The assimilation of ammonium into organic nitrogen catalyzed by the enzyme glutamine synthetase (GS; EC 6.3.1.2) has been suggested to be the limiting step for plant nitrogen utilization (H-M. Lam et al. 1995, Plant Cell 7: 887-898). We have developed a molecular approach to increase glutamine production in transgenic poplar by the overexpression of a conifer GS gene. A chimeric construct consisting of the cauliflower mosaic virus 35S promoter fused to pine cytosolic GS cDNA and nopaline synthetase polyadenylation region was transferred into pBin19 for transformation of a hybrid poplar clone (INRA 7171-B4, Populus tremula x P. alba) via Agrobacterium tumefaciens. Transformed poplar lines were selected by their ability to grow on selective medium containing kanamycin. The presence of the introduced gene in the poplar genome was verified by Southern blotting and polymerase chain reaction analysis. Transgene expression was detected in all selected poplar lines at the mRNA level. The detection of the corresponding polypeptide (41 kDa) and increased GS activity in the transgenics suggest that pine transcripts are correctly processed by the angiosperm translational machinery and that GS1 subunits are assembled in functional holoenzymes. Expression of the pine GS1 gene in poplar was associated with an increase in the levels of total soluble protein and an increase in chlorophyll content in leaves of transformed trees. Furthermore, the mean net growth in height of GS-overexpressing clones was significantly greater than that of non-transformed controls, ranging from a 76% increase in height at 2 months to a 21.3% increase at 6 months. Our results suggest that the efficiency of nitrogen utilization may be engineered in trees by genetic manipulation of glutamine biosynthesis.

BackgroundGlutamine 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.ResultsThe 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.ConclusionsOur 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.

Immature inflorescences of oil palm (Elaeis guineensis) var. Pisifera were inoculated onto modified MS medium containing 0.3% (w/v) activated charcoal and 475 μM 2,4-D. After 2-3 months of culture, a hard yellow callus proliferated at the base of the shoot-like structures. The high incidence of phenolic oxidation required the use of increased levels of activated charcoal (0.5% w/v) and 2,4-D (500 μM). Development of floral structures from inflorescence expiants was frequently observed during the culture period. After 81 weeks of culture, an embryogenic tissue characterized by compact consistency and pearly white color was observed in tissues derived from very young inflorescences. This compact embryogenic tissue differentiated into normal somatic embryos when transferred onto regeneration medium containing NAA (15 μM) and ABA (2 μM). Normal plantlets were recovered from these somatic embryos after 8 weeks on regeneration medium.

Summary• 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 m M ) and high (10 m M ) 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.

BackgroundGlutamine 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.ConclusionsBoth 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.

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.

Immature zygotic embryos at different developmental stages were used for callus induction and regeneration studies. Immature embryos excised from fruits 77, 91, 100, 114, 128, 140 and 193 days after pollination and mature embryos were cultured on modified Y3 medium containing 500 mg 1 -~ cysteine, 0.5% (w/v) PVP-40, 500 p~M 2,4-D and 0.3% (w/v) charcoal. Compact embryogenic tissue began differentiating directly from embryo explants after 2 weeks of culture. The percentage of embryos forming compact embryogenic tissue ranged from 28.6% for 91-day-old embryos to 0% for 140-day-old and older embryos. Friable embryogenic tissue was observed in callus cultures derived from 100-day-old embryos. Although both compact and friable embryogenic tissues were successfully isolated, normal embryo and plantlet development was observed only from friable embryogenic tissue.Abbreviations: ABA-abscisic acid, 2,4-D-2,4-dichlorophenoxyacetic acid, NAA-naphthaleneacetic acid, PVP -polyvinylpyrollidone

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.