Contrasted responses to carbohydrate limitation in tomato fruit at two stages of development

The metabolic consequences of long‐term carbohydrate depletion have been well documented in many sink organs but not extensively in fruit. Therefore, in the present study the response to sugar limitation in tomato fruit (Lycopersicon esculentum Mill.) was investigated at two developmental stages; during the cell division and cell expansion phases. First, the response in excised fruit cultured in vitro was characterized. Sugar depletion caused an arrest of growth and an exhaustion of carbon reserves. The proteins that were degraded and the nitrogen released was transiently stored as asparagine and glutamine in both developmental stages and also as γ ‐aminobutyric acid (GABA) in expanding fruit. Fruit at the cell division stage appeared to be more sensitive to sugar limitation. The response to sugar depletion was then characterized in fruit from plants submitted to extended darkness. In planta, the effects of sugar‐limitation were similar to those described in vitro but much more attenuated, especially in expanding fruit, which still accumulated dry matter. The expression of cell cycle genes, sugar‐ and nitrogen‐related genes was reduced by darkness. Only asparagine synthetase gene expression was induced in both dark‐treated fruit. Together the present data revealed that the effects of the carbon limitation are more pronounced in the youngest fruits as it is probably controlled by the relative sink strength of the fruit.     

龙柚果肉糖积累与蔗糖代谢相关酶活性的研究

本文探讨龙柚果实发育过程中果肉糖积累与蔗糖代谢相关酶活性的变化。结果表明,在龙柚果实发育过程中,3种可溶性糖含量同步上升,在果实膨大期和成熟期,以蔗糖积累为主。在龙柚糖积累过程中,蔗糖合成酶(SS)和蔗糖磷酸合成酶(SPS)活性较高;而蔗糖中性转化酶(NI)活性则随着蔗糖的积累而降低。     

外源γ-氨基丁酸（GABA）对低氧胁迫下甜瓜幼苗根系GABA代谢及氨基酸含量的影响

采用水培法,通过准确控制营养液溶氧浓度,研究了外源γ-氨基丁酸（GABA）对低氧胁迫0~8 d ‘西域一号’甜瓜幼苗根系GABA代谢及氨基酸含量的影响.结果表明：与通气对照相比,低氧处理的甜瓜幼苗正常生长受到严重抑制,其根系谷氨酸脱羧酶（GAD）、谷氨酸脱氢酶（GDH）、谷氨酸合成酶（GOGAT）、谷氨酰胺合成酶（GS）、丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）活性以及GABA、丙酮酸、丙氨酸、天冬氨酸含量均显著提高,而谷氨酸和α 酮戊二酸含量在处理4~8 d均显著降低.与低氧处理相比,外源GABA处理有效缓解了低氧胁迫对幼苗根系生长的抑制作用,同时甜瓜根系内源GABA、谷氨酸、α-酮戊二酸、天冬氨酸含量显著提高,但GAD、GDH、GOGAT、GS、ALT、AST活性在整个处理过程中均显著降低,丙酮酸和丙氨酸含量也显著降低.低氧同时添加GABA和γ-乙烯基 γ-氨基丁酸（VGB）处理显著降低了低氧胁迫下GABA的缓解效应.低氧胁迫下外源GABA被植物根系吸收后,通过反馈抑制GAD活性维持较高的Glu含量,保持植物体内碳、氮代谢平衡,维持正常生理代谢,从而缓解低氧胁迫对甜瓜幼苗的伤害.     

Regulation of gamma-aminobutyric acid degradation in Escherichia coli by nitrogen metabolism enzymes.

The possible role of glutamate dehydrogenase, glutamate synthase, and glutamine synthetase in the regulation of enzyme formation in the gamma-aminobutyric acid (GABA) catabolic pathway of Escherichia coli K-12 was investigated. Evidence is presented indicating that glutamine synthetase acts as a positive regulator in the E. coli GABA control system. Mutations impairing glutamate synthase activity prevent the depression of the enzymes of the GABA pathway in ammonia-limited glucose media. However, mutations resulting in constitutive synthesis of glutamine synthetase (GlnC) restore the ability of the glutamate synthase-less mutants to grow in glucose-GABA media and result in depressed synthesis of the GABA enzymes. It is suggested that the loss of glutamate synthesis activity affects the GABA control system indirectly by lowering glutamine synthetase levels.     

Physiological and metabolic enzymes activity changes in transgenic rice plants with increased phosphoenolpyruvate carboxylase activity during the flowering stage

To compare the differences in physiology and metabolism between phosphoenolpyruvate carboxylase (PEPC) transgenic rice and its control, untransformed wild rice, dry matter accumulation, soluble sugar, starch and protein contents and enzyme activities were determined in different plant parts during flowering. Results revealed that PEPC transgenic rice had higher dry weights for leaf, stem and sheath as well as panicle than the untransformed wild rice did, with the largest increase in the panicle. Soluble sugar and protein content in the grains of PEPC transgenic rice were significantly enhanced while starch content changed less. PEPC transgenic rice exhibited high levels of PEPC activity, manifesting in high net photosynthetic rates during flowering. Moreover, transgenic rice with high PEPC expression levels also had elevated levels of the enzymes such as sucrose-p-synthase and sucrose synthase, which may confer a higher capacity to assimilate CO₂ into sucrose. Little increase in grain starch content was observed in transgenic plants due to the stable activities of starch synthase and Q enzyme. However, the PEPC transgenic rice plant induced the activities of nitrate reductase, glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, glutamine synthetase, and asparagine synthase to high levels, as compared with the untransformed rice plant. PEPC activity was correlated with protein content in grains and the enzymes of nitrogen metabolism, suggesting that high PEPC activity in transgenic rice might be able to redirect carbon and nitrogen flow by regulating some enzymes related to carbon or nitrogen metabolisms. These results may help to understand how the C₃ plants possessing a C₄-like photosynthesis pathway worked by expression of PEPC.     

Sink development, sucrose metabolising enzymes and carbohydrate status in turnip (Brassica rapa L.)

Accumulation of 60–70 % of biomass in turnip root takes place between 49–56 days after sowing. To understand the phenomenon of rapid sink filling, the activities of sucrose metabolising enzymes and carbohydrate composition in leaf blades, petiole and root of turnip from 42–66 days of growth were determined. An increase (2–3 folds) in glucose and fructose contents of roots accompanied by an increase in activities of acid and alkaline invertases was observed during rapid biomass accumulating phase of roots. The observed decrease in the activities of acid and alkaline invertases along with sucrose synthase (cleavage) in petiole during this period could facilitate unrestricted transport of sucrose from leaves to the roots. During active root filling period, a decrease in sucrose synthase (cleavage) and alkaline invertase activities was also observed in leaf blades. A rapid decline in the starch content of leaf blades was observed during the phase of rapid sink filling. These metabolic changes in the turnip plant led to increase in hexose content (35–37 %) of total dry biomass of roots at maturity. High hexose content of the roots appears to be due to high acid invertase activity of the root.     

Enzymes of Nitrogen Assimilation Undergo Seasonal Fluctuations in the Roots of the Persistent Weedy Perennial Cichorium intybus

Chicory (Cichorium intybus), a deep rooted weed, grows in regions with temperate climates. Seasonal partitioning of compounds between the root and shoot results in fluctuations in the soluble carbohydrate, nitrate, amino acid, and protein pools within the roots. The activities of nitrate reductase (NR) (EC 1.6.6.1), glutamine synthetase (EC 6.3.1.2), NADH (EC 1.4.1.14), ferrodoxin glutamate synthase (EC 1.4.7.1), and glutamate dehydrogenase (GDH) (EC 1.4.1.2-4) vary throughout the year and coincide with seasonal alterations in nitrate, fructose, and sucrose. During the winter, NR, glutamine synthetase and ferrodoxin glutamate synthase activities increase in the root, while GDH displays the opposite trend with elevated activity in the summer months. All of these enzymes exhibit seasonal alterations in abundance as detected by Western blot analysis, increasing during the winter and, therefore, contributing to the seasonally dynamic protein pool. Extensive fluctuations in abundance and activity of these enzymes in the root occur during the spring and fall and coincide with shoot growth and senescence, respectively. Several observations indicate that posttranslational modifications of NR and GDH are taking place throughout the year; for example, NR is particularly unstable during the spring and fall, and seasonal GDH activity does not correlate with protein abundance.     

Manipulation Through Liquid Culture in the Contents of Sucrose and Glutamine and Their Transformation to Starch and Protein in Wheat Grain

Detached ears of wheat were cultured in liquid medium manipulated for sucrose and glutamine contents, and the accumulation of starch and protein in relation to the activities of sucrose cleaving—, ammonia assimilating—, and transaminating enzymes was studied in the grain. With an increase in the concentrations of sucrose from 44 to 176 mM and glutamine from 6.4 to 25.7 mM (keeping their ratio at a constant value of 7:1), the contents of starch and protein increased in the grains. However, when the grains were cultured in the medium containing 8.5 to 34 mM glutamine and a fixed concentration of 117 mM sucrose, there was a gradual increase in protein and decrease in starch content in the grain. By such manipulation in the liquid medium, the content of free amino acids also increased in the grain up to 12 days culturing. Amongst sucrose cleaving enzymes, the activities of sucrose-UDP glucosyl transferase and soluble alkaline invertase were much lower than the activity of soluble acid invertase. At high concentration (34 mM) of glutamine in the medium, containing 117 mM sucrose, there was drastic decrease in the activities of soluble acid invertase and UDPG pyrophosphorylase but the activities of ADPG pyrophosphorylase, alkaline inorganic pyrophosphatase, glutamate dehydrogenase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase increased in the grain with increase in glutamine concentration in the culture medium. Evidently, an increase in the level of amino nitrogen, coupled with an optimum sucrose concentration in the grain raised through liquid culturing enhances the conversion of sucrose to protein at the cost of starch accumulation in wheat.     

Assimilation of Ammonium Nitrogen by Heterotrophic and Symbiotrophic White Lupine Plants

The activities of glutamate dehydrogenase, asparagine synthetase, and total glutamine synthetase in the organs of the white lupine (Lupinus albus L.) plants were measured during plant growth and development. In addition, the dynamics of free amino acids and amides in plant organs was followed. It was shown that the change in the nutrition type was important for controlling enzyme activities in the organs examined and, consequently, for directing the pathway of ammonium nitrogen assimilation. As long as the plants remained heterotrophic, glutamine-dependent asparagine synthetase of cotyledons and glutamine synthetase of leaves apparently played a major role in the assimilation of ammonium nitrogen. In symbiotrophic plants, root nodules became an exclusive site of asparagine synthesis, and the role of leaf glutamine synthetase increased. Unlike glutamine synthetase and asparagine synthetase, glutamate dehydrogenase activity was present in all organs examined and was less dependent on the nutrition type. This was also indicated by a weak correlation of glutamate dehydrogenase activity with the dynamics of free amino acid and amide content in these organs. It is supposed that glutamine synthetase plays a leading role in both the primary assimilation of ammonium, produced during symbiotic fixation of molecular nitrogen in root nodules, and in its secondary assimilation in cotyledons and leaves. On the other hand, secondary nitrogen assimilation in the axial organs occurs via an alternative glutamate dehydrogenase pathway.     

Ammonia assimilation by rhizobium cultures and bacteroids.

The enzymes involved in the assimilation of ammonia by free-living cultures of Rhizobium spp. are glutamine synthetase (EC. 6.o.I.2), glutamate synthase (L-glutamine:2-oxoglutarate amino transferase) and glutamate dehydrogenase (ED I.4.I.4). Under conditions of ammonia or nitrate limitation in a chemostat the assimilation of ammonia by cultures of R. leguminosarum, R. trifolii and R. japonicum proceeded via glutamine synthetase and glutamate synthase. Under glucose limitation and with an excess of inorganic nitrogen, ammonia was assimilated via glutamate dehydrogenase, neither glutamine synthetase nor glutamate synthase activities being detected in extracts. The coenzyme specificity of glutamate synthase varied according to species, being linked to NADP for the fast-growing R. leguminosarum, R. melitoti, R. phaseoli and R. trifolii but to NAD for the slow-growing R. japonicum and R. lupini. Glutamine synthetase, glutamate synthase and glutamate dehydrogenase activities were assayed in sonicated bacteroid preparations and in the nodule supernatants of Glycine max, Vicia faba, Pisum sativum, Lupinus luteus, Medicago sativa, Phaseolus coccineus and P. vulgaris nodules. All bacteroid preparations, except those from M. sativa and P. coccineus, contained glutamate synthase but substantial activities were found only in Glycine max and Lupinus luteus. The glutamine synthetase activities of bacteroids were low, although high activities were found in all the nodule supernatants. Glutamate dehydrogenase activity was present in all bacteroid samples examined. There was no evidence for the operation of the glutamine synthetase/glutamate synthase system in ammonia assimilation in root nodules, suggesting that ammonia produced by nitrogen fixation in the bacteroid is assimilated by enzymes of the plant system.     

Investigation of the site of synthesis of chIcoroplastic enzymes of nitrogen metabolism by the use of heat-treated 70S ribosomedeficient rye leaves

The activities of the enzymes nitrate reductase (EC 1.6.6.1), nitrite reductase (EC 1.6.6.4), glutamine synthetase (EC 6.3.1.2), glutamate synthase (GOGAT; EC 1.4.7.1), glutamate-oxaloacetate aminotransferase (EC 2.6.1.1), and glutamate dehydrogenase (EC 1.4.1.2) were compared in light-grown green or etiolated leaves of rye seedlings ( Secale cereale L. cv. Halo) raised at 22°C, and in the bleached 70S ribosome-deficient leaves of rye seedlings grown at a non-permissive high temperature of 32°C. Under normal permissive growth conditions the activities of most of the enzymes were higher in light-grown, than in dark-grown, leaves. All enzyme activities assayed were also observed in the heat-treated 70S ribosome-deficient leaves. Glutamine synthetase, glutamate synthase, and glutamate-oxaloacetate aminotransferase occurred in purified ribosome-deficient plastids separated on sucrose gradients. For glutamate-oxaloacetate aminotransferase four multiple forms were separated by polyacrylamide gel electrophoresis from leaf extracts. The chloroplastic form of this enzyme was also present in 70S ribosome-deficient leaves. It is concluded that the chloroplast-localized enzymes nitrite reductase, glutamine synthetase, glutamate synthase and glutamate-oxaloacetate aminotransferase, or their chloroplast-specific isoenzyme forms, are synthesized on cytoplasmic 80S ribosomes.     

The Role of Ammonium Assimilating Enzymes in Lentil Roots and Nodules

Activities of ammonium assimilating enzymes glutamate dehydrogenase (GDH), glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) as well as the amino acid content were higher in nodules compared to roots. Their activities increased at 40 and 60 d after sowing, with a peak at 90 d, a time of maximum nitrogenase activity. The GS/GOGAT ratio had a positive correlation with the amino acid content in nodules. Higher activities of AST than ALT may be due to lower glutamine and higher asparagine content in xylem. The data indicated that glutamine synthetase and glutamate synthase function as the main route for the assimilation of fixed N, while NADH-dependent glutamate dehydrogenase may function at higher NH₄ ⁺ concentration in young and senescing nodules. Enzyme activities in lentil roots reflected a capacity to assimilate N for making the amino acids they may need for both growth and export to upper parts of the plant. This revised version was published online in July 2006 with corrections to the Cover Date.     

The regulation of ammonia assimilating enzymes in Lemma minor

Summary Lemna minor has the potential to assimilate ammonia via either the glutamine or glutamate pathways. A 3-4 fold variation in the level of ferredoxindependent glutamate synthase may occur, when plants are grown on different nitrogen sources, but these changes show no simple relationship to changes in the endogenous pool of glutamate. High activities of glutamate synthase and glutamine synthetase at low ammonia availability suggests that these two enzymes function in the assimilation of low ammonia concentrations. Increasing ammonia availability leads to a reduction in level of glutamate synthase and glutamine synthetase and an increase in the level of glutamate dehydrogenase. Glutamine synthetase and glutamate dehydrogenase are subject to concurrent regulation, with glutamine rather than ammonia, exerting negative control on glutamine synthetase and positive control on glutamate dehydrogenase. The changes in the ratio of these two enzymes in response to the internal pool of glutamine could regulate the direction of the flow of ammonia into amino acids via the two alternative routes of assimilation.Abbreviations GS Glutamine synthetase - GDH Glutamate dehydrogenase - GOGAT Glutamate synthase     

Carbon and nitrogen metabolism in barley (Hordeum vulgare L.) mutants lacking ferredoxin-dependent glutamate synthase

Five mutant lines of barley (Hordeum vulgare L.), which are only able to grow at elevated levels of CO₂, contain less than 5% of the wild-type activity of ferredoxin-dependent glutamate synthase (EC 1.4.7.1). Two of these lines (RPr 82/1 and RPr 82/9) have been studied in detail. Leaves and roots of both lines contain normal activities of NADH-dependent glutamate synthase (EC 1.4.1.14) and the other enzymes of ammonia assimilation. Under conditions that minimise photorespiration, both mutants fix CO₂ at normal rates; on transfer to air, the rates drop rapidly to 15% of the wild-type. Incorporation of ¹⁴CO₂ into sugar phosphates and glycollate is increased under such conditions, whilst incorporation of radioactivity into serine, glycine, glycerate and sucrose is decreased; continuous exposure to air leads to an accumulation of ¹⁴C in malate. The concentrations of malate, glutamine, asparagine and ammonia are all high in air, whilst aspartate, alanine, glutamate, glycine and serine are low, by comparison with the wild-type parent line (cv. Maris Mink), under the same conditions. The metabolism of [¹⁴C]glutamate and [¹⁴C]glutamine by leaves of the mutants indicates a very much reduced ability to convert glutamine to glutamate. Genetic analysis has shown that the mutation in RPr 82/9 segregates as a single recessive nuclear gene.Abbreviations GDH glutamate dehydrogenase (EC 1.4.1.2) - GS glutamine synthetase (EC 6.3.1.2) - RuBP ribulose 1,5-bisphosphate     

Rapid assimilation of recently fixed N2 in root nodules of Myrica gale

In Myrica gale L. plants the assimilation of ammonia released by symbiotic Frankia was observed by ¹⁵N₂ labelling and subsequent analysis of the isotopic enrichment of nodule amino acids over time by single ion monitoring gas chromatography-mass spectrometry. In detached nodules of Myrica , glutamine was the first amino acid labelled at 30 s and subsequently the amino acids glutamate, aspartate, alanine and γ-amino butyric acid (GABA) became labelled. This pattern of labelling is consistent with the incorporation of ammonium via glutamine synthetase [GS; EC 6.3.1.2]. No evidence for the ammonium assimilation via glutamate dehydrogenase [GDH; EC 1.4.1.2] was observed as glutamate became labelled only after glutamine. Using attached nodules and pulse-chase labelling, we observed synthesis of glutamine, glutamate, aspartate, alanine, GABA and asparagine, and followed the transport of fixed nitrogen in the xylem largely as glutamine and asparagine. Estimation of the cost of nitrogen fixation and asparagine synthesis in Myrica nodules suggests a minimum of one sucrose required per asparagine produced. Rapid translocation of recently fixed nitrogen was observed in Myrica gale nodules as 80% of the nitrogen fixed during a 1-h period was translocated out of the nodules within 9 h. The large pool of asparagine that is present in nodules may buffer the transport of nitrogen and thus act to regulate nitrogen fixation via a feedback mechanism.     

Sink metabolism in tomato fruit : I. Developmental changes in carbohydrate metabolizing enzymes

In developing tomato (Lycopersicon esculentum Mill.) fruit, starch levels reach a peak early in development with soluble sugars (hexoses) gradually increasing in concert with starch degradation. To determine the enzymic basis of this transient partitioning of carbon to starch, the activities of key carbohydrate-metabolizing enzymes were investigated in extracts from developing fruits of three varieties (cv VF145-7879, cv LA1563, and cv UC82B), differing in final soluble sugar accumulation. Of the enzymes analyzed, ADPglucose pyrophosphorylase and sucrose synthase levels were temporally correlated with the transient accumulation of starch, having highest activities in cv LA1563, the high soluble sugar accumulator. Of the starch-degrading enzymes, phosphorylase levels were fivefold higher than those of amylase, and these activities did not increase during the period of starch degradation. Fiften percent of the amylase activity and 45 to 60% of the phosphorylase activity was localized in the chloroplast in cv VF145-7879. These results suggest that starch degradation in tomato fruit is predominantly phosphorolytic. The results suggest that starch biosynthetic capacity, as determined by levels of ADPglucose pyrophosphorylase rather than starch degradative capacity, regulate the transient accumulation of starch that occurs early in tomato fruit development. The results also suggest that ADPglucose pyrophosphorylase and sucrose synthase levels correlated positively with soluble sugar accumulation in the three varieties examined.     

N2 fixation and NH 4 + assimilation in the thermophilic anaerobes Clostridium thermosaccharolyticum and Clostridium thermoautotrophicum

Inorganic nitrogen metabolism in the obligate anaerobic thermophiles Chlostridium thermosaccharolyticum and Clostridium thermoautotrophicum differs in several respects. C. thermosaccharolyticum contains a nitrogenase as inferred from NH ₄ ⁺ repressible C₂H₂ reduction, a glutamine synthetase which is partially repressed by ammonium, very labile glutamate synthase activities with both NADH and NADPH, NADPH-dependent glutamate dehydrogenase, and NH ₄ ⁺ -dependent asparagine synthetase. C. thermoautotrophicum contains no nitrogenase, but glutamine synthetase, no glutamate synthase, no glutamate dehydrogenase, but a NADH-dependent alanine dehydrogenase and a NH ₄ ⁺ -dependent asparagine synthetase.Abbreviation GOGAT glutamine-oxoglutarate amidotransferase amidotransferase (glutamate synthase)     

Enzymes involved in starch synthesis in the developing mung bean seed

The levels of free sugars, starch and enzymes involved in starch metabolism—sucrose synthetase, UDP and ADP glucose pyrophosphorylase, phosphorylase and starch synthetase—were assayed during seed development of three cultivars of mung bean (Vigna radiata). Free sugars and starch increased with increasing seed weight. Changes in levels of sucrose synthetase, UDP- and ADP-glucose pyrophosphorylases, and phosphorylase were paralleled by changes in starch accumulation. After the maximum activity levels of these enzymes had been reached, maximum activities of soluble starch synthetase and starch granule-bound starch synthetase occurred. There were high activities of sucrose synthetase and phosphorylase at maximum rates of starch accumulation. Thus, starch could be synthesized via the ADP glucose pathway in mung bean seeds. However, phosphorylase may account for the starch accumulation in the early stages of mung bean seed development.     

去果河套蜜瓜源叶碳水化合物及其相关酶昼夜变化特征

以河套蜜瓜为试材,在果实迅速膨大期通过去果处理改变库源关系,研究源叶净光合速率,蔗糖、还原糖和淀粉含量及其代谢相关酶活性的昼夜变化规律。结果表明:(1)源叶的净光合速率为单峰曲线,无明显的"光合午休"现象,去果处理对其无影响。(2)源叶中蔗糖和还原糖含量的昼夜变化为单峰曲线,蔗糖磷酸合成酶和蔗糖合成酶合成方向活性的昼夜变化为双峰曲线,蔗糖合成酶分解方向、酸性转化酶和中性转化酶活性的昼夜变化无明显规律,改变库源关系对这些指标均无显著影响;蔗糖含量升高受蔗糖磷酸合成酶和蔗糖合成酶合成方向正调控,而蔗糖含量降低则受多种酶的共同调节。(3)源叶中淀粉含量和腺苷二磷酸葡萄糖焦磷酸化酶活性的昼夜变化为单峰曲线,去果处理可以显著提高淀粉含量和腺苷二磷酸葡萄糖焦磷酸化酶活性,淀粉含量升高受腺苷二磷酸葡萄糖焦磷酸化酶正调控。