Unraveling the involvement of ABA in the water deficit-induced modulation of nitrogen metabolism in Medicago truncatula seedlings

Effects of water deficit and/or abscisic acid (ABA) were investigated on early seedling growth of Medicago truncatula, and on glutamate metabolism under dark conditions. Water deficit (simulated by polyethylene glycol, PEG), ABA and their combination resulted in a reduction in growth rate of the embryo axis, and also in a synergistic increase of free amino acid (AA) content. However, the inhibition of water uptake retention induced by water deficit seemed to occur in an ABA-independent manner. Expression of several genes involved in glutamate metabolism was induced during water deficit, whereas ABA, in combination or not with PEG, repressed them. The only exception came from a gene encoding 1-pyrroline-5-carboxylate synthetase (P5CS) which appeared to be induced in an ABA-dependent manner under water deficit. Our results demonstrate clearly the involvement of an ABA-dependent and an ABA-independent regulatory system, governing growth and glutamate metabolism under water deficit

cDNA-AFLP profiling in the embryo axes during common bean germination

cDNA-AFLP fingerprinting was used to identify genes with modulated expression during germination in common bean (Phaseolus vulgaris L.). The analysis was performed on the embryo axes. Nine time points covering the whole germination were considered and 800 transcript-derived fragments (PvTDFs) were scored. Among them, 80 % showed no changes during germination. The 97 PvTDFs showing differential expressions during germination were sequenced along with 14 constant transcripts that were randomly chosen. The expression of seven variable PvTDFs was confirmed by real-time RT-PCR. We observed that 92 % of the transcript changes, including 35 % of appearing mRNAs, took place before radicule protrusion, 0–17 h after imbibition (HAI). A major shift in gene expression was observed between 9 and 14 HAI, suggesting a key moment of cell re-programming. Sequence homologies were found for 52 % of the sequenced PvTDFs. The identified transcripts encode proteins belonging to several functional groups including transcription factors, proteins involved in storage compound hydrolysis, cell elongation or oxidative stress protection

Differential regulation of alanine aminotransferase homologues by abiotic stresses in wheat (Triticum aestivum L.) seedlings

Wheat (Triticum aestivum L.) seedlings contain four alanine aminotransferase (AlaAT) homologues. Two of them encode AlaAT enzymes, whereas two homologues act as glumate:glyoxylate aminotransferase (GGAT). To address the function of the distinct AlaAT homologues a comparative examination of the changes in transcript level together with the enzyme activity and alanine and glutamate content in wheat seedlings subjected to low oxygen availability, nitrogen and light deficiency has been studied. Shoots of wheat seedlings were more tolerant to hypoxia than the roots as judging on the basis of enzyme activity and transcript level. Hypoxia induced AlaAT1 earlier in roots than in shoots, while AlaAT2 and GGAT were unaffected. The increase in AlaAT activity lagged behind the increase in alanine content. Nitrogen deficiency has little effect on the activity of GGAT. In contrast, lower activity of AlaAT and the level of mRNA for AlaAT1 and AlaAT2 in wheat seedlings growing on a nitrogen-free medium seems to indicate that AlaAT is regulated by the availability of nitrogen. Both AlaAT and GGAT activities were present in etiolated wheat seedlings but their activity was half of that observed in light-grown seedlings. Exposure of etiolated seedlings to light caused an increase in enzyme activities and up-regulated GGAT1. It is proposed that hypoxia-induced AlaAT1 and light-induced peroxisomal GGAT1 appears to be crucial for the regulation of energy availability in plants grown under unfavourable environmental conditions

Nitrogen metabolism responses to water deficit act through both abscisic acid (ABA)-dependent and independent pathways in Medicago truncatula during post-germination

The modulation of primary nitrogen metabolism by water deficit through ABA-dependent and ABA-independent pathways was investigated in the model legume Medicago truncatula. Growth and glutamate metabolism were followed in young seedlings growing for short periods in darkness and submitted to a moderate water deficit (simulated by polyethylene glycol; PEG) or treated with ABA. Water deficit induced an ABA accumulation, a reduction of axis length in an ABA-dependent manner, and an inhibition of water uptake/retention in an ABA-independent manner. The PEG-induced accumulation of free amino acids (AA), principally asparagine and proline, was mimicked by exogenous ABA treatment. This suggests that AA accumulation under water deficit may be an ABA-induced osmolyte accumulation contributing to osmotic adjustment. Alternatively, this accumulation could be just a consequence of a decreased nitrogen demand caused by reduced extension, which was triggered by water deficit and exogenous ABA treatment. Several enzyme activities involved in glutamate metabolism and genes encoding cytosolic glutamine synthetase (GS1b; EC 6.3.1.2.), glutamate dehydrogenase (GDH3; EC 1.4.1.1.), and asparagine synthetase (AS; EC 6.3.1.1.) were up-regulated by water deficit but not by ABA, except for a gene encoding Δ1-pyrroline-5-carboxylate synthetase (P5CS; EC not assigned). Thus, ABA-dependent and ABA-independent regulatory systems would seem to exist, differentially controlling development, water content, and nitrogen metabolism under water deficit

Analysis of alanine aminotransferase in various organs of soybean (Glycine max) and in dependence of different nitrogen fertilisers during hypoxic stress

Alanine aminotransferase (AlaAT) catalyses the reversible conversion of pyruvate and glutamate into alanine and oxoglutarate. In soybean, two subclasses were identified, each represented by two highly similar members. To investigate the role of AlaAT during hypoxic stress in soybean, changes in transcript level of both subclasses were analysed together with the enzyme activity and alanine content of the tissue. Moreover, the dependency of AlaAT activity and gene expression was investigated in relation to the source of nitrogen supplied to the plants. Using semi-quantitative PCR, GmAlaAT genes were determined to be highest expressed in roots and nodules. Under normal growth conditions, enzyme activity of AlaAT was detected in all organs tested, with lowest activity in the roots. Upon waterlogging-induced hypoxia, AlaAT activity increased strongly. Concomitantly, alanine accumulated. During re-oxygenation, AlaAT activity remained high, but the transcript level and the alanine content decreased. Our results show a role for AlaAT in the catabolism of alanine during the initial period of re-oxygenation following hypoxia. GmAlaAT also responded to nitrogen availability in the solution during waterlogging. Ammonium as nitrogen source induced both gene expression and enzyme activity of AlaAT more than when nitrate was supplied in the nutrient solution. The work presented here indicates that AlaAT might not only be important during hypoxia, but also during the recovery phase after waterlogging, when oxygen is available to the tissue again

Tuning the thermoelectric properties of A-site deficient SrTiO3 ceramics by vacancies and carrier concentration

Ceramics based on Sr0.8La0.067Ti0.8Nb0.2O3-δ have been prepared by the mixed oxide route. The La1/3NbO3 component generates ∼13.4% A-site vacancies; this was fixed for all samples. Powders were sintered under air and reducing conditions at 1450 to 1700 K; products were of high density (>90% theoretical). Processing under reducing conditions led to the formation of a Ti1-xNbxO2-y second phase, core-shell structures and oxygen deficiency. X-ray diffraction (XRD) confirmed a simple cubic structure with space group Pm3[combining macron]m. Transmission electron microscopy revealed a high density of dislocations while analytical scanning transmission electron microscopy at atomic resolution demonstrated a uniform distribution of La, Nb and vacancies in the lattice. X-ray photoemission spectroscopy and thermogravimetry showed the oxygen deficiency (δ value) to be ∼0.08 in reduced samples with enhanced carrier concentrations ∼2 × 1021 cm-3. Both carrier concentration and carrier mobility increased with sintering time, giving a maximum figure of merit (ZT) of 0.25. Selective additional doping by La or Nb, with no additional A site vacancies, led to the creation of additional carriers and reduced electrical resistivity. Together these led to enhanced ZT values of 0.345 at 1000 K. The contributions from oxygen vacancies and charge carriers have been investigated independently

Reversible Integration of Microfluidic Devices with Microelectrode Arrays for Neurobiological Applications

The majority of current state-of-the-art microfluidic devices are fabricated via replica molding of the fluidic channels into PDMS elastomer and then permanently bonding it to a Pyrex surface using plasma oxidation. This method presents a number of problems associated with the bond strengths, versatility, applicability to alternative substrates, and practicality. Thus, the aim of this study was to investigate a more practical method of integrating microfluidics which is superior in terms of bond strengths, reversible, and applicable to a larger variety of substrates, including microfabricated devices. To achieve the above aims, a modular microfluidic system, capable of reversible microfluidic device integration, simultaneous surface patterning and multichannel fluidic perfusion, was built. To demonstrate the system’s potential, the ability to control the distribution of A549 cells inside a microfluidic channel was tested. Then, the system was integrated with a chemically patterned microelectrode array, and used it to culture primary, rat embryo spinal cord neurons in a dynamic fluidic environment. The results of this study showed that this system has the potential to be a cost effective and importantly, a practical means of integrating microfluidics. The system’s robustness and the ability to withstand extensive manual handling have the additional benefit of reducing the workload. It also has the potential to be easily integrated with alternative substrates such as stainless steel or gold without extensive chemical modifications. The results of this study are of significant relevance to research involving neurobiological applications, where primary cell cultures on microelectrode arrays require this type of flexible integrated solution

SirT3 suppresses hypoxia inducible factor 1α and tumor growth by inhibiting mitochondrial ROS production

It has become increasing clear that alterations in cellular metabolism have a key role in the generation and maintenance of cancer. Some of the metabolic changes can be attributed to the activation of oncogenes or loss of tumor suppressors. Here, we show that the mitochondrial sirtuin, SirT3, acts as a tumor suppressor via its ability to suppress reactive oxygen species (ROS) and regulate hypoxia inducible factor 1α (HIF-1α). Primary mouse embryo fibroblasts (MEFs) or tumor cell lines expressing SirT3 short-hairpin RNA exhibit a greater potential to proliferate, and augmented HIF-1α protein stabilization and transcriptional activity in hypoxic conditions. SirT3 knockdown increases tumorigenesis in xenograft models, and this is abolished by giving mice the anti-oxidant N-acetyl cysteine. Moreover, overexpression of SirT3 inhibits stabilization of HIF-1α protein in hypoxia and attenuates increases in HIF-1α transcriptional activity. Critically, overexpression of SirT3 decreases tumorigenesis in xenografts, even when induction of the sirtuin occurs after tumor initiation. These data suggest that SirT3 acts to suppress the growth of tumors, at least in part through its ability to suppress ROS and HIF-1α