GS-GOGAT Cycle Research Articles

Increasing activity of the GS-GOGAT cycle highlights the compensation of N-assimilation in the absence of nitrogen and its metabolic effects in cyanobacteria

Comparative studies of proteome analysis, photosynthetic activity and fatty acid biosynthesis of the glnA- and glsF-overexpressing strains and the wild-type (WT) were carried out to elucidate the possible role(s) of the two enzymes, glutamine synthetase (GS) and glutamate synthase (GOGAT) in the GS-GOGAT cycle under nitrogen starvation. The evidence indicated the role of GS and GOGAT in the regulatory mechanism of carbon (C-) and nitrogen (N-) assimilation and the induction of photosynthesis and lipid/fatty acid biosynthesis pathway under N-stress. The fatty acid profile of the GS-overexpressing strain (WT + GlnA) under the stress showed a drastic increase in the levels of C18:1Δ9 and C18:1Δ11, whereas only C18:1Δ9 was significantly induced in the GOGAT-overexpressing strain (WT + GlsF). Moreover, the proteome analysis and oxygen evolution data showed that the extra-GS activity in the mutant (MT) cells led to the increasing of proteins involved in photosynthesis as well as the rate of oxygen evolution in the first 24 h after nitrogen depletion, suggesting its role in promoting conversion of light energy to chemical energy under the stress.

Physiological response and tolerance of Myriophyllum aquaticum to a wide range of ammonium concentrations.

Myriophyllum aquaticum (M. aquaticum) can be used in constructed wetlands (CWs) to effectively purify swine wastewater with high-ammonia nitrogen (NH3-N and NH4+-N) concentrations. However, the understanding of its tolerance mechanism to ammonia nitrogen is limited. The physiological response and tolerance mechanism of M. aquaticum to a wide range of NH4+ concentrations (0-35mM) were investigated in the present study. The results indicated that M. aquaticum can tolerate NH4+ concentrations of up to 30mM for 21 days and grow well with high nutrient (N, P) uptake. A suitable concentration of NH4+ for a better growth of M. aquaticum was 0.5-20mM. The free NH4+ content was no obviously increase at NH4+ concentration below 15mM, indicated there was no obviously ammonium accumulation. Exogenous NH4+ inhibited K+ absorption and improved Ca2+ absorption, indicating mineral cation could mediate NH4+ homeostasis under NH4+ stress. Moreover, comparison with those in the control group, the activities of glutamine synthetase (GS), glutamate synthetase (GOGAT) in M. aquaticum increased by 52.7%-115% at 1-20mM NH4+, and superoxide dismutase (SOD) increased by 29.2-143% at 1-35mM NH4+. This indicated that the high NH4+ tolerance of M. aquaticum was mainly due to the balance of free NH4+ content in tissues, as well as improved nitrogen metabolism and antioxidant system. This could be attributed to the role of the GS-GOGAT cycle and SOD. In conclusion, M. aquaticum, which tolerates high NH4+ concentration and has a high N uptake ability, can be used as a good candidate specie to help develop more efficient management strategies for treating high-NH4+ wastewater in CW systems.

Effect and function of β-N-methylamino-L-alanine in the diatom Phaeodactylum tricornutum

The neurotoxin β-N-methylamino-L-alanine (BMAA) is an environmental factor connected to neurodegenerative diseases. BMAA can be produced by various microorganisms (e.g. bacteria, cyanobacteria, dinoflagellates and diatoms) present in diverse ecosystems. No previous study has revealed the function of BMAA in diatoms. In the present study, we combined physiological data with metabolomic and transcriptional data in order to investigate the effect and function of BMAA in the diatom Phaeodactylum tricornutum. P. tricornutum, exposed to different concentrations of exogenous BMAA, showed concentration dependent responses. When the concentration of supplemented BMAA was sufficient to arrest the growth of P. tricornutum, oxidative stress and obstructed carbon fixation were obtained from the specific metabolite and transcriptional data. Results also indicated increased concentration of intracellular chlorophyll a and alterations in the GS-GOGAT cycle, whereas the urea cycle was suppressed. We therefore conclude that BMAA represents a toxic metabolite able to control the growth of P. tricornutum by triggering oxidative stress, and further influencing photosynthesis and nitrogen metabolisms.

Nitrogen Availability Affects the Metabolic Profile in Cyanobacteria.

Nitrogen is essential for the biosynthesis of various molecules in cells, such as amino acids and nucleotides, as well as several types of lipids and sugars. Cyanobacteria can assimilate several forms of nitrogen, including nitrate, ammonium, and urea, and the physiological and genetic responses to these nitrogen sources have been studied previously. However, the metabolic changes in cyanobacteria caused by different nitrogen sources have not yet been characterized. This study aimed to elucidate the influence of nitrate and ammonium on the metabolic profiles of the cyanobacterium Synechocystis sp. strain PCC 6803. When supplemented with NaNO3 or NH4Cl as the nitrogen source, Synechocystis sp. PCC 6803 grew faster in NH4Cl medium than in NaNO3 medium. Metabolome analysis indicated that some metabolites in the CBB cycle, glycolysis, and TCA cycle, and amino acids were more abundant when grown in NH4Cl medium than NaNO3 medium. 15N turnover rate analysis revealed that the nitrogen assimilation rate in NH4Cl medium was higher than in NaNO3 medium. These results indicate that the mechanism of nitrogen assimilation in the GS-GOGAT cycle differs between NaNO3 and NH4Cl. We conclude that the amounts and biosynthetic rate of cyanobacterial metabolites varies depending on the type of nitrogen.

Integrated transcript and metabolite profiling reveals coordination between biomass size and nitrogen metabolism in Arabidopsis F1 hybrids.

Heterosis refers to the improved agronomic performance of F1 hybrids relative to their parents. Although this phenomenon is widely employed to increase biomass, yield, and stress tolerance of plants, the underlying molecular mechanisms remain unclear. To dissect the metabolic fluctuations derived from genomic and/or environmental differences contributing to the improved biomass of F1 hybrids relative to their parents, we optimized the growth condition for Arabidopsis thaliana F1 hybrids and their parents. Modest but statistically significant increase in the biomass of F1 hybrids was observed. Plant samples grown under the optimized condition were also utilized for integrated omics analysis to capture specific changes in the F1 hybrids. Metabolite profiling of F1 hybrids and parent plants was performed using gas chromatography-mass spectrometry. Among the detected 237 metabolites, 2-oxoglutarate (2-OG) and malate levels were lower and the level of aspartate was higher in the F1 hybrids than in each parent. In addition, microarray analysis revealed that there were 44 up-regulated and 12 down-regulated genes with more than 1.5-fold changes in expression levels in the F1 hybrid compared to each parent. Gene ontology (GO) analyses indicated that genes up-regulated in the F1 hybrids were largely related to organic nitrogen (N) process. Quantitative PCR verified that glutamine synthetase 2 (AtGLN2) was upregulated in the F1 hybrids, while other genes encoding enzymes in the GS-GOGAT cycle showed no significant differences between the hybrid and parent lines. These results suggested the existence of metabolic regulation that coordinates biomass and N metabolism involving AtGLN2 in F1 hybrids.

Localization, Gene Expression, and Functions of Glutamine Synthetase Isozymes in Wheat Grain (Triticum aestivum L.).

Glutamine synthetase (GS) plays a major role in plant nitrogen metabolism, but the roles of individual GS isoforms in grains are unknown. Here, the localization and expression of individual TaGS isozymes in wheat grain were probed with TaGS isoenzyme-specific antibodies, and the nitrogen metabolism of grain during the grain filling stage were investigated. Immunofluorescence revealed that TaGS1;1, TaGS1;3, and TaGS2 were expressed in different regions of the embryo. In grain transporting tissues, TaGS1;2 was localized in vascular bundle; TaGS1;2 and TaGS1;1 were in chalaza and placentochalaza; TaGS1;1 and TaGS1;3 were in endosperm transfer cells; and TaGS1;3 and TaGS2 were in aleurone layer. GS exhibited maximum activity and expression at 8 days after flowering (DAF) with peak glutamine content in grains; from then, increased largely from reduction, glutamate dehydrogenase (GDH) aminating activity increased continuously, and the activities of GS and glutamate synthase (GOGAT) decreased, while only TaGS1;3 kept a stable expression in different TaGS isozymes. Hence, GS-GOGAT cycle and GDH play different roles in assimilation of grain in different stages of grain development; TaGS1;3, located in aleurone layer and endosperm transfer cells, plays a key role in Gln into endosperm for gluten synthesis. At 30 DAF, grain amino acids are mainly transported from maternal phloem.

Nanoselenium foliar application enhances biosynthesis of tea leaves in metabolic cycles and associated responsive pathways.

An emerging stress of pesticides in plant and soil is closely watched as it affects crop antioxidant systems, nutritional quality, and flavor. Although selenium (Se) can enhance the resistance of plants, the protective mechanism of nanoselenium is still not known under the long-term pesticide stress in tea trees. In this study, we investigated the potential effects of foliar application of nanoselenium for a two-year field experiment on tea plants under pesticide-induced oxidative stress. Compared to control, nano-Se (10mg/L) markedly enhanced the protein, soluble sugar, carotenoid, tea polyphenols, and catechins contents. High levels of theanine, glutamic acid, proline, and arginine were found to be induced most likely by adjusting the GS-GOGAT cycle. Se-supplementation may promote tea leaves' secondary metabolism, thus increasing the accumulation of total phenols and flavonoids (apigenin, kaempferol, quercetin, myricetin, and rutin). It also minimized the accumulation of malondialdehyde, hydrogen peroxide, and superoxide anion by activating the antioxidants enzymes including in the AsA-GSH cycle. Selenium-rich tea also showed better fragrance and flavor. In summary, nano-Se can ameliorate the nutrients quality and abiotic stresses resistance of crops.

Genome Wide Phosphoproteome Analysis of Zymomonas mobilis Under Anaerobic, Aerobic, and N2-Fixing Conditions.

Protein phosphorylation is a post-translational modification with widespread regulatory roles in both eukaryotes and prokaryotes. Using mass spectrometry, we performed a genome wide investigation of protein phosphorylation in the non-model organism and biofuel producer Zymomonas mobilis under anaerobic, aerobic, and N2-fixing conditions. Our phosphoproteome analysis revealed 125 unique phosphorylated proteins, belonging to major pathways such as glycolysis, TCA cycle, electron transport, nitrogen metabolism, and protein synthesis. Quantitative analysis revealed significant and widespread changes in protein phosphorylation across growth conditions. For example, we observed increased phosphorylation of nearly all glycolytic enzymes and a large fraction of ribosomal proteins during aerobic and N2-fixing conditions. We also observed substantial changes in the phosphorylation status of enzymes and regulatory proteins involved in nitrogen fixation and ammonia assimilation during N2-fixing conditions, including nitrogenase, the Rnf electron transport complex, the transcription factor NifA, GS-GOGAT cycle enzymes, and the PII regulatory protein. This suggested that protein phosphorylation may play an important role at regulating all aspects of nitrogen metabolism in Z. mobilis. This study provides new knowledge regarding the specific pathways and cellular processes that may be regulated by protein phosphorylation in this important industrial organism and provides a useful road map for future experiments that investigate the physiological role of specific phosphorylation events in Z. mobilis.

Molecular characterization of a novel algal glutamine synthetase (GS) and an algal glutamate synthase (GOGAT) from the colorful outer mantle of the giant clam, Tridacna squamosa, and the putative GS-GOGAT cycle in its symbiotic zooxanthellae

Giant clams harbor symbiotic zooxanthellae (Symbiodinium), which are nitrogen-deficient, mainly in the fleshy and colorful outer mantle. This study aimed to sequence and characterize the algal Glutamine Synthetase (GS) and Glutamate Synthase (GLT), which constitute the glutamate synthase cycle (or GS-GOGAT cycle, whereby GOGAT is the protein acronym of GLT) of nitrogen assimilation, from the outer mantle of the fluted giant clam, Tridacna squamosa. We had identified a novel GS-like cDNA coding sequence of 2325 bp, and named it as T. squamosa Symbiodinium GS1 (TSSGS1). The deduced TSSGS1 sequence had 774 amino acids with a molecular mass of 85 kDa, and displayed the characteristics of GS1 and Nucleotide Diphosphate Kinase. The cDNA coding sequence of the algal GLT, named as T. squamosa Symbiodinium GLT (TSSGLT), comprised 6399 bp, encoding a protein of 2133 amino acids and 232.4 kDa. The zooxanthellal origin of TSSGS1 and TSSGOGAT was confirmed by sequence comparison and phylogenetic analyses. Indeed, TSSGS1 and TSSGOGAT were expressed predominately in the outer mantle, which contained the majority of the zooxanthellae. Immunofluorescence microscopy confirmed the expression of TSSGS1 and TSSGOGAT in the cytoplasm and the plastids, respectively, of the zooxanthellae in the outer mantle. It can be concluded that the symbiotic zooxanthellae of T. squamosa possesses a glutamate synthase (TSSGS1-TSSGOGAT) cycle that can assimilate endogenous ammonia produced by the host clam into glutamate, which can act as a substrate for amino acid syntheses. Thus, our results provide insights into why intact giant clam-zooxanthellae associations do not excrete ammonia under normal circumstances.

Feathermoss and epiphytic Nostoc cooperate differently: expanding the spectrum of plant\u2013cyanobacteria symbiosis

Dinitrogen (N2)-fixation by cyanobacteria in symbiosis with feathermosses is the primary pathway of biological nitrogen (N) input into boreal forests. Despite its significance, little is known about the cyanobacterial gene repertoire and regulatory rewiring needed for the establishment and maintenance of the symbiosis. To determine gene acquisitions and regulatory changes allowing cyanobacteria to form and maintain this symbiosis, we compared genomically closely related symbiotic-competent and -incompetent Nostoc strains using a proteogenomics approach and an experimental set up allowing for controlled chemical and physical contact between partners. Thirty-two gene families were found only in the genomes of symbiotic strains, including some never before associated with cyanobacterial symbiosis. We identified conserved orthologs that were differentially expressed in symbiotic strains, including protein families involved in chemotaxis and motility, NO regulation, sulfate/phosphate transport, and glycosyl-modifying and oxidative stress-mediating exoenzymes. The physical moss–cyanobacteria epiphytic symbiosis is distinct from other cyanobacteria–plant symbioses, with Nostoc retaining motility, and lacking modulation of N2-fixation, photosynthesis, GS-GOGAT cycle and heterocyst formation. The results expand our knowledge base of plant–cyanobacterial symbioses, provide a model of information and material exchange in this ecologically significant symbiosis, and suggest new currencies, namely nitric oxide and aliphatic sulfonates, may be involved in establishing and maintaining the cyanobacteria–feathermoss symbiosis.

Enantioselective disturbance of chiral herbicide dichlorprop to nitrogen metabolism of Arabidopsis thaliana: Regular analysis and stable isotope attempt

As the most essential element, nitrogen play a pivotal role in plant physiological process, which is susceptible to contaminants. However, the enantioselective effects of chiral herbicides on nitrogen metabolism have not been comprehensively understood. In this study, effects of chiral herbicide dichlorprop (DCPP) on the nitrogen behaviors in Arabidopsis thaliana were investigated. The results revealed that the uptakes of inorganic nitrogen (NO3− and NH4+) were preferentially impacted by herbicidal active (R)-DCPP, where the transportation of beneficial NO3− was inhibited but the toxic NH4+ was accumulated. As for nitrogen assimilation, enantioselectivity was disappeared and reoccurred when NO3− was assimilated into NH4+. Furthermore, the pathways of NH4+ assimilation differed in Arabidopsis exposed to DCPP enantiomers, where (S)-DCPP remained almost the same to the control relying on GS-GOGAT cycle, but the dominant pathway in (R)-DCPP group was shifted to glutamate dehydrogenase route. Consequently, a profound enantioselectivity was also observed in the effects on amino acids synthesis. As for nitrogen stable isotope fractionation, (R)-DCPP led to more negative δ15N values than (S)-DCPP at 0.3μM, indicating the selective uptake of toxic NH4+ and different discrimination of δ15N was also observed between DCPP enantiomers. Moreover, stable isotopic evidence also revealed the disturbance caused by DCPP to the balance of nitrogen demand and consumption in A. thaliana. All these results may contribute to the final enantioselective toxicity, providing a new sight into the better understanding the action mechanism of chiral herbicides.

Exploring the role of GS-GOGAT cycle in microcystin synthesis and regulation - a model based analysis.

Toxic cyanobacteria blooms populate water bodies by consuming external nutrients and releasing cyanotoxins that are detrimental for other aquatic species, producing a significant impact on the plankton ecosystem and food web. To exercise population-level control of toxin production, understanding the biochemical mechanisms that explain cyanotoxin regulation within a bacterial cell is of utmost importance. In this study, we explore the mechanistic events to investigate the dependence of toxin microcystin on external nitrogen, a known regulator of the toxin, and for the first time, propose a kinetic model that analyzes the intracellular conditions required to ensure nitrogen dependence on microcystin. We hypothesize that the GS-GOGAT cycle is manipulated by variable influx of different intracellular metabolites that can either disturb or promote the balance between the enzyme microcystin synthetase and substrate glutamate to produce variable microcystin levels. As opposed to the popular notion that nitrogen starvation increases microcystin synthesis, our analyses suggest that under certain intracellular metabolite regimes, this relationship can either be completely lost or reversed. External nitrogen can only complement the conditions fixed by intracellular glutamate, glutamine and 2-oxoglutarate. This mechanistic understanding can provide an experimentally testable hypothesis for exploring the less-known biology of microcystin synthesis and designing specific interventions.

Compound-Specific Isotope Analysis of Amino Acid Labeling with Stable Isotope Nitrogen (15N) in Higher Plants

Individual free amino acid δ15N values in plant tissue reflect the metabolic pathways involved in their biosynthesis and catabolism and could thus aid understanding of environmental stress and anthropogenic effects on plant metabolism. In this study, compound-specific nitrogen isotope analysis of amino acid by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) was carried out to determine individual free amino acid δ15N values. High correlations were observed between the δ15N values obtained by GC-C-IRMS and elemental analyzer-isotope ratio mass spectrometry (EA-IRMS) determinations, and the mean precision measured was better than 1 ‰. Cation-exchange chromatography was employed to purify the sample, and the difference between prior to and following passage through the resin was within 1 ‰. The amino acid δ15N values of plant leave samples following incubation in 15N-nitrate at different time points were determined. A typical foliar free amino acid 15N-enrichment pattern was found, and glutamine was the most rapidly labeled amino acid; other amino acids derived from the GS-GOGAT cycle were also enriched. The pyruvate family amino acids were labeled less quickly followed by the aromatic amino acids. This study highlighted that amino acid metabolism pathways had a major effect on the δ15N values. With the known amino acid metabolism pathways and δ15N values determined by the presented method, the influence of various external factors on the metabolic cycling of amino acid can be understood well.

Kinetic flux profiling dissects nitrogen utilization pathways in the oleaginous green alga Chlorella protothecoides.

As a promising candidate for biodiesel production, the green alga Chlorella protothecoides can efficiently produce oleaginous biomass and the lipid biosynthesis is greatly influenced by the availability of nitrogen source and corresponding nitrogen assimilation pathways. Based on isotope-assisted kinetic flux profiling (KFP), the fluxes through the nitrogen utilization pathway were quantitatively analyzed. We found that autotrophic C. protothecoides cells absorbed ammonium mainly through glutamate dehydrogenase (GDH), and partially through glutamine synthetase (GS), which was the rate-limiting enzyme of nitrogen assimilation process with rare metabolic activity of glutamine oxoglutarate aminotransferase (GOGAT, also known as glutamate synthase); whereas under heterotrophic conditions, the cells adapted to GS-GOGAT cycle for nitrogen assimilation in which GS reaction rate was associated with GOGAT activity. The fact that C. protothecoides chooses the adenosine triphosphate-free and less ammonium-affinity GDH pathway, or alternatively the energy-consuming GS-GOGAT cycle with high ammonium affinity for nitrogen assimilation, highlights the metabolic adaptability of C. protothecoides exposed to altered nitrogen conditions.

Identifying essential genes/reactions of the rice photorespiration by in silico model-based analysis

BackgroundPhotorespiration, a highly wasteful process of energy dissipation, depresses the productivity of C3 plants such as rice (Oryza sativa) under dry and hot conditions. Thus, it is highly required to understand the cellular physiology and relevant metabolic states under photorespiration using systems approaches, thereby devising strategies for improving rice production.FindingsIn silico model-driven gene deletion analysis was performed on photorespiring leaf cells under ambient and stressed environmental conditions using our central metabolic network of rice cells. As a result, we identified a number of essential genes for the cell growth across various functional pathways such as photorespiratory cycle, Calvin cycle, GS-GOGAT cycle and sucrose metabolism as well as certain inter-compartmental transporters, which are mostly in good agreement with previous experiments. Synthetic lethal (SL) screening was also performed to identify the pair of non-essential genes whose simultaneous deletion become lethal, revealing the existence of more than 220 pairs of SLs on rice central metabolism.ConclusionsThe gene deletion and synthetic lethal analyses highlighted the rigid nature of rice photosynthetic pathways and characterized functional interactions between central metabolic genes, respectively. The biological roles of such reported essential genes should be further explored to better understand the rice photorespiration in future.Electronic supplementary materialThe online version of this article (doi:10.1186/1939-8433-6-20) contains supplementary material, which is available to authorized users.

Growth Phase-dependent Activation of Nitrogen-related Genes by a Control Network of Group 1 and Group 2 σ Factors in a Cyanobacterium

It has been reported that an RNA polymerase sigma factor, SigC, mainly contributes to specific transcription from the promoter PglnB-54,-53 under nitrogen-deprived conditions during the stationary phase of cell growth in the cyanobacterium Synechocystis sp. strain PCC 6803 (Asayama, M., Imamura, S., Yoshihara, S., Miyazaki, A., Yoshida, N., Sazuka, T., Kaneko, T., Ohara, O., Tabata, S., Osanai, T., Tanaka, K., Takahashi, H., and Shirai, M. (2004) Biosci. Biotechnol. Biochem. 68, 477-487). In this study, we further examined the functions of group 2 sigma factors of RNA polymerase in NtcA-dependent nitrogen-related gene expression in PCC 6803. Results indicated that SigB and SigC contribute to the transcription from PglnB-54,-53 with a sigma factor replaced in a growth phase-dependent manner. We also confirmed the contribution of SigB and SigC to the transcription of other NtcA-dependent genes, glnA, sigE, and amt1, as in the case of glnB. On the other hand, the transcription of glnN was dependent on SigB and SigE. In the SigB and SigC-based regulation, the level of SigB increased, but that of SigC was constant under conditions of nitrogen deprivation. Furthermore, it was found that SigC negatively and positively regulates the level of SigB in the log and stationary phase, respectively. SigC also had a positive effect on the level of sigB transcript during the stationary phase. In contrast, SigB acts positively on SigC levels in both growth phases. These results and previous findings indicated that multiple group 2 sigma factors take part in the control of NtcA-dependent nitrogen-related gene expression in cooperation with a group 1 sigma factor, SigA.

Effect of exogenous ammonium on glutamine synthetase, glutamate synthase, and glutamate dehydrogenase in the root of rice seedling

Root biomass of rice seedlings was increased at lower concentration of exogenous NH 4 + , but it was decreased at higher concentration of exogenous NH 4 + . The level of free NH 4 + in the roots was accumulated gradually with the increase of NH 4 + concentration in the nutrient solution. The content of the soluble proteins was essentially constant at higher NH 4 + . The activities of glutamine synthetase (GS), NADH-dependent glutamate synthase (NADH-GOGAT), and NADH-dependent glutamate dehydrogenase (NADH-GDH) were risen with exogenous NH 4 + concentration at the lower NH 4 + concentration range. But the activities of GS and NADH-GOGAT were declined, and the level of NADH-GDH activity was kept constant under higher NH 4 + concentration. The GS/GDH ratio suggested that NH 4 + was assimilated by GS-GOGAT cycle under lower NH 4 + concentration, but NADH-GDH was more important for NH 4 + assimilation and detoxifying NH 4 + to the tissue cells at the higher NH 4 + level. According to the growth and the activity changes of these ammonium-assimilating enzymes of rice seedling roots, 10. 0 μg/mL NH 4 + -N in nutrient solution was more suitable to the rice growth.

Effect of the arbuscular mycorrhizal fungus Glomus fasciculatum on the uptake of amino nitrogen by Lolium perenne.

The ability of ryegrass (Lolium perenne L.) to take up and utilize aspartic acid (Asp) and serine (Ser,), and the effect of colonization of the roots by the arbuscular mycorrhizal (AM) fungus Glomus fasciculatum (Thax. sensu Gerd.) were studied. The seedlings were grown under controlled conditions in a series of micro-lysimeters. All plants were fed with a nutrient solution containing either nitrate, Asp or Ser as the sole N source. After 49 d, they were supplied with 15 N labeled nitrate. Asp or Ser for 1 h and harvested. AM colonization increased the growth and total N content of the plants in all cases. Similarly, the amount of Asp or Ser taken up was higher in AM than in control plants. There were no differences in biomass production between the nitrate and Ser-fed plants. However uptake rates were lower for Ser than for nitrate. Growth of the Asp-fed plants was significantly less than the other two treatments, and uptake of 15 N-Asp was lower than uptake of 15 N-Ser. Analysis of 15 N incorporation into the amino acids extracted from the roots suggests the hydrolysis of Ser followed by re-assimilation of the resulting ammonia via the GS-GOGAT cycle. There were no differences in the patterns of accumulation of amino acids in the root-zone of control and AM-ryegrass. The implication of these results for the pathway of nitrogen transfer between plants is discussed.