Gisela Mäck

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 (NO3−nutrition) and also in the lateral roots (NH4+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 β-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 (NO3−-fed plants) or even increased (NH4+-fed plants).

Glutamine synthetase isoenzymes, oligomers and subunits from hairy roots of Beta vulgaris L. var. lutea

Abstract. A cytosolic and a plastidic isoenzyme of glutamine synthetase (GS; EC 6.3.1.2) were separated from hairy roots of Beta vulgaris L. var. lutea. The predominant activity was that of cytosolic GS 1; the relative proportion of plastidic GS 2 activity changed, however, depending on the growth conditions. Maximum activity of both isoenzymes was measured after growth with NO−3 as the major N-source. Growth with NH+4 as the sole N-source or growth in constant darkness resulted in a significant decrease in GS 1 activity, whereas GS 2 activity was much less effected and thus contributed as much as 25% of total root GS activity. The isoenzymes GS 1 and GS 2 were active both in the octameric and tetrameric states. Both oligomers of GS 2 and octameric GS 1 were active under all growth conditions applied whereas tetrameric GS 1 was not active when the roots were grown under light-dark changes with NO−3 as the major N-source. The molecular masses of the subunits were identical for both isoenzymes. Glutamine synthetase 1 was composed of up␣to four different 38-kDa subunits and two different 41-kDa subunits; GS 2 was assembled from one type of 38-kDa subunit and one type of 41-kDa subunit. The GS␣2 subunits were most probably identical to two of the GS␣1 subunits. The subunit composition of GS 1, but not of GS 2, changed depending on the growth conditions of the roots. Changes in GS 1 subunit composition were correlated with changes in GS 1 activity. The different growth conditions induced the specific assembly of different GS 1 isoenzymes which could, however, not be separated by anion-exchange chromatography but became evident only after two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Changes in nitrate reductase activity and the protective effect of molybdenum during cold stress in winter wheat grown on acid soil

Summary Two cultivars (frost resistant Sadovo 1 and frost susceptible San Pastore ) of winter wheat ( Triticum aestivum L.) were used to study the protective effect of molybdenum during freezing of plants grown on acid soil. We followed the effect of cold acclimation (2°C for 7 days) and freezing (−5 °C for 16 h) on the activities of nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS) in the leaves of both wheat cultivars grown on soil with pH 6.0 and pH 4.5. No difference was observed between the cultivars in the temperature induced changes of enzyme activities. During cold acclimation the activities of the three enzymes increased irrespective of the soils pH; this increase, which was most pronounced for NADH: NR activity, was paralleled by an increase in NR protein. Freezing affected only NR activity in plants grown on acid soil; the sharpest decline was found for NADH : NR activity and FADH : NR partial activity. Molybdenum supplied to winter wheat cv. Sadovo 1 grown on soil with pH 4.5 prevented the frost-induced decline of NADH : NR activity. The possible mechanism of the protective effect is discussed.

Constitutive and Inducible net NH+4 Uptake of Barley (Hordeum vulgare L.) Seedlings

Summary High initial NH+4 depletion rates were measured immediately after exposure of N-free grown and NH+4-induced barley to NH+4. We provide evidence that this rapid initial NH+4 depletion was due primarily to the filling of the Apparent Free Space of the root cell wall. The high initial depletion rates depended on the NH+4 concentration of the uptake solution and on the degree of prior NH+4 saturation of the Apparent Free Space. N-free grown barley seedlings were able to take up NH+4 constitutively with a Vmax of 3.7 ·mol · (g root FW)-1·h-1 and a K5, of 153 γLM. However, an induction of NH+4 uptake by the NH+4 reserves of the grain (105 nmole per dry grain; 74 % of which was stored in the dormant seed) during early germination cannot be excluded. Exposure of N-free grown seedlings to external NH+4 (1 mM) resulted in an induction of NH+4 uptake after a 80 min lag; the rates were higher (Vmax= 6-8.3 μmol·(g root FW)-1·h-1) than the constitutive rates, but K5, remained unchanged (154 to 166 μLM after induction and 153 gM prior to induction). This indicates that the same NH+4 uptake system (low-capacity uptake system) was operating in N-free and NH+4-induced barley and that during induction the amount of transport proteins was increased. A linear uptake component (high-capacity system) was measured at high external (> 1 mM) NH+4 concentrations with NH+4-induced and N-free grown plants. The steady-state rates were about 10-fold higher than that of the low-capacity system although no additional protein synthesis was required.

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.

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 NO3-·(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 NO3-·(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.

Changes in the Isoform Pattern and Subunit Composition of GS-1 in Sugar Beet Leaves Dependent on Leaf Age

Summary This investigation reports on changes in the subunit composition of glutamine synthetase 1 (GS-1, E.C. 6.3.1.2) in sugar beet (Beta vulgaris L.) leaves dependent on leaf age. The highest GS-1 activity was found in young and in senescing leaves of sugarbeet. Dependent on the ontogeny of the leaves, the isoform pattern of GS-1 and the subunit composition were changed. In young leaves GS-1 was found to be a heterooctamer, while in senescent leaves this enzyme was functional as a homo-octamer. This different subunit composition of GS-1 is discussed with respect to changes in the physiological role of leaves from sink to source.

The activity of nitrate reductase, glutamine synthetase and phosphoenolpyruvate carboxylase in leaf blades of developing Zea mays

Summary The specific activities of nitrate reductase, glutamine synthetase and phosphoenolpyruvate carboxylase in the leaf canopy of 2-, 3-, 5-, and 8-week-old Zea mays (Forla) were recorded from early vegetative growth to anthesis. A regulation of the ratio between the activity of the N assimilating enzymes and that of phosphoenolpyruvate carboxylase has been found in the leaf blades as follows: a) A temporal separation of the maximum of the N assimilating enzymes (3rd week after sowing) and that of phosphoenolpyruvate carboxylase (2nd and 8th week after sowing); b) a spatial separation of the maximum of the N assimilating enzymes (uppermost leaves of the plant) and that of phosphoenolpyruvate carboxylase (middle leaves); and c) a spatial separation of the maximum of the N assimilating enzymes (in the base and inner sections of the leaf blade) and that of phosphoenolpyruvate carboxylase (in the midsections of the leaf blade). The activity of the N assimilating enzymes was closely related to the protein and chlorophyll content, but negatively correlated to the nitrate and ammonium content of the leaf blade. Phosphoenolpyruvate carboxylase activity was associated with enlargement of the leaf blade and with stem elongation.