RESUMEN

The utilization of high amounts of nitrate fertilizers for crop yield leads to nitrate pollution of ground and surface waters. In this study, we report the assimilation and utilization of nitrate luxuriant levels, 20 times more than the highest N fertilizer application in Europe, by transgenic poplars overexpressing a cytosolic glutamine synthetase (GS1). In comparison with the wild-type controls, transgenic plants grown under high N levels exhibited increased biomass (171.6%) and accumulated higher levels of proteins, chlorophylls and total sugars such as glucose, fructose and sucrose. These plants also exhibited greater nitrogen-use efficiency particularly in young leaves, suggesting that they are able to translocate most of the resources to the above-ground part of the plant to produce biomass. The transgenic poplar transcriptome was greatly affected in response to N availability with 1237 genes differentially regulated in high N, while only 632 genes were differentially expressed in untransformed plants. Many of these genes are essential in the adaptation and response against N excess and include those involved in photosynthesis, cell wall formation and phenylpropanoid biosynthesis. Cellulose production in the transgenic plants was fivefold higher than in control plants, indicating that transgenic poplars represent a potential feedstock for applications in bioenergy. In conclusion, our results show that GS transgenic poplars can be used not only for improving growth and biomass production but also as an important resource for potential phytoremediation of nitrate pollution.

Asunto(s)

Biocombustibles , Nitratos/metabolismo , Populus/metabolismo , Árboles/metabolismo , Biodegradación Ambiental/efectos de los fármacos , Biomasa , Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Metabolismo de los Hidratos de Carbono/genética , Carbono/metabolismo , Clorofila/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Glutamato-Amoníaco Ligasa/metabolismo , Lignina/metabolismo , Nitrógeno/metabolismo , Nitrógeno/farmacología , Fenotipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Populus/efectos de los fármacos , Populus/genética , Populus/crecimiento & desarrollo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Solubilidad , Transcriptoma/genética , Árboles/efectos de los fármacos , Árboles/genética , Árboles/crecimiento & desarrollo

RESUMEN

Transgenic hybrid poplars engineered to express ectopically the heterologous pine cytosolic GS1a display a number of significant pleiotropic phenotypes including enhanced growth, enhanced nitrogen use efficiency, and resistance to drought stress. The present study was undertaken in order to assess mechanisms whereby ectopic expression of pine GS1a in transgenic poplars results in enhanced agronomic phenotypes. Microarray analysis using the Agilent Populus whole genome array has allowed identification of genes differentially expressed between wild type (WT) and GS transgenics in four tissues (sink leaves, source leaves, stems, and roots) under three growth conditions (well-watered, drought, and recovery). Analysis revealed that differentially expressed genes in functional categories related to nitrogen metabolism show a trend of significant down-regulation in GS poplars compared to the WT, including genes encoding nitrate and nitrite reductases. The down-regulation of these genes was verified using qPCR, and downstream effects were further tested using NR activity assays. Results suggest that higher glutamine levels in GS transgenics regulate nitrate uptake and reduction. Transcript levels of nitrogen-related genes in leaves, including GS/GOGAT cycle enzymes, aspartate aminotransferase, GABA shunt enzymes, photorespiration enzymes, asparagine synthetase, phenylalanine ammonia lyase, isocitrate dehydrogenase, and PII, were also assessed using qPCR revealing significant differences between GS poplars and the WT. Moreover, metabolites related to these differentially expressed genes showed alterations in levels, including higher levels of GABA, hydroxyproline, and putrescine in the GS transgenic. These alterations in nitrogen homeostasis offer insights into mechanisms accounting for drought tolerance observed in GS poplars.

Asunto(s)

Glutamato-Amoníaco Ligasa/biosíntesis , Nitrógeno/metabolismo , Pinus/genética , Proteínas de Plantas/biosíntesis , Plantas Modificadas Genéticamente/enzimología , Populus/enzimología , Estrés Fisiológico , Glutamato-Amoníaco Ligasa/genética , Pinus/enzimología , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Populus/genética

RESUMEN

BACKGROUND: Glutamine synthetase (GS) plays a central role in plant nitrogen assimilation, a process intimately linked to soil water availability. We previously showed that hybrid poplar (Populus tremula X alba, INRA 717-1B4) expressing ectopically a pine cytosolic glutamine synthetase gene (GS1a) display enhanced tolerance to drought. Preliminary transcriptome profiling revealed that during drought, members of the superoxide dismutase (SOD) family were reciprocally regulated in GS poplar when compared with the wild-type control, in all tissues examined. SOD was the only gene family found to exhibit such patterns. RESULTS: In silico analysis of the Populus genome identified 12 SOD genes and two genes encoding copper chaperones for SOD (CCSs). The poplar SODs form three phylogenetic clusters in accordance with their distinct metal co-factor requirements and gene structure. Nearly all poplar SODs and CCSs are present in duplicate derived from whole genome duplication, in sharp contrast to their predominantly single-copy Arabidopsis orthologs. Drought stress triggered plant-wide down-regulation of the plastidic copper SODs (CSDs), with concomitant up-regulation of plastidic iron SODs (FSDs) in GS poplar relative to the wild type; this was confirmed at the activity level. We also found evidence for coordinated down-regulation of other copper proteins, including plastidic CCSs and polyphenol oxidases, in GS poplar under drought conditions. CONCLUSIONS: Both gene duplication and expression divergence have contributed to the expansion and transcriptional diversity of the Populus SOD/CCS families. Coordinated down-regulation of major copper proteins in drought-tolerant GS poplars supports the copper cofactor economy model where copper supply is preferentially allocated for plastocyanins to sustain photosynthesis during drought. Our results also extend previous findings on the compensatory regulation between chloroplastic CSDs and FSDs, and suggest that this copper-mediated mechanism represents a common response to oxidative stress and other genetic manipulations, as in GS poplars, that affect photosynthesis.

Asunto(s)

Sequías , Glutamato-Amoníaco Ligasa/metabolismo , Proteínas de Plantas/metabolismo , Populus/enzimología , Superóxido Dismutasa/metabolismo , Regulación de la Expresión Génica de las Plantas , Glutamato-Amoníaco Ligasa/genética , Proteínas de Plantas/genética , Superóxido Dismutasa/genética

RESUMEN

Hybrid poplar (Populus tremula X P. alba) genetically engineered to express the pine cytosolic glutamine synthetase gene (GS1a) has been previously shown to display desirable field performance characteristics, including enhancements in growth and nitrogen use efficiency. Analysis of wood samples from a 3-year-old field trial of three independently transformed GS1a transgenic hybrid poplar lines revealed that, when compared with wild-type controls, ectopic expression of GS1a resulted in alterations in wood properties and wood chemistry. Included were significant enhancements in wood fibre length, wood density, microfibre angle, per cent syringyl lignin and elevated concentrations of wood sugars, specifically glucose, galactose, mannose and xylose. Total extractive content and acid-insoluble lignin were significantly reduced in wood of GS1a transgenics when compared with wild-type trees. Together, these cell wall characteristics resulted in improved wood pulping attributes, including improved lignin solubilization with no concurrent decrease in yield. Trees with increased GS1a expression have improved characteristics for pulp and paper production and hold potential as a feedstock for biofuels production.

Asunto(s)

Glutamato-Amoníaco Ligasa/metabolismo , Populus/enzimología , Populus/crecimiento & desarrollo , Madera/química , Pared Celular/metabolismo , Cruzamientos Genéticos , Glutamato-Amoníaco Ligasa/genética , Hibridación Genética , Lignina/metabolismo , Plantas Modificadas Genéticamente , Populus/genética

RESUMEN

BACKGROUND: Glutamine synthetase (GS; EC: 6.3.1.2, L-glutamate: ammonia ligase ADP-forming) is a key enzyme in ammonium assimilation and metabolism of higher plants. The current work was undertaken to develop a more comprehensive understanding of molecular and biochemical features of GS gene family in poplar, and to characterize the developmental regulation of GS expression in various tissues and at various times during the poplar perennial growth. RESULTS: The GS gene family consists of 8 different genes exhibiting all structural and regulatory elements consistent with their roles as functional genes. Our results indicate that the family members are organized in 4 groups of duplicated genes, 3 of which code for cytosolic GS isoforms (GS1) and 1 which codes for the choroplastic GS isoform (GS2). Our analysis shows that Populus trichocarpa is the first plant species in which it was observed the complete GS family duplicated. Detailed expression analyses have revealed specific spatial and seasonal patterns of GS expression in poplar. These data provide insights into the metabolic function of GS isoforms in poplar and pave the way for future functional studies. CONCLUSIONS: Our data suggest that GS duplicates could have been retained in order to increase the amount of enzyme in a particular cell type. This possibility could contribute to the homeostasis of nitrogen metabolism in functions associated to changes in glutamine-derived metabolic products. The presence of duplicated GS genes in poplar could also contribute to diversification of the enzymatic properties for a particular GS isoform through the assembly of GS polypeptides into homo oligomeric and/or hetero oligomeric holoenzymes in specific cell types.

Asunto(s)

Glutamato-Amoníaco Ligasa/genética , Familia de Multigenes , Populus/enzimología , Hibridación Genómica Comparativa , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes Duplicados , Isoenzimas/genética , Filogenia , Populus/genética , Populus/crecimiento & desarrollo , Estaciones del Año

RESUMEN

The initial reaction in the pathway leading to the production of indole-3-acetic acid (IAA) in plants is the reaction between chorismate and glutamine to produce anthranilate, catalysed by the enzyme anthranilate synthase (ASA; EC 4.1.3.27). Compared with non-transgenic controls, leaves of transgenic poplar with ectopic expression of the pine cytosolic glutamine synthetase (GS1a; EC 6.3.1.2) produced significantly greater glutamine and significantly enhanced ASA α-subunit (ASA1) transcript and protein (approximately 130% and 120% higher than in the untransformed controls, respectively). Similarly, tobacco leaves fed with 30 mM glutamine and 2 mM chorismate showed enhanced ASA1 transcript and protein (175% and 90% higher than controls, respectively). Furthermore, free IAA was significantly elevated both in leaves of GS1a transgenic poplar and in tobacco leaves fed with 30 mM glutamine and 2 mM chorismate. These results indicated that enhanced cellular glutamine may account for the enhanced growth in GS transgenic poplars through the regulation of auxin biosynthesis.

Asunto(s)

Antranilato Sintasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Glutamina/metabolismo , Ácidos Indolacéticos/metabolismo , Pinus/enzimología , Hojas de la Planta/enzimología , Populus/crecimiento & desarrollo , Antranilato Sintasa/metabolismo , Ácido Corísmico/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Glutamato-Amoníaco Ligasa/genética , Glutamina/farmacología , Immunoblotting , Pinus/efectos de los fármacos , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/genética , Plantas Modificadas Genéticamente , Populus/efectos de los fármacos , Populus/enzimología , Populus/genética , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Nicotiana/efectos de los fármacos , Nicotiana/metabolismo

RESUMEN

Glutamine synthetase (GS) is the main enzyme involved in ammonia assimilation in plants and is the target of phosphinothricin (PPT), an herbicide commonly used for weed control in agriculture. As a result of the inhibition of GS, PPT also blocks photorespiration, resulting in the depletion of leaf amino acid pools leading to the plant death. Hybrid transgenic poplar (Populus tremula x P. alba INRA clone 7171-B4) overexpressing cytosolic GS is characterized by enhanced vegetative growth [Gallardo, F., Fu, J., Cantón, F.R., García-Gutiérrez, A., Cánovas, F.M., Kirby, E.G., 1999. Expression of a conifer glutamine synthetase gene in transgenic poplar. Planta 210, 19-26; Fu, J., Sampalo, R., Gallardo, F., Cánovas, F.M., Kirby, E.G., 2003. Assembly of a cytosolic pine glutamine synthetase holoenzyme in leaves of transgenic poplar leads to enhanced vegetative growth in young plants. Plant Cell Environ. 26, 411-418; Jing, Z.P., Gallardo, F., Pascual, M.B., Sampalo, R., Romero, J., Torres de Navarra, A., Cánovas, F.M., 2004. Improved growth in a field trial of transgenic hybrid poplar overexpressing glutamine synthetase. New Phytol. 164, 137-145], increased photosynthetic and photorespiratory capacities [El-Khatib, R.T., Hamerlynck, E.P., Gallardo, F., Kirby, E.G., 2004. Transgenic poplar characterized by ectopic expression of a pine cytosolic glutamine synthetase gene exhibits enhanced tolerance to water stress. Tree Physiol. 24, 729-736], enhanced tolerance to water stress (El-Khatib et al., 2004), and enhanced nitrogen use efficiency [Man, H.-M., Boriel, R., El-Khatib, R.T., Kirby, E.G., 2005. Characterization of transgenic poplar with ectopic expression of pine cytosolic glutamine synthetase under conditions of varying nitrogen availability. New Phytol. 167, 31-39]. In vitro plantlets of GS transgenic poplar exhibited enhanced resistance to PPT when compared with non-transgenic controls. After 30 days exposure to PPT at an equivalent dose of 275 g ha(-1), growth of GS transgenic poplar plantlets was 5-fold greater than controls. The response of young leaves to PPT treatment depends on physiological state as indicated by GS and Rubisco (LSU) levels. Young leaves from control plants, typically in a low differentiation state, respond to the herbicide showing up-regulation of GS and LSU. In contrast, young leaves from transgenic lines, with higher initial GS and LSU levels compared to control, display up-regulation of NADP(+)-isocitrate dehydrogenase. Differences between control and GS transgenics in their response to PPT are discussed in relation to their differences in photosynthetic and photorespiratory capacities (El-Khatib et al., 2004).

Asunto(s)

Aminobutiratos/farmacología , Citosol/efectos de los fármacos , Citosol/enzimología , Regulación Enzimológica de la Expresión Génica , Populus/enzimología , Regulación de la Expresión Génica de las Plantas , Glutamato-Amoníaco Ligasa , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Plantas Modificadas Genéticamente , Populus/efectos de los fármacos , Populus/genética , Populus/crecimiento & desarrollo , Plantones/efectos de los fármacos , Plantones/enzimología , Plantones/genética

RESUMEN

The present study addresses the hypothesis that enhanced expression of glutamine synthetase (GS) in transgenic poplar, characterized by the ectopic expression of pine cytosolic GS, results in an enhanced efficiency of nitrogen (N) assimilation and enhanced growth. Transgenic and control poplar were supplied with low and high N levels and the role of ectopic expression of the pine GS in growth and N assimilation was assessed by using amino acid analysis, (15)N enrichment, biochemical analyses, and growth measurements. While leaves of transgenic poplar contained 85% less (P < 0.01) free ammonium than leaves of nontransgenic control plants, leaves of transgenics showed increases in the levels of free glutamine and total free amino acids. Transgenic poplar lines also displayed significant increases in growth parameters when compared with controls grown under both low (0.3 mm) and high (10 mm) nitrate conditions. Furthermore, (15)N-enrichment experiments showed that 27% more (P < 0.05) (15)N was incorporated into structural compounds in transgenic lines than in nontransgenic controls. Using the methods described here, we present direct evidence for increased N assimilation efficiency and growth in GS transgenic lines.

Asunto(s)

Glutamato-Amoníaco Ligasa/metabolismo , Nitrógeno/metabolismo , Populus/genética , Populus/metabolismo , Aminoácidos/metabolismo , Glutamato-Amoníaco Ligasa/genética , Pinus/enzimología , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente

RESUMEN

Physiological responses to water stress in hybrid poplar (INRA 7171-B4, Populus tremula L. x P. alba L.) lines transformed to overexpress a pine cytosolic glutamine synthetase (GS1) gene were compared with those of non-transgenic plants. Before, during and after a drought treatment, net photosynthetic rates (Anet) were higher in transgenic than in non-transgenic plants. Stomatal conductance (gs) was higher in transgenic than in non-transgenic plants before, but not after exposure to drought. Before drought treatment, a sudden reduction in photosynthetic photon flux caused a greater burst of CO2 efflux in transgenic than non-transgenic plants, indicating greater photorespiratory activity. Drought caused greater reductions in photochemical quenching, photosystem II (PSII) antennae transfer efficiency (Fv'/Fm') and light-adapted PSII yield (PhiPSII) in non-transgenic than in transgenic plants, especially at low irradiances. Antennae-based thermal dissipation was higher in transgenic plants than in non-transgenic plants both during the imposition of drought and 1 or 3 days after the relief of drought. Under severe water stress and subsequently, transgenic plants maintained a higher expression of glutamine synthetase, glutamate synthase and Rubisco and higher concentrations of chlorophyll and glycine than non-transgenic plants. These findings indicate that overexpression of pine cytosolic GS1 enhanced sustained photosynthetic electron transport capacity during severe stomatal limitation. The data also suggest that ectopic expression of cytosolic GS increases photorespiratory activity, and that this serves as a protective sink for electrons from photosynthetic reaction centers.

Asunto(s)

Glutamato-Amoníaco Ligasa/genética , Populus/fisiología , Árboles/fisiología , Clorofila/fisiología , Deshidratación , Regulación Enzimológica de la Expresión Génica/genética , Fotosíntesis/fisiología , Transpiración de Plantas/fisiología , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/fisiología , Populus/enzimología , Populus/genética , Árboles/enzimología , Árboles/genética