Molecular characterization of transgenic maritime pine somatic plants overexpressing a cytosolic glutamine synthetase gene (PsGS1a) involved in nitrogen assimilation.
IUFRO subdivision 2.4 Conference, Genomics and Forest Tree Genetics, May 30 – June 3, 2016, Arcachon, France, S6-10, pp. 29.
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Ávila C, Rueda-López M, Canales J, Cánovas FM, Michel R, Pillet-Emanuel H, Canlet F, Debille S, Trontin JF
Collaboration FCBA, Université de Malaga
Nitrogen (N) is a nutrient usually available at extremely low levels in forest ecosystems. Thus, efficient mechanisms are required for the assimilation, storage, mobilization and recycling of inorganic and organic forms. Ammonium is the predominant N source in forest soil, and it is well documented that conifers, unlike herbaceous plants, have a preference for ammonium over nitrate as an inorganic N source. In conifer, the cytosolic glutamine synthetase GS1 is involved in ammonium assimilation and amino acid biosynthesis in both photosynthetic (GS1a holoenzyme) and non-photosynthetic cells (GS1b). Different approaches have shown that GS is a key component of plant N use efficiency, supporting a role of GS in plant development, growth and biomass production.
A pine GS1a gene was constitutively overexpressed under the control of CaMV35S (P35S) or maize ubiquitin (Pubi) promoters in maritime pine following Agrobacterium-mediated genetic transformation of embryogenic tissue. Plants were regenerated from cryopreserved transgenic lines and appropriate non-transgenic (NT) controls through somatic embryogenesis.
PCR analyses confirmed the transgenic status of somatic plants with a few T-DNA copies (1-3) integrated into the genome. Two-years-old plants from different lines were sampled for estimating GS1a transcript content, total protein content, GS1a enzymatic activity and also for GS1a protein detection. Results are highly variable between lines and even between plants from the same clone suggesting pleiotropic and/or (epi)genetic regulation effects. GS1a transcript detection was similar to NT controls. Increased total protein content and GS1a activity were observed in some lines and plants for both constructs. Two Pubi- or P35S-GS1a plants with high GS1a activity were selected for further transcriptomic profiling (9K PINARRAY2). These plants were confirmed to be upregulated for GS1a (RT-qPCR) and differentially expressed 527 and 685 genes compared to NT controls. Gene ontology terms could be assigned to 301 and 360 unigenes, respectively. A good concordance in functional categories was observed between both constructs with high preponderance of the “protein” category but also “photosynthesis” and “amino acid metabolism”. More stringent selection criteria highlighted 12 commonly deregulated genes involved in stress resistance and growth. Strikingly, GS1a overexpression resulted in slightly improved plant growth behaviour in some lines but only during the first season at the GMO glasshouse containment.
This research received funding from the European Community’s FP7/2007-2013 programme (289841-PROCOGEN), The French National Research Agency and the Spanish Ministry of Science and Innovation (SUSTAINPINE: ANR-09-KBBE-007, BIO2012-33797). This work also benefited from equipments of the XYLOBIOTECH technical facility (XYLOFOREST platform, ANR-10-EQPX-16).