Metabolic and genetic perturbations accompany the modification of galactomannan in seeds of Medicago truncatula expressing mannan synthase from guar (Cyamopsis tetragonoloba L.)

Galactomannan gums are widely used in the food and oil industries, and there is considerable interest in applying biotechnological approaches to improve their physical properties. A mannan synthase from guar ( Cyamopsis tetragonoloba ) was expressed under the control of a bean β-phaseolin promoter in transgenic Medicago truncatula . Although the expression of exogenous mannan synthase caused a slight decrease in galactomannan levels in Medicago , the molecular weight and viscosity of the polymer were significantly increased, although the mannose to galactose ratio and degree of polydispersity remained unchanged. At the same time, expression of about 2.8% of the genes was altered significantly in the seeds of transgenic Medicago lines analysed by Affymetrix genome chip, with a particularly striking induction of putative trehalose phosphate synthase genes. Mannan synthase expression also caused large alterations in the levels of a number of sugars and sugar alcohols, suggesting that over-expression of a processive glycosyltransferase perturbs the mechanisms of sugar sensing and/or homeostasis, possibly involving signalling via trehalose-6-phosphate.     

Production of transgenic barrel medic (Medicago truncatula Gaernt.) using the ipt-type MAT vector system and impairment of Recombinase-mediated excision events

Expression of the uidA reporter gene was tested in transformation experiments of barrel medic (Medicago truncatula Gaertn.) with the ipt-type control vectors pIPT5, pIPT10 and pIPT20 and distinct in vitro culture conditions. The highest GUS expression levels were obtained with the pIPT10 construct carrying the ipt gene under the control of the native ipt promoter and using kanamycin as selective agent. The ipt-shooty transformants, characterized by the absence of both rooting ability and apical dominance associated with vitrification, were easily identified by visual selection. Using only the ipt gene as selectable marker, we obtained a stable transformation frequency of 9.8% with pIPT10 construct. The ipt-type MAT vector pEXM2 was then used to monitor the excision events mediated by the yeast Recombinase and the consequent production of ipt marker-free transgenic plants. Transgenic ipt-shooty lines were recovered at a frequency of 7.9% in the absence of kanamycin-based selection. The ipt-shooty phenotype was maintained in all the transgenic lines and no reversion to the normal phenotype occurred. PCR analysis revealed the presence of the ‘hit and run’ cassette in the genome of all the regenerated ipt-shooty lines while RT-PCR experiments confirmed the expression of the R gene, encoding the yeast Recombinase. A detailed molecular investigation, carried out to verify the integrity of the RS sites, revealed that these regions were intact in most cases. Our results with barrel medic suggest that the MAT system must be carefully evaluated and discussed on a case by case basis. L. Scaramelli, A. Balestrazzi and M. Confalonieri have contributed equally to this work.     

Enhanced levels of free and protein-bound threonine in transgenic alfalfa (Medicago sativa L.) expressing a bacterial feedback-insensitive aspartate kinase gene

Threonine, lysine, methionine, and tryptophan are essential amino acids for humans and monogastric animals. Many of the commonly used diet formulations, particularly for pigs and poultry, contain limiting amounts of these amino acids. One approach for raising the level of essential amino acids is based on altering the regulation of their biosynthetic pathways in transgenic plants. Here we describe the first production of a transgenic forage plant, alfalfa (Medicago sativa L.) with modified regulation of the aspartate-family amino acid biosynthetic pathway. This was achieved by over-expressing the Escherichia coli feedback-insensitive aspartate kinase (AK) in transgenic plants. These plants showed enhanced levels of both free and protein-bound threonine. In many transgenic plants the rise in free threonine was accompanied by a significant reduction both in aspartate and in glutamate. Our data suggest that in alfalfa, AK might not be the only limiting factor for threonine biosynthesis, and that the free threonine pool in this plant limits its incorporation into plant proteins.     

A tandem Kunitz protease inhibitor (KPI106)–serine carboxypeptidase (SCP1) controls mycorrhiza establishment and arbuscule development in Medicago truncatula

Plant proteases and protease inhibitors are involved in plant developmental processes including those involving interactions with microbes. Here we show that a tandem between a Kunitz protease inhibitor (KPI106) and a serine carboxypeptidase (SCP1) controls arbuscular mycorrhiza development in the root cortex of Medicago truncatula. Both proteins are only induced during mycorrhiza formation and belong to large families whose members are also mycorrhiza‐specific. Furthermore, the interaction between KPI106 and SCP1 analysed using the yeast two‐hybrid system is specific, indicating that each family member might have a defined counterpart. In silico docking analysis predicted a putative P1 residue in KPI106 (Lys173) that fits into the catalytic pocket of SCP1, suggesting that KPI106 might inhibit the enzyme activity by mimicking the protease substrate. In vitro mutagenesis of the Lys173 showed that this residue is important in determining the strength and specificity of the interaction. The RNA interference (RNAi) inactivation of the serine carboxypeptidase SCP1 produces aberrant mycorrhizal development with an increased number of septated hyphae and degenerate arbuscules, a phenotype also observed when overexpressing KPI106. Protease and inhibitor are both secreted as observed when expressed in Nicotiana benthamiana epidermal cells. Taken together we envisage a model in which the protease SCP1 is secreted in the apoplast where it produces a peptide signal critical for proper fungal development within the root. KPI106 also at the apoplast would modulate the spatial and/or temporal activity of SCP1 by competing with the protease substrate.     

Molecular cloning and characterization of a Candida albicans gene (EFB1) coding for the elongation factor EF-1β

Abstract The formation of H₂ by chemolithoautrophically growing Oligotropha carboxidovorans has been identified as the result of the oxidation of CO mediated by the cytoplasmic species of the molybdenum-containing CO dehydrogenase multienzyme complex as follows: CO + H ₂ O → CO ₂+ H ₂. Purified CO dehydrogenase was shown to carry hydrogen uptake and formation activities in addition to its catabolic function which is the oxidation of CO. Among the electron donors supporting H₂ formation were CO, NADH, reduced flavins and reduced viologen dyes. The reduction of protons to H₂ by cytoplasmic CO dehydrogenase is interpreted as a detoxification reaction for electrons to prevent cell damage in O. carboxidovorans .     

An (E,E)‐α‐farnesene synthase gene of soybean has a role in defence against nematodes and is involved in synthesizing insect‐induced volatiles

Plant terpene synthase genes (TPSs) have roles in diverse biological processes. Here, we report the functional characterization of one member of the soybean TPS gene family, which was designated GmAFS. Recombinant GmAFS produced in Escherichia coli catalysed the formation of a sesquiterpene (E,E)‐α‐farnesene. GmAFS is closely related to (E,E)‐α‐farnesene synthase gene from apple, both phylogenetically and structurally. GmAFS was further investigated for its biological role in defence against nematodes and insects. Soybean cyst nematode (SCN) is the most important pathogen of soybean. The expression of GmAFS in a SCN‐resistant soybean was significantly induced by SCN infection compared with the control, whereas its expression in a SCN‐susceptible soybean was not changed by SCN infection. Transgenic hairy roots overexpressing GmAFS under the control of the CaMV 35S promoter were generated in an SCN‐susceptible soybean line. The transgenic lines showed significantly higher resistance to SCN, which indicates that GmAFS contributes to the resistance of soybean to SCN. In soybean leaves, the expression of GmAFS was found to be induced by Tetranychus urticae (two‐spotted spider mites). Exogenous application of methyl jasmonate to soybean plants also induced the expression of GmAFS in leaves. Using headspace collection combined with gas chromatography–mass spectrometry analysis, soybean plants that were infested with T. urticae were shown to emit a mixture of volatiles with (E,E)‐α‐farnesene as one of the most abundant constituents. In summary, this study showed that GmAFS has defence roles in both below‐ground and above‐ground organs of soybean against nematodes and insects, respectively.     

The effects of the herbicide quinclorac on shoot growth in tomato is alleviated by inhibitors of ethylene biosynthesis and by the presence of an antisense construct to the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene in transgenic plants

Reduction of shoot growth, leaf epinasty and chlorosis in young tomato plants (Lycopersicon esculentum Mill. cv. Hellfrucht/Frühstamm) treated hydroponically with 10^-7 M of the herbicide quinclorac were partially compensated when the plants were simultaneously sprayed with salicyclic acid or the oxime ether derivative PACME. Since salicyclic acid and PACME are known inhibitors of ethylene biosynthesis, it is suggested that this pathway is implicated in quinclorac action. Further support for this hypothesis was obtained in experiments with transgenic tomato plants containing an antisense gene to 1-aminocyclopropane-1-carboxylic acid (ACC) synthase in ethylene biosynthesis. When quinclorac was applied via the root antisense plants showed reduced phenotypical alterations compared to those of wild-type plants.