRM210, which enabledEscherichia coli glnA, ntrB, ntrC deletion mutants to utilize (NH₄)₂SO₄ as a sole source of nitrogen. TheV. alginolyticus glnA gene was expressed from a regulatory region contained within the cloned fragment.V. alginolyticus glnA expression from pRM210 was subject to regulation by temperature, oxygen and nitrogen levels. GS specific activity in anE. coli wild-type strain was not affected by temperature or oxygen. pRM211 was a deletion derivative of pRM210 and GS production by pRM211 was not regulated by temperature, oxygen or nitrogen levels inE. coli.Abbreviation GS glutamine synthetase     

Changes in the activities of chloroplast and cytosolic isoenzymes of glutamine synthetase during normal leaf growth and plastid development in wheat

Soluble protein extracts and chloroplasts from a serial sequence of transverse sections of a 7-d-old wheat leaf (Triticum aestivum cv. Maris Huntsman) were used to study changes in the activity of glutamine synthetase (GS; EC 6.3.1.2) during cell and chloroplast development. Glutamine synthetase activity increased more than 50-fold per cell from the base to the tip of the wheat leaf. Two isoenzymes of GS were separated using fast protein liquid chromatography (FPLC). Glutamine synthetase localized in the cytoplasm (GS1) eluted at about 0.21 M NaCl, and the isoenzyme localized in the chloroplast (GS2) eluted at about 0.33 M NaCl. The increase in GS activity during leaf development was found to be caused primarily by an increase in the activity of the chloroplast GS2. The activity of the cytoplasmic GS1 remained constant as the cells were displaced from the base to the tip of the leaf, whereas GS2 activity increased within the chloroplast throughout development. At the base of the leaf, 26% of total GS activity was cytoplasmic; the remaining 74% was in the chloroplast. At 10 cm from the base, only 4% of the activity was cytoplasmic, and 96% was in the chloroplast. The results indicate that the chloroplast GS2 is probably responsible for most of the ammonia assimilation in the mature wheat leaf, whereas cytoplasmic GS1 may serve a role in immature developing leaf cells.Abbreviations FPLC fast protein liquid chromatography - GS glutamine synthetase - GS1 cytoplasmic glutamine synthetase - GS2 chloroplast glutamine synthetase     

Changes in mRNA level rhythmicity in the leaves ofSinapis alba during a lengthening of the photoperiod which induces flowering

A previous study has shown that mRNAs exhibit complex patterns of diurnal rhythms in their quantity in the leaves ofSinapis alba during an 8 h light/16 h dark short day (SD). In order to determine whether this situation is rapidly modified in plants subjected to an extended light treatment, we have usedin vitro translation and two-dimensional polyacrylamide gel electrophoresis, together with a strict gel comparison procedure giving aP=0.03 certitude level, to analyse the mRNA complement at different times during a 22 h light/2 h dark long day (LD).During this LD, complex changes affected about 10% of the mRNAs. Thirty-four different patterns were observed. Some diurnal rhythms present in SD are not modified by the lengthening of the light period, but most are affected. Moreover, we have shown that some mRNAs presenting a constant quantity under a SD regime show an increase or a decrease during the first hours of the photoperiod lengthening.InSinapis, this LD also induces flowering. All the changes in mRNA quantity detected thus parallel the photoperiodic induction of flowering in the leaves and are quantitative; no mRNA was shown to appear or to disappear.     

Extracellular ice and cell shape in frost-stressed cereal leaves: A low-temperature scanning-electron-microscopy study

Low-temperature scanning electron microscopy was used to examine transverse fracture faces through cereal leaf pieces subjected to frost. Specimens were studied before and after sublimation of the ice. The position of extracellular ice in the leaf was inferred from the difference between the specimen before and after sublimation and from ridges and points which occurred in the extracellular ice during sublimation. Steps in the fracture surface indicated that the fracture plane passed through the extracellular ice crystals as well as through cells and also helped identify extracellular ice. The cells in controls were turgid and extracellular ice was absent. Leaf pieces from hardened rye were excised and frost-stressed to-3.3°,-21° and-72°C, cooling at 2–12°·h^-1. Cell collapse and extracellular ice were evident at-3.3°C and increased considerably by-21° C. At-21° and-72°C the leaf pieces were mainly filled with extracellular ice and there were few remaining gas spaces. The epidermal and mesophyll cells were laterally flattened, perpendicular to their attachment to adjacent cells, and phloem and vascular sheath cells were more irregularly deformed. Leaf pieces from tender barley were cooled at 2°C·min^-1 to-20° C; they were then mainly filled with extracellular ice, and the cells were highly collapsed as in the rye. In rye leaves frozen to-3.6° C before excision, ice crystals occurred in peri-vascular, sub-epidermal and intervening mesophyll spaces. In rye leaf pieces frozen to-3.3° C after excision or to-3.6° C before excision, mesophyll cells were partly collapsed even when not covered by ice, indicating that collapse of the cell wall, as well as the enclosed protoplast, was driven by dehydration. No gas or ice-filled spaces were found between wall and the enclosed protoplast. It is suggested that this can be explained without invoking chemical bonding between cell wall and plasma membrane: when the wall pores are filled by water, the pore size would reduce vapour pressure so making penetration of the wall by ice or gas less likely.Abbreviations SEM scanning electron microscopy     

The effect of chemical stress on the polypeptide composition of the intercellular fluid of barley leaves

The effect of chemical stress on the polypeptide composition of the intercellular fluid of barley (Hordeum vulgare L.) and tomato (Lycopersicon esculentum Mill.) leaves has been studied. In some dicotyledonous plant species, including tomato, exposure to chemical stress leads to the denovo synthesis of intercellular proteins known as pathogenesis-related proteins which have been implicated to be part of a defence mechanism. In barley, however, no such changes in the polypeptide composition of the intercellular fluid could be detected. On the other hand, similar stress conditions induce in barley a strong accumulation of mRNA encoding leaf-specific thionins. These barley thionins represent a novel class of cell-wall proteins toxic to phytopathogenic fungi and are possibly involved in the defence mechanism. These proteins could not be detected in tomato plants. In contrast to the pathogenesis-related proteins of dicotyledonous plants, the leaf-specific thionins of barley are not present in the intercellular fluid of leaves. These results indicate that barley may have evolved a different mechanism to cope with the presence of stress.Abbreviations PAGE polyacrylamide gel electrophoresis - PR pathogenesis-related - SDS sodium dodecyl sulfate     

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

The -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 -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).Abbreviations GS glutamine synthetase (E.C. 6.3.1.2.) - IEF isoelectric focussing - PAGE polyacrylamide gel electrophoresis This work was supported by a grant from Bundesministerium für Forschung und Technologie and by Kleinwanzlebener Saatzucht AG, Einbeck.