Fernando Gallardo

Expression of a conifer glutamine synthetase gene in transgenic poplar

Abstract. 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 × 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.

Accumulation of glutamine synthetase during early development of maritime pine (Pinus pinaster) seedlings

Seedlings of Pinus pinaster Alton accumulated chlorophyll (Chl) when grown in complete darkness. Contents of Chl a and Chl b increased during germination, reaching similar levels in light- and dark-grown plants. Glutamine-synthetase (GS; EC 6.3.1.2) activity was detected in the embryo and its level increased markedly in cotyledons of dark-germinated seedlings. Similar levels of GS activity were observed when the seeds were germinated in the presence of white light. Only one GS form, which eluted at about 0.1 M KCl, was found by ion-exchange chromatography. A predominant GS polypeptide of 43 kDa was detected in cotyledons, and its steady-state level increased with development in a lightindependent fashion. In roots and needles, a related GS polypeptide of 43 kDa was the unique species detectable by western blot analysis. Immunoblots of soluble proteins from isolated chloroplasts showed low abundance of GS protein, indicating that glutamine synthesis in pine cotyledons occurs mainly in the cytosol. Nitrogen-feeding experiments carried out with detached shoots indicated that neither NO3−nor NH4+regulate GS levels and the polypeptide pattern. Our results indicate that environmental factors, such as light and nitrogen supply, have a limited role in GS accumulation during pine development.

Changes in NADP+-linked isocitrate dehydrogenase during tomato fruit ripening

The activity of NADP+-specific isocitrate dehydrogenase (NADP+-IDH, EC 1.1.1.42) was investigated during the ripening of tomato (Lycopersicon esculentum Mill.) fruit. In the breaker stage, NADP+-IDH activity declined but a substantial recovery was observed in the late ripening stages when most lycopene synthesis occurs. These changes resulted in higher NADP+-IDH activity and specific polypeptide abundance in ripe than in green fruit pericarp. Most of the enzyme corresponded to the predominant cytosolic isoform which was purified from both green and ripe fruits. Fruit NADP+-IDH seems to be a dimeric enzyme having a subunit size of 48 kDa. The Km values of the enzymes from green and ripe pericarp for NADP+, isocitrate and Mg2+ were not significantly different. The similar molecular and kinetic properties and chromatographic behaviour of the enzymes from the two kinds of tissue strongly suggest that the ripening process is not accompanied by a change in isoenzyme complement. The increase in NADP+-IDH in the late stage of ripening also suggests that this enzyme is involved in the metabolism of C6 organic acids and in glutamate accumulation in ripe tissues.

Molecular characterization of a cDNA clone encoding glutamine synthetase from a gymnosperm, Pinus sylvestris

A full-length cDNA clone (pGSP114) encoding glutamine synthetase was isolated from a λgt11 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.

Genetic modification of amino acid metabolism in woody plants

Forest trees comprise a large group of angiosperm and gymnosperm species of economic importance that play a crucial role in the ecosystems. Nitrogen is frequently a limiting factor for growth of forest trees, thus development of a fundamental understanding of nitrogen assimilation and metabolism is particularly important in broadening our understanding of fundamental tree biology. There are a number of fundamental ways in which woody plants differ from herbaceous species, including seed dormancy and germination, growth habit and enhanced secondary development, management of reduced nitrogen during dormancy, and the metabolic requirements for secondary growth, a major sink for both reduced nitrogen and carbon. Poplar species (Populus spp.) have emerged as model systems for research in woody angiosperms. Modification of metabolism using genetic engineering approaches has recently focussed on altering the biosynthesis of glutamine, polyamines, glutathione, and lignin. These approaches potentially affect plant development and stress tolerance. The aim of this minireview is to integrate the experimental genetic engineering approaches in the context of developing an increased understanding of overall nitrogen and amino acid metabolism in trees.

Expression of ferredoxin-dependent glutamate synthase in dark-grown pine seedlings

Pine seedlings are able to accumulate chlorophylls and develop green plastids in a light-independent manner. In this work, we have characterized ferredoxin-dependent glutamate synthase (EC 1.4.7.1; Fd-GOGAT), a key enzyme in nitrogen interconversion during this process. Fd-GOGAT has been purified about 170-fold from cotyledons of maritime pine (Pinus pinaster). As occurs in angiosperms, the native enzyme is a single polypeptide with an apparent molecular mass of 163–168 kDa that is confined to the chloroplast stroma. Polyclonal antibodies generated against the purified enzyme were used to immunoscreen a λgt11 expression library from Scots pine (Pinus sylvestris) seedlings and partial cDNA clones were isolated and characterized. The clone with the longest cDNA insert (pGOP44) contained the codification for the C-terminal (550 amino acids) of the pine Fd-GOGAT polypeptide. Immunological cross-reactivity and comparative amino sequence analysis revealed that Fd-GOGAT is a well conserved protein in higher plants. Western blot analyses showed that protein was expressed in chloroplast-containing pine tissues and this expression pattern was not affected by exogenously supplied nitrogen. Fd-GOGAT mRNA, polypeptide and enzyme activity accumulated in substantial amounts in dark-grown pine seedlings. The presence of a functional Fd-GOGAT may be important to provide the required glutamate for the biosynthesis of nitrogen compounds during chloroplast biogenesis in the dark.

The occurrence of glutamine synthetase isoenzymes in tomato plants is correlated to plastid differentiation

Summary Two glutamine synthetase (GS; EC 6.3.1.2) isoenzymes, GS1 and GS2, have been separated by ion exchange chromatography from several tissues of tomato plants, a typical specie containing only chloroplastic GS in its leaves. In etiolated seedlings, as occurs in other plants, only GSl was detected in roots and a similar isoenzyme represented the major form in cotyledons. After 40 h of continuous light exposure the isoform pattern changed and GS2 replaced GS1 in the roots; meanwhile both isoenzymes showed similar levels of activity in cotyledons. The distribution of GS isoforms in different tissues of tomato plants indicates that GS1 and GS2 can only be detected during plastid differentiation.

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 Ca2+/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.

European discussion forum on transgenic tree biosafety

37 Food and Drug Administration approval. The Aea-TMOF expressing B. bassiana strain was effective against adults and larvae, causing a decrease in fecundity and abnormal development, respectively. Whether these effects would meet the standard for commercial application is at present unknown. Further experiments examining impacts on feeding and disease transmission as well as using combinations of host molecules may lead to additional products with greater exploitability. The recent expression of a malarial sporozoiteagglutinating antibody and antimicrobial toxin in the entomopathogenic fungus M. anisopliae has expanded the utility of fungal biological control in limiting the spread of diseases15. In theory, the approach described in this report can be combined with the expression of such factors, leading to biopesticides with greater efficacy, specificity and safety. Even so, concerns regarding the field application and release of transgenic organisms and the constraints to adoption, whether economic or related to efficacy, warrant further examination.

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

Main conclusionA 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.Abstractγ-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.