Glutamate, Ornithine, Arginine, Proline, and Polyamine Metabolic Interactions: The Pathway Is Regulated at the Post-Transcriptional Level

The metabolism of glutamate into ornithine, arginine, proline, and polyamines is a major network of nitrogen-metabolizing pathways in plants, which also produces intermediates like nitric oxide, and γ-aminobutyric acid (GABA) that play critical roles in plant development and stress. While the accumulations of intermediates and the products of this network depend primarily on nitrogen assimilation, the overall regulation of the interacting sub-pathways is not well understood. We tested the hypothesis that diversion of ornithine into polyamine biosynthesis (by transgenic approach) not only plays a role in regulating its own biosynthesis from glutamate but also affects arginine and proline biosynthesis. Using two high putrescine producing lines of Arabidopsis thaliana (containing a transgenic mouse ornithine decarboxylase gene), we studied the: (1) effects of exogenous supply of carbon and nitrogen on polyamines and pools of soluble amino acids; and, (2) expression of genes encoding key enzymes in the interactive pathways of arginine, proline and GABA biosynthesis as well as the catabolism of polyamines. Our findings suggest that: (1) the overall conversion of glutamate to arginine and polyamines is enhanced by increased utilization of ornithine for polyamine biosynthesis by the transgene product; (2) proline and arginine biosynthesis are regulated independently of polyamines and GABA biosynthesis; (3) the expression of most genes (28 that were studied) that encode enzymes of the interacting sub-pathways of arginine and GABA biosynthesis does not change even though overall biosynthesis of Orn from glutamate is increased several fold; and (4) increased polyamine biosynthesis results in increased assimilation of both nitrogen and carbon by the cells

Understanding polyamine metabolism through transgenic manipulation in poplar suspension cultures

Polyamines are low molecular weight aliphatic amines that are obligatory requirements for cell survival and growth. The commonly occurring polyamines in plants are putrescine, spermidine, and spermine. Suspension cultures of poplar (Populus nigra x maximowiczii), transformed with a mouse ornithine decarboxylase gene (under the control of a 2X 35S CaMV promoter) were used to study the impact of up-regulation of putrescine biosynthesis (and consequent enhanced catabolism) on several aspects of cellular metabolism. The transgenic cells were compared with a control cell line that was transformed with the beta-glucuronidase (GUS) gene. It was observed that enhanced putrescine metabolism resulted in: (i) increased expression of arginine decarboxylase gene, along with enhanced activity of the corresponding enzyme, (ii) decreased expression of S-adenosylmethionine gene and activity of the enzyme, (iii) changes in the cellular contents of almost all amino acids, (iv) a compromise in cell health due to increased oxidative stress, (v) better tolerance towards Aluminum toxicity, (vi) increased susceptibility to glutamate decarboxylase inhibition, (vii) greater assimilation of carbon from sucrose in the growth medium, and (viii) small changes in the expression of ornithine aminotransferase, proline dehydrogenase and Delta 1-pyrroline-5-carboxylate reductase genes, and an increase in ornithine aminotransferase activity

Regulation of polyamine metabolism in transgenic poplar cell cultures

Polyamines (PA) are naturally occurring low molecular weight aliphatic amines found in all living organisms and essential for their growth and development. The present study uses the tool of transgenic manipulation to elucidate the regulation of PA pathway in poplar cells. This study was divided into two segments; the first segment provides insight into biochemistry of a putrescine overproducing cell line (HP) that overexpresses a mouse ornithine decarboxylase (mODC) under 35S-CaMV promoter, with respect to different treatments. The second segment focused on creation and biochemical characterization of a cell line (SOS1) that overexpresses ODC/SAMDC from Plasmodium falciparum controlled by the same 35S-CaMV promoter producing a bifunctional protein. From the results it is concluded that: (i) nutrient stress in the form of Ca deficiency in the growth medium is more detrimental to HP cells than control cells, (ii) the tested PA analogues and inhibitors at the tested concentrations have no major observable effect on either HP or the control cells, (iii) Cysteine and methionine although consumed faster in HP cells, are not limiting either for protein synthesis or for growth, (iv) uptake of sucrose is higher in the control cells but its incorporation into PAs is higher in HP cells, (v) SOS1 cells after stable integration of PfODC/SAMDC produce significantly increased amounts of Spd and Spm than control cells, and (vi) the simultaneous expression of both SAMDC and ODC from Plasmodium has greater impact on production of Spd/Spm than on Put in SOS1 cells

Unravelling collembolan life belowground: Stoichiometry, metabolism and release of carbon and nitrogen

This thesis investigated carbon and nitrogen dynamics of soil dwelling Collembola by using direct measurements and stable isotope additions. In an isotope change experiment, collembolans exchanged between 6 and 10% of carbon and nitrogen in their body tissue per day to metabolism and between 0.5 and 2% to reproduction. When collembolans on low and high protein diets were compared, animals on the low protein quality depleted their tissue 15N values relative to those on high quality diet indicating that the nitrogen turnover decreased on the low protein quality diet. In a wheat microcosm investigating source contributions from soil, roots and isotope labelled green manure the mixing model analysis indicated that photosynthate (root derived C) was the main carbon source for collembolans (54–79% of total C) indicating that the rhizosphere channel is very important for collembolans in addition to the detritus based channel

Identification and characterization of vacuolar nucleotide phosphatases and characterization of an engineered catabolic pathway for urea in Arabidopsis thaliana

Plants have the remarkable ability to remobilize nutrients by degrading cellular components such as damaged organelles, proteins, and RNA in vacuoles. According to the current model of RNA degradation in vacuoles, the RNA is degraded in three steps. After the transport of the RNA into vacuoles, it is first degraded to 3’-mononucleotides (3'-NMPs) by the intercellular ribonuclease RNS2. The RNA degradation products are then dephosphorylated by vacuolar acid phosphatases (AP), releasing nucleosides. These nucleosides are eventually transported by the Equilibrative Nucleoside Transporter 1 (ENT1) from vacuoles to the cytosol, where they are either degraded or salvaged to nucleotides, thereby either releasing nutrients or conserving metabolic resources. RNS2 and ENT1 were studied and characterized several years ago, but the vacuolar nucleotide phosphatases have not yet been identified. This study focuses on the identification and characterization of the nucleotide-dephosphorylating APs from vacuoles. Three protein families, the Haloacid Dehalogenase IIIB (HADIIIB) family, the Purple Acid Phosphatase (PAP) family, and the Endonuclease (Endo) S1/P1-type nucleases were considered based on their previous identification in the vacuolar proteome or their potential to localize in the vacuole and based on their potential nucleotidase activities. In total 44 candidate proteins were then screened employing a set of selection criteria, including protein conservation in other plants, protein localization, substrate preferences, and expression profiles. Several mutant combinations were generated for the most promising candidates using T-DNA insertion lines and the CRISPR/Cas9 technique. When the final candidates (HADIIIB4 renamed to Vacuolar Nucleoside Phosphate Phosphatase 1 (VNPP1) and PAP26)) were identified, they were fully characterized in vitro and in vivo. The function of VNPP1 and PAP26 in vacuolar mononucleotide dephosphorylation could be demonstrated by the accumulation of 3'-NMPs in the corresponding mutants. Additional tissue-specific vacuolar and non-vacuolar NMP phosphatases were also discovered in the course of this work, opening the door to new research directions for investigating the significance of mononucleotide catabolism in seeds, pollen, and roots.Nitrogen is one of the most important nutrients for plants. Nitrogen deficiency leads to reduced plant growth and productivity. Urea is the most used nitrogen fertilizer in the world because it has a high nitrogen content and is easy to transport and apply in the field. However, fertilizing exclusively with urea results in reduced growth due to the toxicity of the ammonia. The urea is immediately hydrolysed to ammonia in the roots, the resulting higher ammonia concentration in the roots is toxic, and the plants excrete the excess ammonia back into the soil. In addition, the nitrogen use efficiency of nitrogen fertilizer in general and urea in particular is generally below 50% and needs improvement. Therefore, a biotechnological solution expressing heterologous transgenes in the model plant Arabidopsis thaliana was assessed with the aim to reduce ammonia toxicity for the plant and improve nitrogen use efficiency. For this purpose two enzymes representing an alternative urea catabolism pathway in various bacteria and fungi were investigated. In this system, urea is not immediately hydrolyzed to ammonia as in organisms employing Urease (like plants), but is first carboxylated by a Urea Carboxylase (UC) and the product allophanate is then hydrolysed by and Allophanate Hydrolase (AH) to ammonia and carbon dioxide. The performance transgenic lines expressing this system in the Urease mutant of Arabidopsis under the control of the 35S promoter was investigated. These lines were able to use urea demonstrating that the heterologous enzymes are functional in the plant. Since urea hydrolysis by UC and AH can occur in two steps, additional lines with wild type background (Urease positive) were investigated where the UC was expressed ubiquitously and the AH was expressed only in the shoot and in the light. In these plants ammonia release from allophanate can only occur in the shoot. Such transgenic plants performed significantly better than the wild-type with pure urea nutrition. The transgenic plants had a higher biomass and leaf area, and a higher nitrogen content. The results raise the hope that low nitrogen use efficiency and ammonia toxicity could be tackled with this system also in crop plants.Pflanzen haben die bemerkenswerte Fähigkeit, Nährstoffe zu remobilisieren, indem sie zelluläre Komponenten wie beschädigte Organellen, Proteine und RNA in den Vakuolen abbauen. Nach dem derzeitigen Modell des RNA-Abbaus in den Vakuolen wird die RNA in drei Schritten abgebaut. Nach dem Transport der RNA in die Vakuolen wird sie zunächst durch die interzelluläre Ribonuklease RNS2 zu 3'-Mononukleotiden (3'-NMPs) katabolisiert. Die RNA-Abbauprodukte werden dann durch saure Vakuolenphosphatasen (AP) dephosphoryliert, wodurch Nukleoside freigesetzt werden. Diese Nukleoside werden schließlich durch den Equilibrative Nucleoside Transporter 1 (ENT1) aus den Vakuolen in das Zytosol transportiert, wo sie entweder weiter abgebaut oder im Salvage-Stoffwechsel zu Nukleotiden recycled werden, wodurch entweder Nährstoffe freigesetzt oder Stoffwechselressourcen geschont werden. RNS2 und ENT1 wurden bereits vor einigen Jahren untersucht und charakterisiert, aber die vakuolären Nukleotidphosphatasen sind noch nicht identifiziert worden. Diese Studie konzentriert sich auf die Identifizierung und Charakterisierung der Nukleotid-dephosphorylierenden APs aus Vakuolen. Es wurden drei Proteinfamilien, die Haloacid Dehalogenase IIIB (HADIIIB)-Familie, die Purple-Acid-Phophatatase (PAP)-Familie und die Endonuklease (Endo) S1/P1-Typ-Nukleasen auf der Grundlage ihrer früheren Identifizierung im Vakuolenproteom oder ihres Potenzials, in der Vakuole zu lokalisieren, und aufgrund ihrer potenziellen Nukleotidase-Aktivitäten in die Untersuchungen einbezogen. Insgesamt wurden 44 Kandidatenproteine anhand einer Reihe von Auswahlkriterien wie Proteinkonservierung in anderen Pflanzen, Proteinlokalisierung, Substratpräferenzen und Expressionsprofilen untersucht. Für die vielversprechendsten Kandidaten wurden mithilfe von T-DNA-Insertionslinien und der CRISPR/Cas9-Technik mehrere Mutantenkombinationen erzeugt. Als die endgültigen Kandidaten (HADIIIB4, umbenannt in Vacuolar Nucleoside Phosphate Phosphatase 1 (VNPP1) und PAP26)) identifiziert waren, wurden diese in vitro und in vivo vollständig charakterisiert. Die Funktion von VNPP1 und PAP26 bei der vakuolären Dephosphorylierung von Mononukleotiden konnte durch die Akkumulation von 3'-NMPs in den entsprechenden Mutanten belegt werden. Weitere gewebespezifische vakuoläre und nicht-vakuoläre NMP-Phosphatasen wurden im Rahmen dieser Arbeit ebenfalls entdeckt, was neue Forschungsrichtungen für die Untersuchung der Bedeutung des Mononukleotid-Katabolismus in Samen, Pollen und Wurzeln eröffnet

Unravelling ties in the nitrogen network: Polyamines and nitric oxide emerging as essential players in signalling roadway

Nitrogen (N) is a central mineral nutrient essential for plant development and growth. It is usually scarcely found in soils, so the knowledge of the overall plant N metabolism deserves substantial attention. Polyamines (PAs) are N-containing low-molecular-weight compounds of polycationic nature involved in essential processes all throughout the life of plants whereas nitric oxide (NO) is a gaseous free radical involved in signalling cascades related to many physiological events. PAs and NO share signalling functions and interact with each other in several biological functions, mainly in stress responses. Biosynthesis pathways of PAs and NO are overlapped; PAs induce NO formation, but it is still not completely defined whether PAs act as substrates, cofactors, or signals for promoting NO synthesis and also, which are the mechanisms involved in NO regulation of PAs metabolism. Polyamine levels are of vital importance in the regulation of the network of N-metabolising pathways in plants, as they are components of the core of the overall N metabolism. In light of the importance of improving the efficiency of N uptake and distribution, it is time to elucidate the intricate relationship among N as a nutrient with PAs and NO as emerging signalling molecules. The close cooperation among these players in the whole N metabolism is an interesting target for the development of biotechnological tools for sustainable agriculture.Fil: Recalde, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Gómez Mansur, Nabila María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Cabrera, Andrea Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Matayoshi, Carolina Lucila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Gallego, Susana Mabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Groppa, María Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Benavides, Maria Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

New isoforms and assembly of glutamine synthetase in the leaf of wheat (Triticum aestivum L.).

Glutamine synthetase (GS; EC 6.3.1.2) plays a crucial role in the assimilation and re-assimilation of ammonia derived from a wide variety of metabolic processes during plant growth and development. Here, three developmentally regulated isoforms of GS holoenzyme in the leaf of wheat (Triticum aestivum L.) seedlings are described using native-PAGE with a transferase activity assay. The isoforms showed different mobilities in gels, with GSII>GSIII>GSI. The cytosolic GSI was composed of three subunits, GS1, GSr1, and GSr2, with the same molecular weight (39.2kDa), but different pI values. GSI appeared at leaf emergence and was active throughout the leaf lifespan. GSII and GSIII, both located in the chloroplast, were each composed of a single 42.1kDa subunit with different pI values. GSII was active mainly in green leaves, while GSIII showed brief but higher activity in green leaves grown under field conditions. LC-MS/MS experiments revealed that GSII and GSIII have the same amino acid sequence, but GSII has more modification sites. With a modified blue native electrophoresis (BNE) technique and in-gel catalytic activity analysis, only two GS isoforms were observed: one cytosolic and one chloroplastic. Mass calibrations on BNE gels showed that the cytosolic GS1 holoenzyme was ~490kDa and likely a dodecamer, and the chloroplastic GS2 holoenzyme was ~240kDa and likely a hexamer. Our experimental data suggest that the activity of GS isoforms in wheat is regulated by subcellular localization, assembly, and modification to achieve their roles during plant development