GOGAT Activity Research Articles

Metabolic response of different osmo-sensitive Sacchromyces cerevisiae to ACA microcapsule

Microencapsulation technology is a convenient method to alter and regulate cell product formation. In order to probe the metabolic response of different osmo-sensitive Sacchromyces cerevisiae to ACA microcapsule, the hyper-osmo-sensitive type S. cerevisiae (Y02724) and wild type S. cerevisiae (BY4741) were encapsulated into liquid core ACA microcapsules. The behavior of cell growth, glucose consumption, ethanol production and the yields of glycerol and organic acids were determined. Free cell culture was used as control. The enzyme activities of NADP +-glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GOGAT) on microencapsulation cells and free cultured cells were measured too. The results demonstrated that the growth of Y02724 in both aerobic and anaerobic conditions was seriously inhibited by ACA microcapsule, while the ethanol and acetatic acid yield of microencapsulation Y02724 in anaerobic condition were significantly higher than that of suspended cultivation. For Y02724, the microencapsulation cultivation significantly increased the GS and GOGAT activities and decreased the GDH activity in comparison with control group. ACA microcapsules did not significantly change the growth behavior and metabolic performance of BY4741, but decreased the GS activity. In conclusion, microcapsules microenvironment significantly changes the metabolism behavior of hyper-osmo-sensitive type S. cerevisiae (Y02724), but nearly had no effect on BY4741.

Amino acid pattern and glutamate metabolism during dehydration stress in the 'resurrection' plant Sporobolus stapfianus: a comparison between desiccation-sensitive and desiccation-tolerant leaves

The present study analyses changes in nitrogen compounds, amino acid composition, and glutamate metabolism in the resurrection plant Sporobolus stapfianus during dehydration stress. Results showed that older leaves (OL) were desiccation-sensitive whereas younger leaves (YL) were desiccation-tolerant. OL lost their soluble protein more rapidly, and to a larger extent than YL. Enzymes of primary nitrogen assimilation were affected by desiccation and the decrease in the glutamine synthetase (GS, EC 6.3.1.2) and ferredoxin-dependent GOGAT (Fd-GOGAT, EC 1.4.7.1) activities was higher in OL than in YL, thus suggesting higher sensibility to dehydration. Moreover, YL showed higher total GS enzyme activity at the end of the dehydration stress and was shown to maintain high chloroplastic GS protein content during the entire stress period. Free amino acid content increased in both YL and OL between 88% and 6% relative water content. Interestingly, OL and YL did not accumulate the same amino acids. OL accumulated large amounts of proline and gamma-aminobutyrate whereas YL preferentially accumulated asparagine and arginine. It is concluded (i) that modifications in the nitrogen and amino acid metabolism during dehydration stress were different depending on leaf development and (ii) that proline and gamma-aminobutyrate accumulation in S. stapfianus leaves were not essential for the acquisition of desiccation tolerance. On the contrary, the accumulation of large amounts of asparagine and arginine in the YL during dehydration could be important and serve as essential nitrogen and carbon reservoirs useful during rehydration. In this context, the role of GS for asparagine accumulation in YL is discussed.

Effects of lanthanum(III) on nitrogen metabolism of soybean seedlings under elevated UV-B radiation

The hydroponic culture experiments of soybean bean seedlings were conducted to investigate the effect of lanthanum (La) on nitrogen metabolism under two different levels of elevated UV-B radiation (UV-B, 280–320 nm). The whole process of nitrogen metabolism involves uptake and transport of nitrate, nitrate assimilation, ammonium assimilation, amino acid biosynthesis, and protein synthesis. Compared with the control, UV-B radiation with the intensity of low level 0.15 W/m 2 and high level 0.45 W/m 2 significantly affected the whole nitrogen metabolism in soybean seedlings ( p < 0.05). It restricted uptake and transport of NO 3 −, inhibited activity of some key nitrogen-metabolism-related enzymes, such as: nitrate reductase (NR) to the nitrate reduction, glutamine systhetase (GS) and glutamine synthase (GOGAT) to the ammonia assimilation, while it increased the content of free amino acids and decreased that of soluble protein as well. The damage effect of high level of UV-B radiation on nitrogen metabolism was greater than that of low level. And UV-B radiation promoted the activity of the anti-adversity enzyme glutamate dehydrogenase (GDH), which reduced the toxicity of excess ammonia in plant. After pretreatment with the optimum concentration of La (20 mg/L), La could increase the activity of NR, GS, GOGAT, and GDH, and ammonia assimilation, but decrease nitrate and ammonia accumulation. In conclusion, La could relieve the damage effect of UV-B radiation on plant by regulating nitrogen metabolism process, and its alleviating effect under low level was better than that under the high one.

EFFECTS OF WATER STRESS ON NUTRIENT QUALITY AND ACCUMULATION OF PROTEIN IN RICE GRAINS

Aims Soil drying is an important factor threatening rice production in China, and there has been much recent research on the development of grain yield and quality in rice grown under shortages of water. The objective of this study is to examine the effects of soil moisture and N supply levels on rice grain N accumulation and nutrient quality. Methods We conducted a pot study using three rice cultivars and varying in N content to examine physiological characteristics for N accumulation and distribution to grains during the grain-filling period in response to water stress and N supply exposure at heading stage. Important findings The activities of glutamine synthetase (GS) and glutamate synthase (GOGAT) involved in N metabolism were enhanced by mild water stress at 10-20 DAA (days after anthesis) under normal N level, and abilities to synthesize amino acids were improved, which promoted the accumulation of N and elevated the protein content in grains. However, the activities of GS and GOGAT were negatively affected by mild water stress with exposure to high N level, which resulted in decreased synthesis of amino acids. The dynamic changes of the ratio of protein to grain weight presented a “V type during grain-filling. Water stress improved the ratio of protein to grain weight from 15 DAA to harvest under normal N level, but the ratio was decreased by water stress under higher N level when compared with well-watered treatments. Changes of four component parts of protein subjected to water stress varied with nitrogen levels and cultivars. Compared to well-watered treatment, the water-stressed treatment had higher contents of prolamine and glutelin under normal N level, but the contents of prolamine and glutelin subjected to water stress were significantly less than those in the well-watered treatment under higher N level. Water stress affected the lysine content of grains, and varied with cultivars and N supplies. Lysine contents of `Shanyou63' under two N levels were decreased by water stress, but were increased in `Yangdao6'. The variation observed in enzymes involved in N metabolism and partitioning at grain filling to grains of the rice imposed by water stress at heading indicated that plant genotypes influence N accumulation and protein components of grains and subsequent nutrient quality.

Effect of hydro- and osmopriming of chickpea (Cicer arietinum L.) seeds on enzymes of sucrose and nitrogen metabolism in nodules

Three year data on the effect of water- and mannitol (4%) priming of chickpea seeds (12 h at 25°C) showed higher number and biomass of nodules in the plants from primed seeds than from non-primed seeds. The biomass of nodules increased to 75 DAS but decreased by 90 DAS. Activities of sucrose metabolism enzymes (sucrose synthase (SS) and alkaline invertase) and of nitrogen metabolism (glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH)) in nodules of primed and non-primed crops during development are reported. SS and alkaline invertase activities increased to 70 DAS and then decreased. In primed plants, the higher SS activity in nodules at 60 and 70 DAS might be responsible for providing more energy and carbon skeleton for nitrogen fixation and for ammonium assimilation in primed plants. At 85 DAS, though the SS activity decreased in comparison with the earlier growth stages, it was still higher in nodules of the primed crops than the non-primed crop. Activity of alkaline invertase was maximum at 70 DAS in the nodules of primed and non-primed crops. Priming increased nodule GS activity at 70 and 85 DAS. GOGAT activity was unaffected by priming but GDH activity was greater in nodules from primed crops at 50 DAS. Elevated SS and GS nodule activities in primed chickpeas might be responsible in increasing nodule biomass and metabolic activity thereby increasing seed fill.

Oxidation of the enzymes involved in nitrogen assimilation plays an important role in the cadmium-induced toxicity in soybean plants

Cadmium causes oxidative damage and hence affects nitrogen assimilation. In the present work we tested the relationship between the inactivation of the enzymes involved in nitrogen assimilation pathway (glutamine synthetase (GS)/glutamate synthase (GOGAT)) and the protein oxidation in nodules of soybean (Glycine max L.) plants under Cd2+ stress. Therefore, the effect of Cd2+ and reduced gluthatione (GSH) on GS and GOGAT activities, and protein abundance and oxidation were analyzed. Under the metal treatment, amino acids oxidative modification occurred, evidenced by the accumulation of carbonylated proteins, especially those of high molecular weight. When Cd2+ was present in the nutrient solution, although a decrease in GS and GOGAT activities was observed (17 and 52%, respectively, compared to controls), the protein abundance of both enzymes remained similar to control nodules. When GSH was added together with Cd2+ in the nutrient medium, it protected the nodule against Cd2+ induced oxidative damage, maintaining GS and GOGAT activities close to control values. These results allow us to conclude that the inactivation of the nitrogen assimilation pathway by Cd2+ in soybean nodules is due to an increment in GS and GOGAT oxidation that can be prevented by the soluble antioxidant GSH.

Partial substitution of NO 3− by NH 4+ fertilization increases ammonium assimilating enzyme activities and reduces the deleterious effects of salinity on the growth of barley

Productivity of cereal crops is restricted in saline soils but may be improved by nitrogen nutrition. In this study, the effect of ionic nitrogen form on growth, mineral content, protein content and ammonium assimilation enzyme activities of barley (Hordeum vulgare cv. Alexis L.) irrigated with saline water, was determined. Leaf and tiller number as well as plant fresh and dry weights declined under salinity (120 mM NaCl). In non-saline conditions, growth parameters were increased by application of NH(4)(+)/NO(3)(-) (25:75) compared to NO(3)(-) alone. Under saline conditions, application of NH(4)(+)/NO(3)(-) led to a reduction of the detrimental effects of salt on growth. Differences in growth between the two nitrogen regimes were not due to differences in photosynthesis. The NH(4)(+)/NO(3)(-) regime led to an increase in total N in control and saline treatments, but did not cause a large decrease in plant Na(+) content under salinity. Activities of GS (EC 6.3.1.2), GOGAT (EC 1.4.1.14), PEPC (EC 4.1.1.31) and AAT (EC 2.6.1.1) increased with salinity in roots, whereas there was decreased activity of the alternative ammonium assimilation enzyme GDH (EC 1.4.1.2). The most striking effect of nitrogen regime was observed on GDH whose salinity-induced decrease in activity was reduced from 34% with NO(3)(-) alone to only 14% with the mixed regime. The results suggest that the detrimental effects of salinity can be reduced by partial substitution of NO(3)(-) with NH(4)(+) and that this is due to the lower energy cost of N assimilation with NH(4)(+) as opposed to NO(3)(-) nutrition.

Effect of glutamate synthase (GOGAT) activity on Bacillus subtilis spore properties.

The role of glutamate as osmoprotector was investigated through the study of a mutation in its biosynthetic pathway. A glt::Tn917-lacZ-cat insertion mutant (N1) conferring glutamate auxotrophy and enhanced beta-galactosidase expression on high-salt media was selected. Co-transformation experiments and PCR analysis allowed locating the insertion into the gltB gene corresponding to the small unit of the glutamate synthase (GOGAT). The N1 mutant strain presented a glutamate requirement for growth and a tenfold decrease in GOGAT activity. Transcriptional activity of GOGAT, measured as beta-galactosidase from the transposon fusion, correlated with enzymatic activity; expression was enhanced at the stationary phase and in high-ionic-strength media. However, osmotolerance of cultures of N1 mutant were as wild-type (wt), at least in semi-rich medium. In contrast, sporulation was slightly reduced (75% of wt), and spores were less resistant to UV, heat, and osmolarity, properties linked to the content of small, acid-soluble proteins (SASP). The content of these proteins was, in fact, reduced, in particular the SASP-gamma type. The peptidoglycan-cortex, however, was not impaired since spores maintained lysozyme resistance. Addition of glutamate during sporulation partially rescued spore resistance, but germination and outgrowth remained impaired. Deficiencies in germination and outgrowth were also observed with spores from a gltA mutant strain. Taken together, these results pointed to the importance of GOGAT activity during sporulation, in particular for the synthesis SASPs.

Aluminum Effects on Nitrogen Uptake and Nitrogen Assimilating Enzymes in Maize Genotypes with Contrasting Tolerance to Aluminum Toxicity

The objective of this study was to investigate the effect of aluminum (Al) on NO3 − and NH4 + uptake and on activity/expression of the N‐assimilating enzymes glutamine synthetase (GS1, GS2, and GSr isoforms, EC 6.3.1.2), glutamate synthase (NADH‐GOGAT, EC 1.4.7.14; ferredoxin‐GOGAT, EC 1.4.7.1), and glutamate dehydrogenase (GDH, EC 1.4.1.2) of maize (Zea mays L.) genotypes contrasting in tolerance to Al toxicity. Seeds were germinated on paper towels, screened for uniformity and allowed to grow for 5–6 d in a modified Steinberg nutrient solution, pH 5.5, in a glasshouse, under natural light. Immediately after transference to a growth chamber (12 h photoperiod, 26°C ± 0.5/18°C ± 0.9 day/night temperature, 72% RH and 540 µ Em−2 s−1 quantum flux density) Al stress was imposed by lowering pH to 4.5 and addition of 166 µmoles L−1 AlCl3. Control plants were grown in the same nutrient solution without Al, pH 4.5. Aluminum inhibited NO3 − and NH4 + uptake and decreased shoot dry matter yield in all genotypes used in this investigation. Irrespective of their tolerance to Al toxicity, Al‐treated plants had less nitrogen (N), phosphorus (P), calcium (Ca), magnesium (Mg), and sulfur (S) in their leaves. In roots, Al treatment did not affect dry matter weight but decreased accumulation of N, Ca, and Mg. Phosphorus and potassium (K) tended to accumulate in roots of Al‐treated plants, suggesting that Al inhibited P translocation to the leaves. In roots, Al induced anaplerotic GDH and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) activities, but inhibited GS activity. Moreover, in the apex (0–5 mm) and in the 6–15 mm segment of the elongation zone of most genotypes, Al tended to inhibit expression of GSr and favor expression of GS1. This increase in GS1 expression concomitantly with decreased N uptake and elevated deaminating GDH activity are indicative of metabolic changes usually associated with plant senescence. In leaves, Al did not influence GOGAT and GS activities and expression of GS isoforms suggesting it does not disturb assimilation of nitrogenous compounds translocated from roots to leaves. Nonetheless, lower leaf PEPC activity suggests that Al intoxicated plants had lower CO2 − fixation rates. The inverse effect of Al treatment on the activities of GDH and PEPC in roots and leaves, indicates that whereas increased activities in roots were induced by stress related stimuli, decreased activities in leaves were caused by a reduced nitrogen supply to the leaves. Because the earliest effects observed in this investigation appeared only after 24 h of exposure to Al and were similar in both tolerant and sensitive genotypes, it is concluded that the changes observed in N uptake and assimilation into plant amino acids were downstream of the initial, primary Al toxicity event.

Regulation of glutamine metabolism during the development of Bombyx mori larvae

Waste ammonia is re-assimilated into amino acids via the amide group of glutamine and the amino group of glutamate (i.e. through glutamine synthetase/glutamate synthase pathway) for silk synthesis in the silkworm, Bombyx mori, in the last larval stadium. Glutamine concentration in hemolymph gradually decreased with the progress of the fifth instar and it remained at very low levels during the spinning stage, then followed by a sharp increase at the larval–pupal ecdysis. The changes in glutamine synthetase (GS) activity in silkworm tissues were relatively small through the larval development, while the changes in glutamate synthase (GOGAT) activity, especially in the posterior silk glands, were more drastic. In addition, activities of GOGAT in the tissues were much higher than those of the other enzymes involved in glutamine utilization, suggesting that glutamine pool was regulated mainly by the changes in GOGAT activity. Western blot analysis indicated that the changes in GOGAT protein level correlated with the changes in GOGAT activity. Topical application of a juvenile hormone analogue, methoprene, induced an accumulation of glutamine in the hemolymph of the fifth instar larvae. The levels of GOGAT protein and activity in the tissues of the methoprene treated larvae were much lower than those of the control larvae, whereas the methoprene treatment had no effect on the levels of GS activity. In conclusion, GOGAT expression promoted by reduction of juvenile hormone titer is quite important for enhanced utilization of nitrogen for synthesis of silk protein during the last larval instar.

Supplemental boron stimulates ammonium assimilation in leaves of tobacco plants (Nicotiana tabacum L.)

The aim of this work was to analyse the response of NH4 assimilation in leaves of tobacco plants (Nicotiana tabacum L. cv. Tennessee 86), to different B applications (B1, 5 MH 3BO3; B2, 10 MH 3BO3; B3, 20 M H3BO3). The plants were grown under controlled environmental conditions and received a complete nutrient solution. In this experiment, we analysed the foliar concentrations of B and NH4 , as well as the corresponding enzymatic activities: GS,GOGAT, GDH, PEPC; the end products of this assimilation, amino acids and proteins; and finally the concentration of non-structural sugars. Our results indicated that the different B treatments influenced the utilization of NH4 by tobacco leaves. The B3 treatment registered the lowest NH4 concentration, and B1 the highest, due probably to the higher GS, GOGAT and GDH activities registered at B3. Conversely, a decline in the concentration of non-structural sugars was recorded at B3. In addition, the high assimilation rate caused a progressive accumulation of amino acids as well as proteins, and boosted biomass production in the leaves.

Nitrogen and carbon regulation of glutamine synthetase and glutamate synthase in Corynebacterium glutamicum ATCC 13032

The effect of nitrogen and carbon status on the regulation of glutamine synthetase (GS) and glutamate synthase (GOGAT) were investigated in Corynebacterium glutamicum 13032. Under carbon-sufficient, nitrogen-limiting conditions, GS and GOGAT activities were five- and seven-fold higher, respectively, and transcription of the corresponding genes ( glnA and gltBD) was similarly induced. GS activity was also induced in complete medium with added glucose, while GOGAT activity was unaffected. Under carbon-limiting, nitrogen-limiting conditions, the level of GS induction was reduced approximately three-fold, whereas GOGAT activity did not respond. Disruption of the hkm gene, encoding a putative histidine kinase upstream of gltBD, reduced the levels of GOGAT activity two-fold under both nitrogen-rich and nitrogen-limiting conditions. Promoter studies using a hkm–chloramphenicol acetylase fusion plasmid revealed that transcription of hkm is moderately induced (ca. 1.5-fold) by nitrogen starvation, indicating that the Hkm protein may play a role in signal transduction of the nutritional status of the growth medium.

Roles of glutamate synthase, gltBD, and gltF in nitrogen metabolism of Escherichia coli and Klebsiella aerogenes.

Mutants of Escherichia coli and Klebsiella aerogenes that are deficient in glutamate synthase (glutamate-oxoglutarate amidotransferase [GOGAT]) activity have difficulty growing with nitrogen sources other than ammonia. Two models have been proposed to account for this inability to grow. One model postulated an imbalance between glutamine synthesis and glutamine degradation that led to a repression of the Ntr system and the subsequent failure to activate transcription of genes required for the use of alternative nitrogen sources. The other model postulated that mutations in gltB or gltD (which encode the subunits of GOGAT) were polar on a downstream gene, gltF, which is necessary for proper activation of gene expression by the Ntr system. The data reported here show that the gltF model is incorrect for three reasons: first, a nonpolar gltB and a polar gltD mutation of K. aerogenes both show the same phenotype; second, K. aerogenes and several other enteric bacteria lack a gene homologous to gltF; and third, mutants of E. coli whose gltF gene has been deleted show no defect in nitrogen metabolism. The argument that accumulated glutamine represses the Ntr system in gltB or gltD mutants is also incorrect, because these mutants can derepress the Ntr system normally so long as sufficient glutamate is supplied. Thus, we conclude that gltB or gltD mutants grow slowly on many poor nitrogen sources because they are starved for glutamate. Much of the glutamate formed by catabolism of alternative nitrogen sources is converted to glutamine, which cannot be efficiently converted to glutamate in the absence of GOGAT activity. Finally, GOGAT-deficient E. coli cells growing with glutamine as the sole nitrogen source increase their synthesis of the other glutamate-forming enzyme, glutamate dehydrogenase, severalfold, but this is still insufficient to allow rapid growth under these conditions.

Ammonium metabolism in the symbiotic sea anemone Anemonia viridis.

The temperate sea anemone Anemonia viridis (Forskal) forms an endosymbiotic association with dinoflagellate algae commonly referred to as zooxanthellae. It is now well established that under appropriate environmental conditions, these associations can be autotrophic for carbon. Under such conditions, many of these symbioses, including A. viridis, not only retain excretory ammonium, but can take up ammonium added to the surrounding seawater. The flux from inorganic to organic nitrogen will be via the free amino acid pools and in A. viridis these were found to be markedly different between zooxanthellae and host with glycine and taurine dominant in the latter. When anemones were maintained with 20 μM ammonium, the concentration of free amino groups increased in the zooxanthellae but appeared not to change in the host. There was no evidence that the ratio of glutamine – glutamate in zooxanthellae changed when anemones were maintained with 20 μM ammonium for 47 days. These ratios imply that zooxanthellae from this temperate symbiosis may not be nitrogen-limited. GDH was detected in both zooxanthellae and host where it was most active with the coenzyme NADPH. In addition, GDH showed activity when glutamine replaced ammonium as the substrate, indicating that the host may have alternative means to assimilate ammonium. Zooxanthellae were shown to possess GOGAT activity in the presence of a ferredoxin analogue. This suggests that in vivo zooxanthellae could assimilate ammonium via the activity of GS linked with ferredoxin-dependent GOGAT. Given evidence from other studies of rapid ammonium assimilation and essential amino acid synthesis in symbiotic host tissue, it appears that the capacity of cnidarians to metabolise nitrogen may at present be underestimated.

Role of CaCl 2 in Ammonium Assimilation in Roots of Tobacco Plants ( Nicotiana tabacum L.)

Summary The aim of this study was to determine the response of NH 4 + assimilation in roots to different CaCl 2 application (Ca1: 1.25 mmol/L CaCl 2 · 2H 2 O, Ca2: 2.5 mmol/L CaCl 2 · 2H 2 O and Ca3: 5 mmol/L CaCh · 2H 2 O). Tobacco plants ( Nicotiana tabacum cv. Tennessee 86) were grown under controlled conditions and submitted to regular fertilization with macro- and micronutrients. The contents of Ca 2+ , Cl - and NH 4 + , the activity of enzymes related to the process of NH 4 + assimilation (GS: glutamine synthetase, EC 6.3.1.2.; GOGAT: glutamate synthase, EC 1.4.1.14; GDH, glutamate dehydrogenase EC 1.4.1.2.), and the end products of this process (amino acids and proteins) were analysed in roots. Our results indicate that the utilization of NH 4 + in the plant was influenced by the different treatments. In the Ca3 treatment the NH 4 + content was lower than in Ca1 and Ca2, apparently due to the higher GS, GOGAT and GDH activities registered in Ca3. However, we found that the concentration of amino acids and protein as well as biomass production in the root was more important in the Ca1 and Ca2 treatments than in Ca3. This is the result of the greater translocation of these products to the shoot, stimulated by the high Ca 2+ dosage applied (5 mmol/L) to the cultivation medium in the Ca3 treatment.

Glutamine synthetase and glutamate synthase activities in relation to nitrogen fixation in Lotus spp.

Lotus corniculatus, L. tenuis, L. pedunculatus, and L. subbiflorus inoculated with Mesorhizobium loti NZP2037 strain were grown in a growth chamber. The plants dry weight (DW), the nodule fresh weight (FW), the nitrogenase activity, the nodule glutamine synthetase (GS) and glutamate synthase (GOGAT) activities, as well as the leghemoglobin content and the amino acid in the stem were measured 28 days after inoculation. The highest DW of plants was measured in L. tenuis and the highest FW of nodules was measured in L. pedunculatus. Nitrogenase activity in L. tenuis, L. pedunculatus and L. subbiflorus was six fold the activity in L. corniculatus. Nodule GS and GOGAT activities did not follow this same pattern. L. tenuis had the highest values of GS and GOGAT activities in the nodule, and a high nitrogenase activity which is consistent with its high plant DW. The four species of Lotus were compared and no correlation between nitrogen fixation parameters and ammonia assimilation enzymes was found, but the GS/GOGAT ratio has a positive and significant correlation (r²=0.82**) with the amino acid content in stems.

Differential accumulation of ferredoxin‐ and NADH‐dependent glutamate synthase activities, peptides, and transcripts in developing soybean seedlings in response to light, nitrogen, and nodulation

Soybean (Glycine max [L.] Merr. cv. Century) seedlings were germinated in vermiculite, sub‐irrigated with a complete nutrient solution, without nitrogen or supplemented with 10 mM KNO3, 5 mM (NH4)2SO4, or 5 mM NH4NO3. After 14 days in the light, or 5 days in the dark, tissues from different organs were harvested separately. Similarly, tissues from different organs from 14‐ or 21‐day‐old nodulated or non‐nodulated soybean seedlings, maintained in the absence of nitrogen, were harvested. Proteins and total RNA were isolated from the different plant organs and used for immunoblot and RNA blot analyses, respectively. Protein or RNA blots were separately incubated with antisera or hybridized with probes specific for either ferredoxin‐dependent glutamate synthase (Fd‐GOGAT) or NADH‐dependent glutamate synthase (NADH‐GOGAT). The specific activity and the abundance of the Fd‐GOGAT peptide and transcript increased, approximately 3–10 times, in cotyledons and hypocotyls/stems in plants germinated in the light compared with those germinated in the dark. Fd‐GOGAT activity, peptide, and transcript were highest in leaves. Except for increases in the specific activity of samples from roots treated with (NH4)2SO4 or NH4NO3, there were minor or no changes in Fd‐GOGAT activity, peptide and transcript among organs of seedlings treated with different nitrogen sources or by nodulation. Low levels of NADH‐GOGAT transcript were detected in all organs. NADH‐GOGAT activity, peptide, and transcript increased in roots of seedlings treated with different nitrogen sources, but these changes were more apparent on RNA blots versus immunoblots. The highest NADH‐GOGAT activity and most abundant amounts of the peptide and transcript were observed in nodules. Despite being induced by different environmental factors, both GOGAT activities are controlled, at least in part, by either gene expression or by RNA stability, because in most instances, both isoenzymes exhibited paralleled changes in specific activity and the abundance of their corresponding peptides and transcripts on immunoblot and RNA blots, respectively. However, there were some exceptions to the parallel increases in specific activities, peptides and transcripts which suggest that post‐translational modification may also regulate the activties of the two GOGAT isoforms. Collectively, the results presented here suggest that the two GOGAT isoforms have distinct physiological functions in soybean.

Enzymic properties and capacities of developing tomato (Lycopersicon esculentum L.) fruit plastids

To characterize the developmental stage of tomato fruits, chlorophyll content, photosynthetic O 2 evolution and CO 2 fixation of pericarp slices were determined. During the first developmental stages a higher expression level of the triose phosphate translocator was detected. Transport measurements revealed that both the hexose phosphate and the triose phosphate translocator are very likely to be active at this time. Plastidic and cytosolic fructose-1,6-bisphosphatase are active in green fruit pericarp, whereas in red pericarp only the cytosolic form is present. Tomato fruit chloroplasts are able to synthesize starch from Glc6P. Starch synthesis is strongly dependent on the addition of 3PGA and ATP and on plastid illumination. Fruit chloroplasts exhibit very low CO 2 fixation rates and so the capacities of green pericarp slices were investigated. In relation to chlorophyll content, pericarp slices show the same capacity of starch synthesis as spinach or potato leaves. To investigate the presence of further reactions consuming the products of photosynthetic electron transport, the GOGAT activity was measured. In the light, glutamine/2-oxoglutarate-dependent formation of glutamate occurred with a high activity. In the presence of Glc6P only 18% of the light activity was obtained. Since the Glc6P-dependent activity is rather low, the release of 14 CO 2 from labelled [1- 14 C]-Glc6P was also measured. In the dark, the formation of glutamate and oxidation of Glc6P are very tightly coupled to each other in fruit chloroplasts.

Arbuscular mycorrhizae and nitrogen assimilation in maize after drought and recovery

In a greenhouse experiment, the effect of arbuscular mycorrhizal (AM) fungus (Glomus intraradices Schenck &amp; Smith) colonization on N assimilation in maize (Zea mays L.) was examined after well‐watered, drought and recovery periods. Seeds of selection cycles C0 (drought‐sensitive) and C8 (drought‐resistant) of the tropical maize cultivar Tuxpeño sequía were used for this study. Maize plants were exposed or not to drought stress for 3 weeks (45‐65 days after sowing, DAS) followed by 3 weeks of recovery (66‐86 DAS) at the preflowering stage. Root and shoot samples harvested at the end of the drought or well‐watered and recovery periods were determined for key enzymes involved in N assimilation (NR, nitrate reductase; NiR, nitrite reductase; GS, glutamine synthetase; GOGAT, glutamate synthase), protein and amino acid concentrations, and total N contents. Drought stress significantly (P ≤ 0.01 or P ≤ 0.001) decreased all the enzyme activities except NiR in the roots and shoots of both cultivars. After 3 weeks of drought, the AM roots of both cultivars had higher activities of NR (C0, 45%; C8, 26%), GS (C0, 76%; C8, 33%) and GOGAT (C0, 41%; C8, 53%) than non‐AM roots and were comparable to well‐watered plants. These enzyme activities were also enhanced in drought‐stressed AM shoots of C0 and C8. Total amino acid concentrations in AM plants of C0 were 4.6 and 1.6 times higher in roots and shoots, respectively, compared to non‐AM plants. The predominant amino acids detected were Ala, Arg, Asn, Asp, Gln and Glu which constituted approximately 56 and 75% of the total pool in roots and shoots, respectively. Soluble proteins and total N contents were also higher in AM plants than non‐AM plants under drought conditions. The enhancement of N‐assimilating enzymes and nitrogenous compounds in maize may indicate a transfer of NO3− through the extraradical mycelium or increased N assimilation due to the AM symbiosis. Our overall results suggest that AM association plays an important role in enhancing N assimilation or N nutritional status which enables the host plant to withstand drought conditions and recover after stress is relieved.

GDH3 encodes a glutamate dehydrogenase isozyme, a previously unrecognized route for glutamate biosynthesis in Saccharomyces cerevisiae.

It has been considered that the yeast Saccharomyces cerevisiae, like many other microorganisms, synthesizes glutamate through the action of NADP+-glutamate dehydrogenase (NADP+-GDH), encoded by GDH1, or through the combined action of glutamine synthetase and glutamate synthase (GOGAT), encoded by GLN1 and GLT1, respectively. A double mutant of S. cerevisiae lacking NADP+-GDH and GOGAT activities was constructed. This strain was able to grow on ammonium as the sole nitrogen source and thus to synthesize glutamate through an alternative pathway. A computer search for similarities between the GDH1 nucleotide sequence and the complete yeast genome was carried out. In addition to identifying its cognate sequence at chromosome XIV, the search found that GDH1 showed high identity with a previously recognized open reading frame (GDH3) of chromosome I. Triple mutants impaired in GDH1, GLT1, and GDH3 were obtained. These were strict glutamate auxotrophs. Our results indicate that GDH3 plays a significant physiological role, providing glutamate when GDH1 and GLT1 are impaired. This is the first example of a microorganism possessing three pathways for glutamate biosynthesis.