Jose Luis Ortega

Biochemical and molecular characterization of transgenic Lotus japonicus plants constitutively over-expressing a cytosolic glutamine synthetase gene

Higher plants assimilate nitrogen in the form of ammonia through the concerted activity of glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS enzyme is either located in the cytoplasm (GS1) or in the chloroplast (GS2). To understand how modulation of GS activity affects plant performance, Lotus japonicus L. plants were transformed with an alfalfa GS1 gene driven by the CaMV 35S promoter. The transformants showed increased GS activity and an increase in GS1 polypeptide level in all the organs tested. GS was analyzed by non-denaturing gel electrophoresis and ion-exchange chromatography. The results showed the presence of multiple GS isoenzymes in the different organs and the presence of a novel isoform in the transgenic plants. The distribution of GS in the different organs was analyzed by immunohistochemical localization. GS was localized in the mesophyll cells of the leaves and in the vasculature of the stem and roots of the transformants. Our results consistently showed higher soluble protein concentration, higher chlorophyll content and a higher biomass accumulation in the transgenic plants. The total amino acid content in the leaves and stems of the transgenic plants was 22–24% more than in the tissues of the non-transformed plants. The relative abundance of individual amino acid was similar except for aspartate/asparagine and proline, which were higher in the transformants.

The 5′ untranslated region of the soybean cytosolic glutamine synthetase β1 gene contains prokaryotic translation initiation signals and acts as a translational enhancer in plants

Glutamine synthetase (GS) catalyzes the synthesis of glutamine from glutamate and ammonia. In plants, it occurs as two major isoforms, a cytosolic form (GS1) and a nuclear encoded chloroplastic form. The focus of this paper is to determine the role of the 5′UTR of a GS1 gene. GS1 gene constructs with and without its 5′ and 3′ UTRs, driven by a constitutive promoter, were agroinfiltrated into tobacco leaves and the tissues were analyzed for both transgene transcript and protein accumulation. The constructs were also tested in an in vitro transcription/translation system and in Escherichia coli. Our results showed that while the 3′UTR functioned in the destabilization of the transcript, the 5′UTR acted as a translation enhancer in plant cells but not in the in vitro translation system. The 5′UTR of the GS1 gene when placed in front of a reporter gene (uidA), showed a 20-fold increase in the level of GUS expression in agroinfiltrated leaves when compared to the same gene construct without the 5′UTR. The 5′UTR-mediated translational enhancement is probably another step in the regulation of GS in plants. The presence of the GS1 5′UTR in front of the GS1 coding region allowed for its translation in E. coli suggesting the commonality of the translation initiation mechanism for this gene between plants and bacteria.

Nodule-enhanced expression of a sucrose phosphate synthase gene member (MsSPSA) has a role in carbon and nitrogen metabolism in the nodules of alfalfa (Medicago sativa L.)

Sucrose phosphate synthase (SPS) catalyzes the first step in the synthesis of sucrose in photosynthetic tissues. We characterized the expression of three different isoforms of SPS belonging to two different SPS gene families in alfalfa (Medicago sativa L.), a previously identified SPS (MsSPSA) and two novel isoforms belonging to class B (MsSPSB and MsSPSB3). While MsSPSA showed nodule-enhanced expression, both MsSPSB genes exhibited leaf-enhanced expression. Alfalfa leaf and nodule SPS enzymes showed differences in chromatographic and electrophoretic migration and differences in Vmax and allosteric regulation. The root nodules in legume plants are a strong sink for photosynthates with its need for ATP, reducing power and carbon skeletons for dinitrogen fixation and ammonia assimilation. The expression of genes encoding SPS and other key enzymes in sucrose metabolism, sucrose phosphate phosphatase and sucrose synthase, was analyzed in the leaves and nodules of plants inoculated with Sinorhizobium meliloti. Based on the expression pattern of these genes, the properties of the SPS isoforms and the concentration of starch and soluble sugars in nodules induced by a wild type and a nitrogen fixation deficient strain, we propose that SPS has an important role in the control of carbon flux into different metabolic pathways in the symbiotic nodules.

Transgenic alfalfa (Medicago sativa) with increased sucrose phosphate synthase activity shows enhanced growth when grown under N2-fixing conditions.

AbstractMain conclusionOverexpression of SPS in alfalfa is accompanied by early flowering, increased plant growth and an increase in elemental N and protein content when grown under N2-fixing conditions. Sucrose phosphate synthase (SPS; EC 2.3.1.14) is the key enzyme in the synthesis of sucrose in plants. The outcome of overexpression of SPS in different plants using transgenic approaches has been quite varied, but the general consensus is that increased SPS activity is associated with the production of new sinks and increased sink strength. In legumes, the root nodule is a strong C sink and in this study our objective was to see how increasing SPS activity in a legume would affect nodule number and function. Here we have transformed alfalfa (Medicago sativa, cv. Regen SY), with a maize SPS gene driven by the constitutive CaMV35S promoter. Our results showed that overexpression of SPS in alfalfa, is accompanied by an increase in nodule number and mass and an overall increase in nitrogenase activity at the whole plant level. The nodules exhibited an increase in the level of key enzymes contributing to N assimilation including glutamine synthetase and asparagine synthetase. Moreover, the stems of the transformants showed higher level of the transport amino acids, Asx, indicating increased export of N from the nodules. The transformants exhibited a dramatic increase in growth both of the shoots and roots, and earlier flowering time, leading to increased yields. Moreover, the transformants showed an increase in elemental N and protein content. The overall conclusion is that increased SPS activity improves the N status and plant performance, suggesting that the availability of more C in the form of sucrose enhances N acquisition and assimilation in the nodules.

An intragenic approach to confer glyphosate resistance in chile (Capsicum annuum) by introducing an in vitro mutagenized chile EPSPS gene encoding for a glyphosate resistant EPSPS protein

Chile pepper (Capsicum annuum) is an important high valued crop worldwide, and when grown on a large scale has problems with weeds. One important herbicide used is glyphosate. Glyphosate inactivates the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the synthesis of aromatic amino acids. A transgenic approach towards making glyphosate resistant plants, entails introducing copies of a gene encoding for glyphosate-resistant EPSPS enzyme into the plant. The main objective of our work was to use an intragenic approach to confer resistance to glyphosate in chile which would require using only chile genes for transformation including the selectable marker. Tobacco was used as the transgenic system to identify different gene constructs that would allow for the development of the intragenic system for chile, since chile transformation is inefficient. An EPSPS gene was isolated from chile and mutagenized to introduce substitutions that are known to make the encoded enzyme resistant to glyphosate. The promoter for EPSPS gene was isolated from chile and the mutagenized chile EPSPS cDNA was engineered behind both the CaMV35S promoter and the EPSPS promoter. The leaves from the transformants were checked for resistance to glyphosate using a cut leaf assay. In tobacco, though both gene constructs exhibited some degree of resistance to glyphosate, the construct with the CaMV35S promoter was more effective and as such chile was transformed with this gene construct. The chile transformants showed resistance to low concentrations of glyphosate. Furthermore, preliminary studies showed that the mutated EPSPS gene driven by the CaMV35S promoter could be used as a selectable marker for transformation. We have shown that an intragenic approach can be used to confer glyphosate-resistance in chile. However, we need a stronger chile promoter and a mutated chile gene that encodes for a more glyphosate resistant EPSPS protein.

THU0179 Prevalence, etiology and risk factors for pulmonary arterial hypertension in a cohort of patients with lupus

Background Pulmonary hypertension (PHT) has been reported between 0.5 and 14% in systemic lupus erythematosus (SLE), but some studies have suggested that a mild degree of PHT may be more common. Only a few studies have been carried out in caucasian using an algorithm for systematic evaluation with confirmation by right heart catheterization (RHC). Objectives To assess the prevalence, etiology and risk factors for PHT in a monocentric SLE cohort. Methods Prospective cross-sectional study of 158 SLE patients (ACR 1997 criteria). Resting doppler echocardiographic (dEcho), diffusing capacity for carbon monoxide (DLCO), NT-proBNP and Borg scale (for dyspnea) was performed in all patients. An exercise test (ET) was conducted in selected patients. We confirm PHT using RHC in the case of sPAP ≥45 mmHg (dEcho) or a +ET (>20mmHg increase in PAPs), excluding patients with obvious cardiac disease. A mean pulmonary pressure >20 mm Hg was accepted as PHT. Cumulative clinical characteristics were collected and damage (SLICC/ACR/DI) (SDI) and severity (Katz index) (IGK) were calculated. Results Mean age: 45 (±12.9) years,94.3% females. Mean SLE duration: 14 (±8.0) years; mean SDI:1.1 (±1.6), IGK: 3.3 (±2.25), smokers any time: 37.2%. 21 patients (13.4%) had dyspnea at the evaluation time. 11 patients (6.9%) were found to have any degree of PAH. Etiologic classification (Dana Point 2008): 3 patients have left cardiac disease and the diagnosis was echocardiographic-based (sPAP>35mmHg), 1 patient had resting and 3 had postcapillary PAH on ET, a total of 7 patients in group 2. 1 patient had thromboembolic d. (group 4). 3 patients (2.5%) had precapillary PAH related with SLE (group 1), two of them had been previously diagnosed. Other conditions associated with PAH were not found. All 11 patients (100%) with PAH had dyspnea vs. 10 of 146 (6.8%) without PAH (p<0.001). PAH patients had more impaired DLCO when comparing with patients without PHT: 59% (±14%) vs. 68% (±15%) (p=0.048). 54% of PAH patients vs 4.1% of controls had Nt-proBNP >395, (p=0.000). Difference in medium Nt-proBNP (8685±663 vs. 191±578), p=0.000 (U Mann-Whitney) was also statistically significant. As expected, cardiac disease was more prevalent among PAH patients: 27.3 vs. 6.8%, p=0.05. There were no differences in SLE clinical characteristics between SLE patients with vs. without PHT. Conclusions Our data confirms the low prevalence of precapillary PHT in SLE. We found a clear preponderance of cardiac etiology, mainly detected using exercise testing. A HTP screening program based on echocardiography, Nt-proBNP and DLCO not seem to be cost-effective and should be restricted to SLE patients with unexplained dyspnea. Disclosure of Interest None Declared

Constitutive and Nodule-Specific Overexpression of Cytosolic Glutamine Synthetase (GS1) Genes in Alfalfa

Glutamine synthetase (GS) catalyzes the first and key reaction in the assimilation of ammonia. Gene constructs consisting of the CaMV 35S promoter driving either a soybean GS1, or an alfalfa GS1, gene have been introduced into alfalfa. Northern analysis of RNA isolated from leaves and nodules of nodulated N2-fixing plants showed significant accumulation of the transcript for the GS1, transgene only in the leaves but not in the nodules. However, significant amount of GUS activity could be detected in nodules of plants containing the CaMV 35S promoter-GUS fusion construct. This would suggest that the transcript for the GS1, transgene is not stable in the nitrogen fixing root nodules. Transformed nonnodulated alfalfa plants when grown in the presence of KNO3, showed a significant decrease in the level of the transcript for the trans-gene when compared to the N-fed plants. The results suggest that a product of GS activity might have a role in destabilizing GS transcript level.