Overexpression of a cytosolic NADP+-isocitrate dehydrogenase causes alterations in the vascular development of hybrid poplars.

Cytosolic NADP+-isocitrate dehydrogenase (ICDH) is one of the major enzymes involved in the production of 2-oxoglutarate for amino acid biosynthesis in plants. In most plants studied, ICDH is encoded by either one gene or a small gene family, and the protein sequence has been highly conserved during evolution, suggesting it plays different and essential roles in metabolism and differentiation. To elucidate the role of ICDH in hybrid poplar (Populus tremula x P. alba), transgenic plants overexpressing the Pinus pinaster gene were generated. Overexpression of ICDH resulted in hybrid poplar (Populus tremula × P. alba) trees with higher expression levels of the endogenous ICDH gene and higher enzyme content than control untransformed plants. Transgenic poplars also showed an increased expression of glutamine synthetase (GS1.3), glutamate decarboxylase (GAD) and other genes associated with vascular differentiation. Furthermore, these plants exhibited increased growth in height, longer internodes and enhanced vascular development in young leaves and the apical region of stem. Modifications in amino acid and organic acid content were observed in young leaves of the transgenic lines, suggesting an increased biosynthesis of amino acids for building new structures and also for transport to other sink organs, as expanding leaves or young stems. Taken together, these results support an important role of ICDH in plant growth and vascular development.

Transgenic poplar expressing the pine GS1a show alterations in nitrogen homeostasis during drought.

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.

A putative role for γ-aminobutyric acid (GABA) in vascular development in pine seedlings.

MAIN CONCLUSION: A model for GABA synthesis in stems of pine seedlings is proposed. The localization of GABA in differentiating tracheids suggests a link between GABA production and vascular development. γ-aminobutyric acid (GABA) is a non-proteinogenic amino acid present in both prokaryotic and eukaryotic organisms. GABA plays a fundamental role as a signal molecule in the central nervous system in animals. In plants, GABA has been correlated with cellular elongation, plant development, gene expression regulation, synthesis of ethylene and other hormones, and signaling. Considering the physiological importance of GABA in plants, the lack of works about GABA localization in this kingdom seems surprising. In this work, the immunolocalization of GABA in root and hypocotyl during seedling development and in bent stem showing compression xylem has been studied. In the seedling root, the GABA signal was very high and restricted to the stele supporting previous evidences indicating a potential role for this amino acid in root growth and nutrient transport. In hypocotyl, GABA was localized in vascular tissues, including differentiating xylem, ray parenchyma and epithelial resin duct cells, drawing also a role for GABA in vascular development, communication and defense. During the production of compression wood, a special lignified wood produced when the stem loss its vertical position, a clear GABA signal was found in the new differentiating xylem cells showing a gradient-like pattern with higher signal in less differentiated elements. The results are in accordance with a previous work indicating that glutamate decarboxylase and GABA production are associated to vascular differentiation in pine Molina-Rueda et al. (Planta 232: 1471-1483, 2010). A model for GABA synthesis in vascular differentiation, communication, and defense is proposed in the stem of pine seedlings.

The Populus superoxide dismutase gene family and its responses to drought stress in transgenic poplar overexpressing a pine cytosolic glutamine synthetase (GS1a).

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.

Characterization and developmental expression of a glutamate decarboxylase from maritime pine.

Glutamate decarboxylase (GAD, EC 4.1.1.15) is a key enzyme in the synthesis of γ-aminobutyric acid (GABA) in higher plants. A complete cDNA encoding glutamate decarboxylase (GAD, EC 4.1.1.15) was characterized from Pinus pinaster Ait, and its expression pattern was studied to gain insight into the role of GAD in the differentiation of the vascular system. Pine GAD contained a C-terminal region with conserved residues and a predicted secondary structure similar to the calmodulin (CaM)-binding domains of angiosperm GADs. The enzyme was able to bind to a bovine CaM-agarose column and GAD activity was higher at acidic pH, suggesting that the pine GAD can be regulated in vivo by Ca(2+)/CaM and pH. A polyclonal antiserum was prepared against the pine protein. GAD expression was studied at activity, protein, and mRNA level and was compared with the expression of other genes during the differentiation of the hypocotyl and induction of reaction wood. In seedling organs, GABA levels closely matched GAD expression, with high levels in the root and during lignification of the hypocotyl. GAD expression was also induced in response to the production of compression wood and its expression matched the pattern of other genes involved in ethylene and 2-oxoglutarate synthesis. The results suggest of a role of GAD in hypocotyl and stem development in pine.

Spatial distribution of cytosolic NADP(+)-isocitrate dehydrogenase in pine embryos and seedlings.

Cytosolic NADP(+)-linked isocitrate dehydrogenase (NADP(+)-IDH) is considered the main enzyme catalyzing the production of 2-oxoglutarate for amino acid biosynthesis in plants. We characterized a full-length cDNA encoding the cytosolic NADP(+)-IDH in the gymnosperm Pinus pinaster Ait. The deduced amino acid sequence exhibited high homology with available sequences in angiosperms. Genomic analysis indicated that only one gene, or two genes with a high degree of homology, encodes the protein in P. pinaster. Cytosolic NADP(+)-IDH is up-regulated during embryo germination concomitantly with glutamine synthetase. Immunohistochemical analysis of germinating seeds and young seedlings showed a broad spatial pattern of NADP(+)-IDH expression. The protein was detected in vascular tissues of germinating embryo and seedling organs, as well as in other cellular types and tissues, including parenchyma and epidermal cells. The spatial pattern of NADP(+)-IDH expression in the embryo and seedling organs did not coincide with the reported spatial patterns for other key enzymes of nitrogen metabolism. Treatment of seedlings with phosphinothricin, an inhibitor of glutamine synthetase (GS), differentially affected GS and NADP-IDH in cotyledons. In response to herbicide treatment, GS was up-regulated in 0.5-cm-long cotyledons, whereas NADP(+)-IDH was up-regulated in 1.5-cm-long cotyledons, suggesting that 2-oxoglutarate is required to overcome the herbicide effect in tissues with a high demand for glutamate. The results indicated that cytosolic NADP(+)-IDH is a housekeeping enzyme that has not undergone functional specialization during evolution. Its spatial pattern in pine tissues suggests that it facilitates metabolism in different ways depending on the characteristics of the particular tissue and cellular type.