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1.
Assembly of a cytosolic pine glutamine synthetase holoenzyme in leaves of transgenic poplar leads to enhanced vegetative growth in young plants 总被引:2,自引:0,他引:2
J. FU R. SAMPALO F. GALLARDO F. M. CÁNOVAS & E. G. KIRBY 《Plant, cell & environment》2003,26(3):411-418
Over‐expression of glutamine synthetase (GS, EC 6.3.1.2), a key enzyme in nitrogen assimilation, may be a reasonable approach to enhance plant nitrogen use efficiency. In this work phenotypic and biochemical characterizations of young transgenic poplars showing ectopic expression of a pine cytosolic GS transgene in photosynthetic tissue (Gallardo et al., Planta 210, 19–26, 1999) are presented. Analysis of 22 independent transgenic lines in a 6 month greenhouse study indicated that expression of the pine GS transgene affects early vegetative growth and leaf morphology. In comparison with non‐transgenic controls, transgenic trees exhibited significantly greater numbers of nodes and leaves (12%), and higher average leaf length and width resulting in an increase in leaf area (25%). Leaf shape was not altered. Transgenic poplars also exhibited increased GS activity (66%), chlorophyll content (33%) and protein content (21%). Plant height was correlated with GS content in young leaves, suggesting that GS can be considered a marker for vegetative growth. Molecular and kinetic characterization of GS isoforms in leaves indicated that poplar GS isoforms are similar to their counterparts in herbaceous plants. A new GS isoenzyme that displayed molecular and kinetic characteristics corresponding to the octomeric pine cytosolic GS1 was identified in the photosynthetic tissues of transgenic poplar leaves. These results indicate that enhanced growth and alterations in biochemistry during early growth are the consequence of transgene expression and assembly of pine GS1 subunits into a new functional holoenzyme in the cytosol of photosynthetic cells. 相似文献
2.
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. 相似文献
3.
Response of transgenic poplar overexpressing cytosolic glutamine synthetase to phosphinothricin 总被引:2,自引:0,他引:2
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). 相似文献
4.
5.
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. 相似文献
6.
Rémi Vincent Vincent Fraisier Sylvain Chaillou M. Anis Limami Eliane Deleens Belinda Phillipson Corinne Douat Jean-Pierre Boutin Bertrand Hirel 《Planta》1997,201(4):424-433
A soybean cytosolic glutamine synthetase gene (GS15) was fused with the constitutive 35S cauliflower mosaic virus (CaMV) promoter in order to direct overexpression in Lotus corniculatus L. plants. Following transformation with Agrobacterium rhizogenes, eight independent Lotus transformants were obtained which synthesized additional cytosolic glutamine synthetase (GS) in the shoots. To eliminate
any interference caused by the T-DNA from the Ri plasmid, three primary transformants were crossed with untransformed plants
and progeny devoid of TL- and TR-DNA sequences were chosen for further analyses. These plants had a 50–80% increase in total leaf GS activity. Plants were
grown under different nitrogen regimes (4 or 12 mM NH4
+) and aspects of carbon and nitrogen metabolism were examined. In roots, an increase in free amino acids and ammonium was
accompanied by a decrease in soluble carbohydrates in the transgenic plants cultivated with 12 mM NH4
+ in comparison to the wild type grown under the same conditions. Labelling experiments using 15NH4
+ were carried out in order to monitor the influx of ammonium and its subsequent incorporation into amino acids. This experiment
showed that both ammonium uptake in the roots and the subsequent translocation of amino acids to the shoots was lower in plants
overexpressing GS. It was concluded that the build up of ammonium and the increase in amino acid concentration in the roots
was the result of shoot protein degradation. Moreover, following three weeks of hydroponic culture early floral development
was observed in the transformed plants. As all these properties are characteristic of senescent plants, these findings suggest
that expression of cytosolic GS in the shoots may accelerate plant development, leading to early senescence and premature
flowering when plants are grown on an ammonium-rich medium.
Received: 17 July 1996 / Accepted: 16 October 1996 相似文献
7.
Anis Limami Belinda Phillipson Rafiqa Ameziane Nicolas Pernollet Qunji Jiang Roselyne Roy Eliane Deleens Muriel Chaumont-Bonnet Peter M. Gresshoff Bertrand Hirel 《Planta》1999,209(4):495-502
To investigate the contribution of root cytosolic glutamine synthetase (GS) activity in plant biomass production, two different
approaches were conducted using the model legume Lotus japonicus. In the first series of experiments, it was found that overexpressing GS activity in roots of transgenic plants leads to
a decrease in plant biomass production. Using 15N labelling it was shown that this decrease is likely to be due to a lower nitrate uptake accompanied by a redistribution
to the shoots of the newly absorbed nitrogen which cannot be reduced due to the lack of nitrate reductase activity in this
organ. In the second series of experiments, the relationship between plant growth and root GS activity was analysed using
a series of recombinant inbred lines issued from the crossing of two different Lotus ecotypes, Gifu and Funakura. It was confirmed that a negative relationship exists between root GS expression and plant biomass
production in both the two parental lines and their progeny. Statistical analysis allowed it to be estimated that at least
13% of plant growth variation can be accounted for by variation in GS activity.
Received: 24 September 1998 / Accepted: 14 April 1999 相似文献
8.
A spontaneous double mutant of Chlamydomonas reinhardtii, designated ARF3, was resistant to L-methionine-S-sulfoximine (MSX), lacked chloroplastic glutamine synthetase (GS2) activity,
and grew very poorly in all media tested. In segregants obtained after genetic crosses, the poor-growth phenotype was always
linked to the lack of GS2 and to a diminished rate of consumption of ammonium, even under conditions where photorespiration
was minimized. The ammonium permeases in mutant ARF3, however, were not altered. This indicates that, unlike in higher plants,
GS2 contributes substantially to the primary assimilation of ammonia in this alga, and that its function cannot be replaced
by the cytosolic glutamine synthetase. In genetic crosses, the MSX resistance and the lack of GS2 segregated independently,
indicating that resistance was not due to an altered form of GS2.
Received: 5 June 1998 / Accepted: 10 September 1998 相似文献
9.
10.
Glutamine synthetase from mesophyll and bundle sheath maize cells: isoenzyme complements and different sensitivities to phosphinothricin 总被引:13,自引:0,他引:13
B. González-Moro A. Mena-Petite M. Lacuesta C. González-Murua A. Muñoz-Rueda 《Plant cell reports》2000,19(11):1127-1134
Anion-exchange FPLC has been used to resolve the isoforms of glutamine synthetase (GS, EC 6.3.1.2) from Zea mays mesophyll (MC) and bundle sheath cells (BSC). Two different isoforms were detected in both types of photosynthetic cells.
The predominantly active isoform was GS1 (61%) in MC and GS2 (67%) in BSC. The relative contribution of GS1 and GS2 to the
overall GS activity in BSC in maize here reported resembles the proportion described for most C3 plants. Differences among
these isoforms in terms of their susceptibility to phosphinothricin (PPT), an analogue of glutamate and known inhibitor of
GS, were found. The GS1 isoenzyme from MC was the most sensitive form, being inhibited by 50% at approximately 2.0 μM DL-PPT, whereas the GS2 from BSC presented the highest tolerance to the inhibitor (I50=30 μM). The transferase-to-semibiosynthetic activity ratio for the MC isoforms, which was higher than the ratio for the BSC isoforms,
and the differences shown by the isoforms in susceptibility to PPT predict important differences in the biochemical properties
and regulation of GS isoenzymes. In this regard, the cytoplasmic isoenzymes, and especially the one in MC, due to its relatively
high contribution to mesophyll cell GS activity, could play a vital role in nitrogen metabolism in maize.
Received: 1 December 1999 / Revised: 7 February 2000 / Accepted: 23 February 2000 相似文献
11.
A. Pérez-García S. Pereira J. Pissarra A. García Gutiérrez F. M. Cazorla R. Salema A. de Vicente F. M. Cánovas 《Planta》1998,206(3):426-434
In tomato (Lycopersicon esculentum Mill.) leaves, the predominant glutamine synthetase (GS; EC 6.3.1.2) is chloroplastic (GS2; 45 kDa) whereas the cytosolic
isoform (GS1; 39 kDa) is represented as a minor enzyme. Following either infection by Pseudomonas syringae pv. tomato (Pst) or treatment with phosphinothricin (PPT), a GS inhibitor, GS1 accumulated in the leaves. In contrast to
healthy control leaves, where GS1 was restricted to the veins, in infected and PPT-treated leaves the GS1 polypeptide was
also detected in the leaf blade; moreover, it was more abundant than GS2. Different immunological approaches were therefore
used to investigate whether or not the GS1 polypeptide expressed in Pst-infected and PPT-treated tomato leaves was distributed
among different tissues and subcellular compartments in the same way as the constitutive GS1 expressed in healthy leaves.
By tissue-printing analysis, a similar GS immunostaining was observed in epidermis, mesophyll and phloem of leaflet midrib
cross-sections of control, infected and PPT-treated leaves. Immunocytochemical localization revealed that GS protein was present
in the chloroplast of mesophyll cells and the cytoplasm of phloem cells in healthy leaves; however, in Pst-infected or PPT-treated
leaves, a strong labelling was observed in the cytoplasm of mesophyll cells. Two-dimensional analysis of GS polypeptides showed
that, in addition to the constitutive GS1, a GS1 polypeptide different in charge was present in tomato leaflets after microbial
infection or herbicide treatment. All these results indicate that a novel cytosolic GS is induced in mesophyll cells of Pst-infected
or PPT-treated leaves. A possible role for this new cytosolic GS in the remobilization of leaf nitrogen during infection is
proposed.
Received: 16 January 1998 / Accepted 21 April 1998 相似文献
12.
Single isolates of a mycobiont isolated from Pisonia grandis R. Br., Pisolithus tinctorius (Pers.) Coker & Couch and Tylospora fibrillosa (Burt.) Donk were compared with regard to their relative abilities to produce key enzymes of inorganic nitrogen assimilation.
Nitrate reductase (NR) activities in the P. grandis mycobiont and T. fibrillosa were significantly lower than in P. tinctorius. While specific activities for glutamate dehydrogenase (GDH) were higher in P. tinctorius than the other two fungi following NH4
+ pre-treatment, glutamine synthetase (GS) activity did not differ significantly between the three fungi. In all three fungi,
specific activities for GS were significantly higher than for GDH. NR activity was expressed in all three fungi regardless
of the nitrogen source in the medium, but in P. tinctorius diminished following continued exposure to either NO3
–, NH4
+, glutamine or NO3
– + glutamine. The data are discussed in relation to nitrogen utilisation by the P. grandis mycobiont.
Accepted: 16 October 1997 相似文献
13.
Francisco R. Cantón Angel García-Gutiérrez Fernando Gallardo Antonio de Vicente Francisco M. Cánovas 《Plant molecular biology》1993,22(5):819-828
A full-length cDNA clone (pGSP114) encoding glutamine synthetase was isolated from a gt11 library of the gymnosperm Pinus sylvestris. Nucleotide sequence analysis showed that pGSP114 contains an open reading frame encoding a protein of 357 amino acid residues with a calculated molecular mass of 39.5 kDa. The derived amino acid sequence was more homologous to cytosolic (GS1) (78–82%) than to chloroplastic (GS2) (71–75%) glutamine synthetase in angiosperms. The lack of N-terminal presequence and C-terminal extension which define the primary structure of GS2, also supports that the isolated cDNA encodes cytosolic GS. Southern blot analysis of genomic DNA from P. sylvestris and P. pinaster suggests that GS may be encoded by a small gene family in pine. GS mRNA was more abundant in cotyledons and stems than in roots of both Scots and maritime pines. Western blot analysis in P. sylvestris seedlings showed that only one GS polypeptide, similar in size to GS1 in P. pinaster, could be detected in several different tissues. Our results suggest that cytosolic GS is mainly responsible for glutamine biosynthesis in pine seedlings.This paper is dedicated to the memory of Dr. Jesús S. Olavarría. 相似文献
14.
Kinetic flux profiling dissects nitrogen utilization pathways in the oleaginous green alga Chlorella protothecoides
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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. 相似文献
15.
The nitrate reductase activity from Chlamydomonas reinhardtii was not altered when extracts were incubated with yeast 14-3-3 proteins in the presence of Mg-ATP. However, the C. reinhardtii extracts contained 14-3-3 proteins capable of inhibiting the spinach nitrate reductase, raising the question of their physiological
substrates. Two C. reinhardtii proteins of about 48 and 35 kDa were eluted from 14-3-3 affinity chromatography columns and bound to 14-3-3s in overlay assays.
The 48-kDa protein corresponded to the cytosolic isoform of glutamine synthetase (GS1). The GS1 was phosphorylated by a Ca2+- and calmodulin-dependent protein kinase partially purified from the alga. However, neither phosphorylation nor 14-3-3 binding
seemed to change GS catalytic activity.
Received: 3 February 2000 / Accepted: 6 May 2000 相似文献
16.
Nitrogen metabolism of the needles of 40-year-old Douglas fir and Scots pine trees, growing in two forest stands on cation-poor and acidic sandy soil with a relatively high atmospheric nitrogen deposition was studied. The composition of the free amino acid (FAA) pool, the concentrations of total nitrogen and soluble protein and the activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were determined in the needles. An excessive nitrogen supply by a high atmospheric nitrogen deposition in both forest stands was indicated by the high concentrations of total nitrogen and the amino acids arginine, glutamic acid, glutamine and aspartic acid in control trees. In addition the effect of optimal nutrition and water supply (fertigation) on the needle nitrogen metabolism was evaluated. The total concentration of the FAA pool in needles of both tree species was lower in the fertigated than in the non-fertigated (control) trees, except for 1-year-old needles of Scots pine, in which the concentration after fertigation did not differ from the control. The lower total FAA concentration in the fertigated trees could be attributed to arginine, the concentration of which was on average 60% lower than in the control. Neither the concentration of soluble protein nor the activity of GS were influenced by fertigation. The activity of GDH in fertigated trees only differed significantly from the control in October. Scots pine needles had higher concentrations of protein (50%) and higher activities of GS (44%) and GDH (25%) than Douglas fir needles. Possible explanations for the lower vitality of Douglas fir compared to Scots pine are given. 相似文献
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18.
Mesophyll cells (MCs) and bundle-sheath cells (BSCs) of leaves of the C4 plant maize (Zea mays L.) were separated by cellulase digestion to determine the relative proportion of the glutamine synthetase (GS; EC 6.3.1.2)
or the NADH-glutamate dehydrogenase (GDH; EC 1.4.1.2) isoforms in each cell type. The degree of cross-contamination between
our MC and BSC preparations was checked by the analysis of marker proteins in each fraction. Nitrate reductase (EC 1.6.6.1)
proteins (110 kDa) were found only in the MC fraction. In contrast, ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC
1.4.7.1) proteins (160 kDa) were almost exclusively present in the BSC fraction. These results are consistent with the known
intercellular distribution of nitrate reductase and Fd-GOGAT proteins in maize leaves and show that the cross-contamination
between our MC and BSC fractions was very low. Proteins corresponding to cytosolic GS (GS-1) or plastidic GS (GS-2) were found
in both the MC and BSC fractions. While equal levels of GS-1 (40 kDa) and GS-2 (44 kDa) polypeptides were present in the BSC
fraction, the GS-1 protein level in the MC fraction was 1.8-fold higher than the GS-2 protein pool. Following separation of
the GS isoforms by anion-exchange chromatography of MC or BSC soluble protein extracts, the relative GS-1 activity in the
MC fraction was found to be higher than the relative GS-2 activity. In the BSC fraction, the relative GS-1 activity was very
similar to the relative GS-2 activity. Two isoforms of GDH with apparent molecular weights of 41 kDa and 42 kDa, respectively,
were detected in the BSC fraction of maize leaves. Both GDH isoenzymes appear to be absent from the MC fraction. In the BSCs,
the level of the 42-kDa GDH isoform was 1.7-fold higher than the level of the 41-kDa GDH isoform. A possible role for GS-1
and GDH co-acting in the synthesis of glutamine for the transport of nitrogen is discussed.
Received: 25 January 2000 / Accepted: 30 March 2000 相似文献
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20.
Suárez MF Avila C Gallardo F Cantón FR García-Gutiérrez A Claros MG Cánovas FM 《Journal of experimental botany》2002,53(370):891-904
Ammonium is assimilated into amino acids through the sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) enzymes. This metabolic pathway is driven by energy, reducing power and requires the net supply of 2-oxoglutarate that can be provided by the reaction catalysed by isocitrate dehydrogenase (IDH). Most studies on the biochemistry and molecular biology of N-assimilating enzymes have been carried out on annual plant species and the available information on woody models is far more limited. This is in spite of their economic and ecological importance and the fact that nitrogen is a common limiting factor for tree growth. GS, GOGAT and IDH enzymes have been purified from several woody species and their kinetic and molecular properties determined. A number of cDNA clones have also been isolated and characterized. Although the enzymes are remarkably well conserved along the evolutionary scale, major differences have been found in their compartmentation within the cell between angiosperms and conifers, suggesting possible adaptations to specific functional roles. The analysis of the gene expression patterns in a variety of biological situations such as changes in N nutrition, development, biotic or abiotic stresses and senescence, suggest that cytosolic GS plays a central and pivotal role in ammonium assimilation and metabolism in woody plants. The modification of N assimilation efficiency has been recently approached in trees by overexpression of a cytosolic pine GS in poplar. The results obtained, suggest that an increase in cytosolic GS might lead to a global effect on the synthesis of nitrogenous compounds in the leaves, with enhanced vegetative growth of transgenic trees. All these data suggest that manipulation of cytosolic GS may have consequences for plant growth and biomass production. 相似文献