Metabolite and light regulation of metabolism in plants: lessons from the study of a single biochemical pathway

We are using molecular, biochemical, and genetic approaches to study the structural and regulatory genes controlling the assimilation of inorganic nitrogen into the amino acids glutamine, glutamate, aspartate and asparagine. These amino acids serve as the principal nitrogen-transport amino acids in most crop and higher plants including Arabidopsis thaliana. We have begun to investigate the regulatory mechanisms controlling nitrogen assimilation into these amino acids in plants using molecular and genetic approaches in Arabidopsis. The synthesis of the amide amino acids glutamine and asparagine is subject to tight regulation in response to environmental factors such as light and to metabolic factors such as sucrose and amino acids. For instance, light induces the expression of glutamine synthetase (GLN2) and represses expression of asparagine synthetase (ASN1) genes. This reciprocal regulation of GLN2 and ASN1 genes by light is reflected at the level of transcription and at the level of glutamine and asparagine biosynthesis. Moreover, we have shown that the regulation of these genes is also reciprocally controlled by both organic nitrogen and carbon metabolites. We have recently used a reverse genetic approach to study putative components of such metabolic sensing mechanisms in plants that may be conserved in evolution. These components include an Arabidopsis homolog for a glutamate receptor gene originally found in animal systems and a plant PII gene, which is a homolog of a component of the bacterial Ntr system. Based on our observations on the biology of both structural and regulatory genes of the nitrogen assimilatory pathway, we have developed a model for metabolic control of the genes involved in the nitrogen assimilatory pathway in plants.

An integrative model of amino acid metabolism in the liver of the lactating dairy cow

The objective of this work was to construct a dynamic model of hepatic amino acid metabolism in the lactating dairy cow that could be parameterized using net flow data from in vivo experiments. The model considers 22 amino acids, ammonia, urea, and 13 energetic metabolites, and was parameterized using a steady-state balance model and two in vivo, net flow experiments conducted with mid-lactation dairy cows. Extracellular flows were derived directly from the observed data. An optimization routine was used to derive nine intracellular flows. The resulting dynamic model was found to be stable across a range of inputs suggesting that it can be perturbed and applied to other physiological states. Although nitrogen was generally in balance, leucine was in slight deficit compared to predicted needs for export protein synthesis, suggesting that an alternative source of leucine (e.g. peptides) was utilized. Simulations of varying glucagon concentrations indicated that an additional 5 mol/d of glucose could be synthesized at the reference substrate concentrations and blood flows. The increased glucose production was supported by increased removal from blood of lactate, glutamate, aspartate, alanine, asparagine, and glutamine. As glucose Output increased, ketone body and acetate release increased while CO2 release declined. The pattern of amino acids appearing in hepatic vein blood was affected by changes in amino acid concentration in portal vein blood, portal blood flow rate and glucagon concentration, with methionine and phenylalanine being the most affected of essential amino acids. Experimental evidence is insufficient to determine whether essential amino acids are affected by varying gluconeogenic demands. (C) 2004 Published by Elsevier Ltd.

Nitrogen Metabolism in leaves of a tank epiphytic bromeliad: Characterization of a spatial and functional division

The leaf is considered the most important vegetative organ of tank epiphytic bromeliads due to its ability to absorb and assimilate nutrients. However, little is known about the physiological characteristics of nutrient uptake and assimilation. In order to better understand the mechanisms utilized by some tank epiphytic bromeliads to optimize the nitrogen acquisition and assimilation, a study was proposed to verify the existence of a differential capacity to assimilate nitrogen in different leaf portions. The experiments were conducted using young plants of Vriesea gigantea. A nutrient solution containing NO(3)(-)/NH(4)(+) or urea as the sole nitrogen source was supplied to the tank of these plants and the activities of urease, nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (NADH-GDH) were quantified in apical and basal leaf portions after 1, 3, 6, 9, 12, 24 and 48 h. The endogenous ammonium and urea contents were also analyzed. Independent of the nitrogen sources utilized, NR and urease activities were higher in the basal portions of leaves in all the period analyzed. On the contrary. GS and GDH activities were higher in apical part. It was also observed that the endogenous ammonium and urea had the highest contents detected in the basal region. These results suggest that the basal portion was preferentially involved in nitrate reduction and urea hydrolysis, while the apical region could be the main area responsible for ammonium assimilation through the action of GS and GDH activities. Moreover, it was possible to infer that ammonium may be transported from the base, to the apex of the leaves. In conclusion, it was suggested that a spatial and functional division in nitrogen absorption and NH(4)(+) assimilation between basal and apical leaf areas exists, ensuring that the majority of nitrogen available inside the tank is quickly used by bromeliad`s leaves. (C) 2011 Elsevier GmbH. All rights reserved.

Rapid and slow modulation of nitrate reductase activity in the red macroalga Gracilaria chilensis (Gracilariales, Rhodophyta): influence of different nitrogen sources

Nitrate is one of the most important stimuli in nitrate reductase (NR) induction, while ammonium is usually an inhibitor. We evaluated the influence of nitrate, ammonium or urea as nitrogen sources on NR activity of the agarophyte Gracilaria chilensis. The addition of nitrate rapidly (2 min) induced NR activity, suggesting a fast post-translational regulation. In contrast, nitrate addition to starved algae stimulated rapid nitrate uptake without a concomitant induction of NR activity. These results show that in the absence of nitrate, NR activity is negatively affected, while the nitrate uptake system is active and ready to operate as soon as nitrate is available in the external medium, indicating that nitrate uptake and assimilation are differentially regulated. The addition of ammonium or urea as nitrogen sources stimulated NR activity after 24 h, different from that observed for other algae. However, a decrease in NR activity was observed after the third day under ammonium or urea. During the dark phase, G. chilensis NR activity was low when compared to the light phase. A light pulse of 15 min during the dark phase induced NR activity 1.5-fold suggesting also fast post-translational regulation. Nitrate reductase regulation by phosphorylation and dephosphorylation, and by protein synthesis and degradation, were evaluated using inhibitors. The results obtained for G. chilensis show a post-translational regulation as a rapid response mechanism by phosphorylation and dephosphorylation, and a slower mechanism by regulation of RNA synthesis coupled to de novo NR protein synthesis.

The GATA-type transcriptional activator Gat1 regulates nitrogen uptake and metabolism in the human pathogen Cryptococcus neoformans

Nitrogen uptake and metabolism are essential to microbial growth. Gat1 belongs to a conserved family of zinc finger containing transcriptional regulators known as GATA-factors. These factors activate the transcription of Nitrogen Catabolite Repression (NCR) sensitive genes when preferred nitrogen sources are absent or limiting. Cryptococcus neoformans GAT1 is an ortholog to the Aspergillus nidulans AreA and Candida albicans GAD genes. In an attempt to define the function of this transcriptional regulator in C. neoformans, we generated null mutants (gat1 Delta) of this gene. The gat 1 mutant exhibited impaired growth on all amino acids tested as sole nitrogen sources, with the exception of arginine and proline. Furthermore, the gat1 mutant did not display resistance to rapamycin, an immunosuppressant drug that transiently mimics a low-quality nitrogen source. Gal is not required for C. neoformans survival during macrophage infection or for virulence in a mouse model of cryptococcosis. Microarray analysis allowed the identification of target genes that are regulated by Gat1 in the presence of proline, a poor and non-repressing nitrogen source. Genes involved in ergosterol biosynthesis, iron uptake, cell wall organization and capsule biosynthesis, in addition to NCR-sensitive genes, are Gat1-regulated in C. neoformans. (C) 2010 Elsevier Inc. All rights reserved.

Altered patterns of maltose and glucose fermentation by brewing and wine yeasts influenced by the complexity of nitrogen source

Maltose and glucose fermentations by industrial brewing and wine yeasts strains were strongly affected by the structural complexity of the nitrogen source. In this study, four Saccharomyces cerevisiae strains, two brewing and two wine yeasts, were grown in a medium containing maltose or glucose supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low sugar concentration for brewing and wine strains, independent of nitrogen supplementation, and the type of sugar. At high sugar concentrations altered patterns of sugar fermentation were observed, and biomass accumulation and ethanol production depended on the nature of the nitrogen source and were different for brewing and wine strains. In maltose, high biomass production was observed under peptone and casamino acids for the brewing and wine strains, however efficient maltose utilization and high ethanol production was only observed in the presence of casamino acids for one brewing and one wine strain studied. Conversely, peptone and casamino acids induced higher biomass and ethanol production for the two other brewing and wine strains studied. With glucose, in general, peptone induced higher fermentation performance for all strains, and one brewing and wine strain produced the same amount of ethanol with peptone and casamino acids supplementation. Ammonium salts always induced poor yeast performance. The results described in this paper suggest that the complex nitrogen composition of the cultivation medium may create conditions resembling those responsible for inducing sluggish/stuck fermentation, and indicate that the kind and concentration of sugar, the complexity of nitrogen source and the yeast genetic background influence optimal industrial yeast fermentation performance.

Nitrogen excretion during embryonic development of the green iguana, Iguana iguana (Reptilia; Squamata)

Development within the cleidoic egg of birds and reptiles presents the embryo with the problem of accumulation of wastes from nitrogen metabolism. Ammonia derived from protein catabolism is converted into the less toxic product urea or relatively insoluble uric acid. The pattern of nitrogen excretion of the green iguana, Iguana iguana, was determined during embryonic development using samples from allantoic fluid and from the whole homogenized egg, and in hatchlings and adults using samples of blood plasma. Urea was the major excretory product over the course of embryonic development. It was found in higher concentrations in the allantoic sac, suggesting that there is a mechanism present on the allantoic membrane enabling the concentration of urea. The newly hatched iguana still produced urea while adults produced uric acid. The time course of this shift in the type of nitrogen waste was not determined but the change is likely to be related to the water relations associated with the terrestrial habit of the adult. The green iguana produces parchment-shelled eggs that double in mass during incubation due to water absorption; the eggs also accumulate 0.02. mM of urea, representing 82% of the total measured nitrogenous residues that accumulate inside the allantois. The increase in egg mass and urea concentration became significant after 55. days of incubation then were unchanged until hatching. © 2012 Elsevier Inc.

Different true-protein sources do not modify the metabolism of crossbred Bos taurus x Bos indicus growing heifers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Nitrogen excretion during embryonic development of the green iguana, Iguana iguana (Reptilia; Squamata)

Development within the cleidoic egg of birds and reptiles presents the embryo with the problem of accumulation of wastes from nitrogen metabolism. Ammonia derived from protein catabolism is converted into the less toxic product urea or relatively insoluble uric acid. The pattern of nitrogen excretion of the green iguana, Iguana iguana, was determined during embryonic development using samples from allantoic fluid and from the whole homogenized egg, and in hatchlings and adults using samples of blood plasma. Urea was the major excretory product over the course of embryonic development. It was found in higher concentrations in the allantoic sac, suggesting that there is a mechanism present on the allantoic membrane enabling the concentration of urea. The newly hatched iguana still produced urea while adults produced uric acid. The time course of this shift in the type of nitrogen waste was not determined but the change is likely to be related to the water relations associated with the terrestrial habit of the adult. The green iguana produces parchment-shelled eggs that double in mass during incubation due to water absorption: the eggs also accumulate 0.02 mM of urea, representing 82% of the total measured nitrogenous residues that accumulate inside the allantois. The increase in egg mass and urea concentration became significant after 55 days of incubation then were unchanged until hatching. (C) 2012 Elsevier Inc. All rights reserved.

Global gene expression under nitrogen starvation in Xylella fastidiosa: contribution of the σ54 regulon

Abstract Background Xylella fastidiosa, a Gram-negative fastidious bacterium, grows in the xylem of several plants causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. Results In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time course experiment (2, 8 and 12 hours) of cultures grown in defined medium under nitrogen starvation revealed many differentially expressed genes, such as those related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes directly or indirectly induced by nitrogen starvation in a σ54-dependent manner. A more complete picture of the σ54 regulon was achieved by combining the transcriptome data with an in silico search for potential σ54-dependent promoters, using a position weight matrix approach. One of these σ54-predicted binding sites, located upstream of the glnA gene (encoding glutamine synthetase), was validated by primer extension assays, confirming that this gene has a σ54-dependent promoter. Conclusions Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the σ54 regulon.

The nitrogen costs of photosynthesis in a diatom under current and future pCO2

With each cellular generation, oxygenic photoautotrophs must accumulate abundant protein complexes that mediate light capture, photosynthetic electron transport and carbon fixation. In addition to this net synthesis, oxygenic photoautotrophs must counter the light-dependent photoinactivation of Photosystem II (PSII), using metabolically expensive proteolysis, disassembly, resynthesis and re-assembly of protein subunits. We used growth rates, elemental analyses and protein quantitations to estimate the nitrogen (N) metabolism costs to both accumulate the photosynthetic system and to maintain PSII function in the diatom Thalassiosira pseudonana, growing at two pCO2 levels across a range of light levels. The photosynthetic system contains c. 15-25% of total cellular N. Under low growth light, N (re)cycling through PSII repair is only c. 1% of the cellular N assimilation rate. As growth light increases to inhibitory levels, N metabolite cycling through PSII repair increases to c. 14% of the cellular N assimilation rate. Cells growing under the assumed future 750 ppmv pCO2 show higher growth rates under optimal light, coinciding with a lowered N metabolic cost to maintain photosynthesis, but then suffer greater photoinhibition of growth under excess light, coincident with rising costs to maintain photosynthesis. We predict this quantitative trait response to light will vary across taxa.

Impacts of groundwater discharge at Myora Springs (North Stradbroke Island, Australia) on the phenolic metabolism of eelgrass, Zostera muelleri, and grazing by the juvenile rabbitfish, Siganus fuscescens

Myora Springs is one of many groundwater discharge sites on North Stradbroke Island (Queensland, Australia). Here spring waters emerge from wetland forests to join Moreton Bay, mixing with seawater over seagrass meadows dominated by eelgrass, Zostera muelleri. We sought to determine how low pH / high CO2 conditions near the spring affect these plants and their interactions with the black rabbitfish (Siganus fuscescens), a co-occurring grazer. In paired-choice feeding trials S. fuscescens preferentially consumed Z. muelleri shoots collected nearest to Myora Springs. Proximity to the spring did not significantly alter the carbon and nitrogen contents of seagrass tissues but did result in the extraordinary loss of soluble phenolics, including Folin-reactive phenolics, condensed tannins, and phenolic acids by ?87%. Conversely, seagrass lignin contents were, in this and related experiments, unaffected or increased, suggesting a shift in secondary metabolism away from the production of soluble, but not insoluble, (poly)phenolics. We suggest that groundwater discharge sites such as Myora Springs, and other sites characterized by low pH, are likely to be popular feeding grounds for seagrass grazers seeking to reduce their exposure to soluble phenolics.

A global two component signal transduction system that integrates the control of photosynthesis, carbon dioxide assimilation, and nitrogen fixation

Photosynthesis, biological nitrogen fixation, and carbon dioxide assimilation are three fundamental biological processes catalyzed by photosynthetic bacteria. In the present study, it is shown that mutant strains of the nonsulfur purple photosynthetic bacteria Rhodospirillum rubrum and Rhodobacter sphaeroides, containing a blockage in the primary CO2 assimilatory pathway, derepress the synthesis of components of the nitrogen fixation enzyme complex and abrogate normal control mechanisms. The absence of the Calvin–Benson–Bassham (CBB) reductive pentose phosphate CO2 fixation pathway removes an important route for the dissipation of excess reducing power. Thus, the mutant strains develop alternative means to remove these reducing equivalents, resulting in the synthesis of large amounts of nitrogenase even in the presence of ammonia. This response is under the control of a global two-component signal transduction system previously found to regulate photosystem biosynthesis and the transcription of genes required for CO2 fixation through the CBB pathway and alternative routes. In addition, this two-component system directly controls the ability of these bacteria to grow under nitrogen-fixing conditions. These results indicate that there is a molecular link between the CBB and nitrogen fixation process, allowing the cell to overcome powerful control mechanisms to remove excess reducing power generated by photosynthesis and carbon metabolism. Furthermore, these results suggest that the two-component system integrates the expression of genes required for the three processes of photosynthesis, nitrogen fixation, and carbon dioxide fixation.

The effect of different levels of dietary crude protein on urea metabolism of rusa deer (Cervus timorensis)

Two experiments were conducted to measure urea recycling and rumen flow dynamics in young rusa deer fed low (LP) or high (HP) protein diets. Pool size and flux rate of labelled urea. into and out of the blood pool were measured using single intravenous (i.v.) injection solutions containing [C-14] - and [N-15]-urea. A curve peeling technique was used to fit the enrichment of N-15 or specific radioactivity (SRA) of C-14 to exponential equations. Body urea-N pool size was significantly greater (P < 0.05) when a HP, compared to a LP diet, was fed. Urea space, expressed as a percent of live weight, total flux rate of urea through the blood pool and the irreversible loss of urea was similar for both diets. The mean (+/- S.E.M.) concentration of plasma urea-N was greater when animals were fed the HP diet compared to the LP diet (2 1.1 +/- 0.3 versus 14.4 +/- 1.4 mg/100 ml, respectively). Voluntary feed intake and digestibility of dietary components were also measured. Daily dry matter intakes were not affected by the crude protein (CP) content of the diet, although apparent DM digestibility was significantly greater for HP diet fed in both experiments. An intraruminal infusion of CrEDTA was used to determine rumen flow dynamics. Ruminal mean retention time, relative net outflow rate of water and passage rate constant (k(w)) were significantly greater (P < 0.05) when the HP diet was fed compared to the LP diet. The extent of urea metabolism and flux rates of urea between the blood and secondary pools appear similar to those previously reported for other ruminants fed diets contrasting in CP content. (c) 2005 Elsevier B.V. All rights reserved.