Overexpression of a pine Dof transcription factor in hybrid poplars: A comparative study in trees growing under controlled and natural conditions.

In this work, the role of the pine transcriptional regulator Dof 5 in carbon and nitrogen metabolism has been examined in poplar trees. The overexpression of the gene and potential effects on growth and biomass production were compared between trees growing in a growth chamber under controlled conditions and trees growing in a field trial during two growth seasons. Ten-week-old transgenic poplars exhibited higher growth than untransformed controls and exhibited enhanced capacity for inorganic nitrogen uptake in the form of nitrate. Furthermore, the transgenic trees accumulated significantly more carbohydrates such as glucose, fructose, sucrose and starch. Lignin content increased in the basal part of the stem likely due to the thicker stem of the transformed plants. The enhanced levels of lignin were correlated with higher expression of the PAL1 and GS1.3 genes, which encode key enzymes involved in the phenylalanine deamination required for lignin biosynthesis. However, the results in the field trial experiment diverged from those observed in the chamber system. The lines overexpressing PpDof5 showed attenuated growth during the two growing seasons and no modification of carbon or nitrogen metabolism. These results were not associated with a decrease in the expression of the transgene, but they can be ascribed to the nitrogen available in the field soil compared to that available for growth under controlled conditions. This work highlights the paramount importance of testing transgenic lines in field trials.

Root and shoot performance of Arabidopsis thaliana exposed to elevated CO2: A physiologic, metabolic and transcriptomic response.

The responsiveness of C3 plants to raised atmospheric [CO2] levels has been frequently described as constrained by photosynthetic downregulation. The main goal of the current study was to characterize the shoot-root relationship and its implications in plant responsiveness under elevated [CO2] conditions. For this purpose, Arabidopsis thaliana plants were exposed to elevated [CO2] (800ppm versus 400ppm [CO2]) and fertilized with a mixed (NH4NO3) nitrogen source. Plant growth, physiology, metabolite and transcriptomic characterizations were carried out at the root and shoot levels. Plant growth under elevated [CO2] conditions was doubled due to increased photosynthetic rates and gas exchange measurements revealed that these plants maintain higher photosynthetic rates over extended periods of time. This positive response of photosynthetic rates to elevated [CO2] was caused by the maintenance of leaf protein and Rubisco concentrations at control levels alongside enhanced energy efficiency. The increased levels of leaf carbohydrates, organic acids and amino acids supported the augmented respiration rates of plants under elevated [CO2]. A transcriptomic analysis allowed the identification of photoassimilate allocation and remobilization as fundamental process used by the plants to maintain the outstanding photosynthetic performance. Moreover, based on the relationship between plant carbon status and hormone functioning, the transcriptomic analyses provided an explanation of why phenology accelerates under elevated [CO2] conditions.

The overexpression of the pine transcription factor PpDof5 in Arabidopsis leads to increased lignin content and affects carbon and nitrogen metabolism.

PpDof 5 is a regulator of the expression of glutamine synthetase (GS; EC 6.3.1.2) genes in photosynthetic and non-photosynthetic tissues of maritime pine. We have used Arabidopsis thaliana as a model system to study PpDof 5 function in planta, generating transgenic lines overexpressing the pine transcription factor. The overexpression of PpDof 5 resulted in a substantial increase of lignin content with a simultaneous regulation of carbon and nitrogen key genes. In addition, partitioning in carbon and nitrogen compounds was spread via various secondary metabolic pathways. These results suggest pleiotropic effects of PpDof 5 expression on various metabolic pathways of carbon and nitrogen metabolism. Plants overexpressing PpDof 5 exhibited upregulation of genes encoding enzymes for sucrose and starch biosynthesis, with a parallel increase in the content of soluble sugars. When the plants were grown under nitrate as the sole nitrogen source, they exhibited a significant regulation of the expression of genes involved mainly in signaling, but similar growth rates to wild-type plants. However, plants grown under ammonium exhibited major induction of the expression of photosynthetic genes and differential expression of ammonium and nitrate transporters. All these data suggest that in addition to controlling ammonium assimilation, PpDof 5 could be also involved in the regulation of other pathways in carbon and nitrogen metabolism in pine trees.

De novo assembly of maritime pine transcriptome: implications for forest breeding and biotechnology.

Maritime pine (Pinus pinasterAit.) is a widely distributed conifer species in Southwestern Europe and one of the most advanced models for conifer research. In the current work, comprehensive characterization of the maritime pine transcriptome was performed using a combination of two different next-generation sequencing platforms, 454 and Illumina. De novo assembly of the transcriptome provided a catalogue of 26 020 unique transcripts in maritime pine trees and a collection of 9641 full-length cDNAs. Quality of the transcriptome assembly was validated by RT-PCR amplification of selected transcripts for structural and regulatory genes. Transcription factors and enzyme-encoding transcripts were annotated. Furthermore, the available sequencing data permitted the identification of polymorphisms and the establishment of robust single nucleotide polymorphism (SNP) and simple-sequence repeat (SSR) databases for genotyping applications and integration of translational genomics in maritime pine breeding programmes. All our data are freely available at SustainpineDB, the P. pinaster expressional database. Results reported here on the maritime pine transcriptome represent a valuable resource for future basic and applied studies on this ecological and economically important pine species.

Novel insights into regulation of asparagine synthetase in conifers.

Asparagine, a key amino acid for nitrogen storage and transport in plants, is synthesized via the ATP-dependent reaction catalyzed by the enzyme asparagine synthetase (AS; EC 6.3.5.4). In this work, we present the molecular analysis of two full-length cDNAs that encode asparagine synthetase in maritime pine (Pinus pinaster Ait.), PpAS1, and PpAS2. Phylogenetic analyses of the deduced amino acid sequences revealed that both genes are class II AS, suggesting an ancient origin of these genes in plants. A comparative study of PpAS1 and PpAS2 gene expression profiles showed that PpAS1 gene is highly regulated by developmental and environmental factors, while PpAS2 is expressed constitutively. To determine the molecular mechanisms underpinning the differential expression of PpAS1, the promoter region of the gene was isolated and putative binding sites for MYB transcription factors were identified. Gel mobility shift assays showed that a MYB protein from Pinus taeda (PtMYB1) was able to interact with the promoter region of PpAS1. Furthermore, transient expression analyses in pine cells revealed a negative effect of PtMYB1 on PpAS1 expression. The potential role of MYB factors in the transcriptional regulation of PpAS1 in vascular cells is discussed.

Identification of genes regulated by ammonium availability in the roots of maritime pine trees.

Conifers have a preference for ammonium over nitrate as the main inorganic nitrogen source. However, it is unknown how changes in nitrogen nutrition may affect transcription profiles. In this study, microarray analysis and suppressive subtraction hybridization were used to identify differentially expressed genes in the roots of maritime pine exposed to changes in ammonium availability. A total of 225 unigenes that were differentially regulated by changes in ammonium nutrition were identified. Most of the unigenes were classified into seven functional categories by comparison with sequences deposited in the databases. A significant proportion of these genes were encoded for ammonium-regulated proteins of unknown functions. The differential expression of selected candidate genes was further validated in plants subjected to ammonium excess/deficiency. The transcript levels of representative genes were compared in maritime pine roots, 1, 15 and 35 days after nutritional treatments. Gene expression patterns suggest the existence of potential links between ammonium-responsive genes and genes involved in amino acid metabolism, particularly in asparagine biosynthesis and utilization. Functional analyses and exploration of the natural variability in maritime pine populations for a number of relevant genes are underway.

Differential regulation of two glutamine synthetase genes by a single Dof transcription factor.

The PpDof5 transcription factor from maritime pine (Pinus pinaster) is a regulator of the expression of glutamine synthetase (GS) genes in photosynthetic and non-photosynthetic tissues. PpDof5 mRNA is detected almost ubiquitously during pine development with low levels of gene expression in green tissues and much higher levels in roots and lignified shoots. The PpDof5 protein expressed in bacteria binds to oligonucleotide probes containing the AAAG core sequence derived from the promoters of GS1a and GS1b genes. Transient expression experiments in agroinfiltrated tobacco leaves and in pine protoplasts demonstrated that PpDof5 is able to trans-regulate differentially the transcription of both GS1a and GS1b. PpDof5 activated transcription of the GS1b promoter and, in contrast, behaved as a transcriptional repressor of the GS1a promoter. These results support a regulatory mechanism for the transcriptional control of the spatial distribution of cytosolic GS isoforms in pine. Considering the precise expression patterns of GS1 genes required to fulfil the ammonium assimilation requirements during tree development, we hypothesize that PpDof5 could have a key role in the control of ammonium assimilation for glutamine biosynthesis in conifers. A regulatory model of GS1 gene expression in pine is proposed.