Effects of sodium arsenite on neurite outgrowth and glutamate AMPA receptor expression in mouse cortical neurons.

There has been broad concern that arsenic in the environment exerts neurotoxicity. To determine the mechanism by which arsenic disrupts neuronal development, primary cultured neurons obtained from the cerebral cortex of mouse embryos were exposed to sodium arsenite (NaAsO2) at concentrations between 0 and 2μM from days 2 to 4 in vitro and cell survival, neurite outgrowth and expression of glutamate AMPA receptor subunits were assessed at day 4 in vitro. Cell survival was significantly decreased by exposure to 2μM NaAsO2, whereas 0.5μM NaAsO2 increased cell survival instead. The assessment of neurite outgrowth showed that total neurite length was significantly suppressed by 1μM and 2μM NaAsO2, indicating that the lower concentration of NaAsO2 impairs neuritogenesis before inducing cell death. Immunoblot analysis of AMPA receptor subunit expression showed that the protein level of GluA1, a specific subunit of the AMPA receptor, was significantly decreased by 1μM and 2μM NaAsO2. When immunocytochemistry was used to confirm this effect by staining for GluA1 expression in neuropeptide Y neurons, most of which contain GluA1, GluA1 expression in neuropeptide Y neurons was found to be significantly suppressed by 1μM and 2μM NaAsO2 but to be increased at the concentration of 0.5μM. Finally, to determine whether neurons could be rescued from the NaAsO2-induced impairment of neuritogenesis by compensatory overexpression of GluA1, we used primary cultures of neurons transfected with a plasmid vector to overexpress either GluA1 or GluA2, and the results showed that GluA1/2 overexpression protected against the deleterious effects of NaAsO2 on neurite outgrowth. These results suggest that the NaAsO2 concentration inducing neurite suppression is lower than the concentration that induces cell death and is the same as the concentration that suppresses GluA1 expression. Consequently, the suppression of GluA1 expression by NaAsO2 seems at least partly responsible for neurite suppression induced by NaAsO2.

Highly inducible synthesis of heterologous proteins in epithelial cells carrying a glucocorticoid-responsive vector.

A glucocorticoid-responsive vector is described which allows for the highly inducible expression of complementary DNAs (cDNAs) in stably transfected mammalian cell lines. This vector, pLK-neo, composed of a variant mouse mammary tumor virus long terminal repeat promoter, containing a hormone regulatory element, a Geneticin resistance-encoding gene in a simian virus 40 transcription unit, and a polylinker insertion site for heterologous cDNAs, was used to express the polymeric immunoglobulin (poly-Ig) receptor and the thymocyte marker, Thy-1, in Madin-Darby canine kidney (MDCK) cells and in murine fibroblast L cells. A high level of poly-Ig receptor or Thy-1 mRNA accumulation was observed in MDCK cells in response to dexamethasone with a parallel ten- to 200-fold increase in protein synthesis depending on the recombinant protein and the transfected cell clone.

Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set.

Plant membrane compartments and trafficking pathways are highly complex, and are often distinct from those of animals and fungi. Progress has been made in defining trafficking in plants using transient expression systems. However, many processes require a precise understanding of plant membrane trafficking in a developmental context, and in diverse, specialized cell types. These include defense responses to pathogens, regulation of transporter accumulation in plant nutrition or polar auxin transport in development. In all of these cases a central role is played by the endosomal membrane system, which, however, is the most divergent and ill-defined aspect of plant cell compartmentation. We have designed a new vector series, and have generated a large number of stably transformed plants expressing membrane protein fusions to spectrally distinct, fluorescent tags. We selected lines with distinct subcellular localization patterns, and stable, non-toxic expression. We demonstrate the power of this multicolor 'Wave' marker set for rapid, combinatorial analysis of plant cell membrane compartments, both in live-imaging and immunoelectron microscopy. Among other findings, our systematic co-localization analysis revealed that a class of plant Rab1-homologs has a much more extended localization than was previously assumed, and also localizes to trans-Golgi/endosomal compartments. Constructs that can be transformed into any genetic background or species, as well as seeds from transgenic Arabidopsis plants, will be freely available, and will promote rapid progress in diverse areas of plant cell biology.

Selective and powerful stress gene expression in Arabidopsis in response to malondialdehyde.

The provenance, half-life and biological activity of malondialdehyde (MDA) were investigated in Arabidopsis thaliana. We provide genetic confirmation of the hypothesis that MDA originates from fatty acids containing more than two methylene-linked double bonds, showing that tri-unsaturated fatty acids are the in vivo source of up to 75% of MDA. The abundance of the combined pool of free and reversibly bound MDA did not change dramatically in stress, although a significant increase in the free MDA pool under oxidative conditions was observed. The half-life of infiltrated MDA indicated rapid metabolic turnover/sequestration. Exposure of plants to low levels of MDA using a recently developed protocol powerfully upregulated many genes on a cDNA microarray with a bias towards those implicated in abiotic/environmental stress (e.g. ROF1 and XERO2). Remarkably, and in contrast to the activities of other reactive electrophile species (i.e. small vinyl ketones), none of the pathogenesis-related (PR) genes tested responded to MDA. The use of structural mimics of MDA isomers suggested that the propensity of the molecule to act as a cross-linking/modifying reagent might contribute to the activation of gene expression. Changes in the concentration/localisation of unbound MDA in vivo could strongly affect stress-related transcription.

Insect eggs suppress plant defence against chewing herbivores.

Plants activate direct and indirect defences in response to insect egg deposition. However, whether eggs can manipulate plant defence is unknown. In Arabidopsis thaliana, oviposition by the butterfly Pieris brassicae triggers cellular and molecular changes that are similar to the changes caused by biotrophic pathogens. In the present study, we found that the plant defence signal salicylic acid (SA) accumulates at the site of oviposition. This is unexpected, as the SA pathway controls defence against fungal and bacterial pathogens and negatively interacts with the jasmonic acid (JA) pathway, which is crucial for the defence against herbivores. Application of P. brassicae or Spodoptera littoralis egg extract onto leaves reduced the induction of insect-responsive genes after challenge with caterpillars, suggesting that egg-derived elicitors suppress plant defence. Consequently, larval growth of the generalist herbivore S. littoralis, but not of the specialist P. brassicae, was significantly higher on plants treated with egg extract than on control plants. In contrast, suppression of gene induction and enhanced S. littoralis performance were not seen in the SA-deficient mutant sid2-1, indicating that it is SA that mediates this phenomenon. These data reveal an intriguing facet of the cross-talk between SA and JA signalling pathways, and suggest that insects have evolved a way to suppress the induction of defence genes by laying eggs that release elicitors. We show here that egg-induced SA accumulation negatively interferes with the JA pathway, and provides an advantage for generalist herbivores.

Over-expression of PHO1 in Arabidopsis leaves reveals its role in mediating phosphate efflux.

Inorganic phosphate (Pi) homeostasis in multi-cellular eukaryotes depends not only on Pi influx into cells, but also on Pi efflux. Examples in plants for which Pi efflux is crucial are transfer of Pi into the xylem of roots and release of Pi at the peri-arbuscular interface of mycorrhizal roots. Despite its importance, no protein has been identified that specifically mediates phosphate efflux either in animals or plants. The Arabidopsis thaliana PHO1 gene is expressed in roots, and was previously shown to be involved in long-distance transfer of Pi from the root to the shoot. Here we show that PHO1 over-expression in the shoot of A. thaliana led to a two- to threefold increase in shoot Pi content and a severe reduction in shoot growth. (31) P-NMR in vivo showed a normal initial distribution of intracellular Pi between the cytoplasm and the vacuole in leaves over-expressing PHO1, followed by a large efflux of Pi into the infiltration medium, leading to a rapid reduction of the vacuolar Pi pool. Furthermore, the Pi concentration in leaf xylem exudates from intact plants was more than 100-fold higher in PHO1 over-expressing plants compared to wild-type. Together, these results show that PHO1 over-expression in leaves leads to a dramatic efflux of Pi out of cells and into the xylem vessel, revealing a crucial role for PHO1 in Pi efflux.

The cuticle : not only a barrier for plant defence: A novel defence syndrome in plants with cuticular defects.

The cuticle is a physical barrier that prevents water loss and protects against irradiation, xenobiotics and pathogens. This classic textbook statement has recently been revisited and several observations were made showing that this dogma falls short of being universally true. Both transgenic Arabidopsis thaliana lines expressing cell wall-targeted fungal cutinase (so-called CUTE plants) or lipase as well as several A. thaliana mutants with altered cuticular structure remained free of symptoms after an inoculation with Botrytis cinerea. The alterations in cuticular structure lead to the release of fungitoxic substances and changes in gene expression that form a multifactorial defence response. Several models to explain this syndrome are discussed.

Bone anabolic potential of soy isoflavones and plant extracts and genomic changes during differentiation of hPOB-tert osteoblast-like cells

Summary For the nutritional management of bone health and the prevention of osteoporosis it is important to identify nutrients that positively influence the bone remodeling process at the cellular level. Soy isoflavones show promising osteoprotective effects in animals and humans but their mechanism of action in bone cells is yet poorly understood. Firstly, soy tissue cultures were characterized as a new and optimized source of isoflavones. A large variability in the isoflavone content was observed and high-producing strains (46.3 mg/g dry wt isoflavones) were identified. In the Ishikawa cells bioassay, the estrogenicity of isoflavones was confirmed to be 1000 to 10000 less than 17Mestradiol and that of the malonyl forms was shown for the first time (EC50 of 350 nM and 1880 nM for malonylgenistin and malonyldaidzin, respectively). The estrogenic activity of soya tissue culture extracts correlated to their isoflavone content. Secondly, the effects of phytonutrients on BMP-2 gene expression and on the mevalonate synthesis pathway, as key mediators of bone formation, were investigated. Dietary achievable concentrations of genistein and daidzein (10vM), and statins (4xM) but not 17M estradiol (10nM), induced BMP-2 gene expression (by up to 3-fold) and inhibited the cholesterol biosynthetic pathway (by up to 50%) in the human osteoblastic cell line hP0B¬tert. In addition, several plant extracts (Cyperus rotundus, Lindera benzoin and Cnidium monnieri) induced BMP-2 gene expression but this induction was not restricted to the inhibition of the cholesterol synthesis pathway neither to the estrogenicity. Finally, the gene expression profiles during hP0B-tert differentiation induced by vitamin D and dexamethasone were analyzed with the Affymetrix human GeneChip. 1665 different genes and 98 ESTs were significantly regulated. The expression profiles of bone-related genes was largely in agreement with previously documented patterns, supporting the physiological relevance of the genomic results and the hP0B-tert cell line as a valid model of human osteoblast differentiation. The expression of alternative differentiation markers during the osteogenic treatment of hP0B-tert cells indicated that the adipocyte and myoblast differentiation pathways were repressed, confirming that these culture conditions allowed only osteoblast differentiation. The gene ontology analysis identified further sub-groups of genes that may be involved in the bone formation process. Aims of the thesis In order to define new strategies for the nutritional management of bone health and for the prevention of osteoporosis the major goal of the present work was to investigate the potential of phytonutrients to positively modulate the bone formation process at the cellular level and, in particular: 1.To select and optimise alternative plant sources containing high levels of isoflavones with estrogenic activity (Chapter 3). 2.To compare the effects of statins and phytonutrients on BMP-2 gene expression and on the mevalonate synthesis pathway and to select new plant extracts with a bone anabolic potential (Chapter 4). 3.To further characterize the new human periosteal cell line, hP0B-tert, as a bone- formation model, by elucidating its gene expression profile during differentiation induced by vitamin D and dexamethasone (Chapter 5).

Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis.

Wounding in multicellular eukaryotes results in marked changes in gene expression that contribute to tissue defense and repair. Using a cDNA microarray technique, we analyzed the timing, dynamics, and regulation of the expression of 150 genes in mechanically wounded leaves of Arabidopsis. Temporal accumulation of a group of transcripts was correlated with the appearance of oxylipin signals of the jasmonate family. Analysis of the coronatine-insensitive coi1-1 Arabidopsis mutant that is also insensitive to jasmonate allowed us to identify a large number of COI1-dependent and COI1-independent wound-inducible genes. Water stress was found to contribute to the regulation of an unexpectedly large fraction of these genes. Comparing the results of mechanical wounding with damage by feeding larvae of the cabbage butterfly (Pieris rapae) resulted in very different transcript profiles. One gene was specifically induced by insect feeding but not by wounding; moreover, there was a relative lack of water stress-induced gene expression during insect feeding. These results help reveal a feeding strategy of P. rapae that may minimize the activation of a subset of water stress-inducible, defense-related genes.

Plasma membrane cyclic nucleotide gated calcium channels control land plant thermal sensing and acquired thermotolerance.

Typically at dawn on a hot summer day, land plants need precise molecular thermometers to sense harmless increments in the ambient temperature to induce a timely heat shock response (HSR) and accumulate protective heat shock proteins in anticipation of harmful temperatures at mid-day. Here, we found that the cyclic nucleotide gated calcium channel (CNGC) CNGCb gene from Physcomitrella patens and its Arabidopsis thaliana ortholog CNGC2, encode a component of cyclic nucleotide gated Ca(2+) channels that act as the primary thermosensors of land plant cells. Disruption of CNGCb or CNGC2 produced a hyper-thermosensitive phenotype, giving rise to an HSR and acquired thermotolerance at significantly milder heat-priming treatments than in wild-type plants. In an aequorin-expressing moss, CNGCb loss-of-function caused a hyper-thermoresponsive Ca(2+) influx and altered Ca(2+) signaling. Patch clamp recordings on moss protoplasts showed the presence of three distinct thermoresponsive Ca(2+) channels in wild-type cells. Deletion of CNGCb led to a total absence of one and increased the open probability of the remaining two thermoresponsive Ca(2+) channels. Thus, CNGC2 and CNGCb are expected to form heteromeric Ca(2+) channels with other related CNGCs. These channels in the plasma membrane respond to increments in the ambient temperature by triggering an optimal HSR, leading to the onset of plant acquired thermotolerance.

The fou2 gain-of-function allele and the wild-type allele of Two Pore Channel 1 contribute to different extents or by different mechanisms to defense gene expression in Arabidopsis.

The fatty acid oxygenation up-regulated 2 (fou2) mutant in Arabidopsis thaliana creates a gain-of-function allele in a non-selective cation channel encoded by the Two Pore Channel 1 (TPC1) gene. This mutant genetically implicates cation fluxes in the control of the positive feedback loop whereby jasmonic acid (JA) stimulates its own synthesis. In this study we observed extensive transcriptome reprogramming in healthy fou2 leaves closely resembling that induced by treatment with methyl jasmonate, biotic stresses and the potassium starvation response. Proteomic analysis of fou2 leaves identified increased levels of seven biotic stress- and JA-inducible proteins. In agreement with these analyses, epistasis studies performed by crossing fou2 with aos indicated that elevated levels of JA in fou2 are the major determinant of the mutant phenotype. In addition, generation of fou2 aba1-5, fou2 etr1-1 and fou2 npr1-1 double mutants showed that the fou2 phenotype was only weakly affected by ABA levels and unaffected by mutations in NPR1 and ETR1. The results now suggest possible mechanisms whereby fou2 could induce JA synthesis/signaling early in the wound response. In contrast to fou2, transcriptome analysis of a loss-of-function allele of TPC1, tpc1-2, revealed no differential expression of JA biosynthesis genes in resting leaves. However, the analysis disclosed reduced mRNA levels of the pathogenesis-related genes PDF1.2a and THI2.1 in healthy and diseased tpc1-2 leaves. The results suggest that wild-type TPC1 contributes to their expression by mechanisms somewhat different from those affecting their expression in fou2.

Jasmonates and related oxylipins in plant responses to pathogenesis and herbivory.

The vigorous production of oxygenated fatty acids (oxylipins) is a characteristic response to pathogenesis and herbivory, and is often accompanied by the substantial release of small and reactive lipid-fragmentation products. Some oxylipins, most notably those of the jasmonate family, have key roles as potent regulators. Recent advances have been made in understanding oxylipin-regulated signal transduction in response to attack. Much jasmonate signaling takes place via a genetically defined signal network that is linked to the ethylene, auxin, and salicylic acid signal pathways, but a second aspect of jasmonate signaling is emerging. Some jasmonates and several newly discovered cyclopentenone lipids can activate or repress gene expression through the activities of a conserved electrophilic atom group.

A rice cis-natural antisense RNA acts as a translational enhancer for its cognate mRNA and contributes to phosphate homeostasis and plant fitness.

cis-natural antisense transcripts (cis-NATs) are widespread in plants and are often associated with downregulation of their associated sense genes. We found that a cis-NAT positively regulates the level of a protein critical for phosphate homeostasis in rice (Oryza sativa). PHOSPHATE1;2 (PHO1;2), a gene involved in phosphate loading into the xylem in rice, and its associated cis-NATPHO1;2 are both controlled by promoters active in the vascular cylinder of roots and leaves. While the PHO1;2 promoter is unresponsive to the plant phosphate status, the cis-NATPHO1;2 promoter is strongly upregulated under phosphate deficiency. Expression of both cis-NATPHO1;2 and the PHO1;2 protein increased in phosphate-deficient plants, while the PHO1;2 mRNA level remained stable. Downregulation of cis-NATPHO1;2 expression by RNA interference resulted in a decrease in PHO1;2 protein, impaired the transfer of phosphate from root to shoot, and decreased seed yield. Constitutive overexpression of NATPHO1;2 in trans led to a strong increase of PHO1;2, even under phosphate-sufficient conditions. Under all conditions, no changes occurred in the level of expression, sequence, or nuclear export of PHO1;2 mRNA. However, expression of cis-NATPHO1;2 was associated with a shift of both PHO1;2 and cis-NATPHO1;2 toward the polysomes. These findings reveal an unexpected role for cis-NATPHO1;2 in promoting PHO1;2 translation and affecting phosphate homeostasis and plant fitness.

Analysis of HIV-1 expression level and sense of transcription by high-throughput sequencing of the infected cell.

Next-generation sequencing offers an unprecedented opportunity to jointly analyze cellular and viral transcriptional activity without prerequisite knowledge of the nature of the transcripts. SupT1 cells were infected with a vesicular stomatitis virus G envelope protein (VSV-G)-pseudotyped HIV vector. At 24 h postinfection, both cellular and viral transcriptomes were analyzed by serial analysis of gene expression followed by high-throughput sequencing (SAGE-Seq). Read mapping resulted in 33 to 44 million tags aligning with the human transcriptome and 0.23 to 0.25 million tags aligning with the genome of the HIV-1 vector. Thus, at peak infection, 1 transcript in 143 is of viral origin (0.7%), including a small component of antisense viral transcription. Of the detected cellular transcripts, 826 (2.3%) were differentially expressed between mock- and HIV-infected samples. The approach also assessed whether HIV-1 infection modulates the expression of repetitive elements or endogenous retroviruses. We observed very active transcription of these elements, with 1 transcript in 237 being of such origin, corresponding on average to 123,123 reads in mock-infected samples (0.40%) and 129,149 reads in HIV-1-infected samples (0.45%) mapping to the genomic Repbase repository. This analysis highlights key details in the generation and interpretation of high-throughput data in the setting of HIV-1 cellular infection.

Subcellular localization, distribution and expression of calmodulin in Zea mays roots.

The subcellular localization, distribution and the steady state level of calmodulin from maize roots (Zea mays L., cv. LG 11) were studied. To analyze the subcellular localization, 2-day old root membranes were fractionated by sucrose density gradient centrifugation and immunoblotting was done with an antibody raised against a vertebrate calmodulin (SWant) which recognized the plant calmodulin. Calmodulin was principally associated with high density fractions and particularly plasmalemma. For studying the distribution of calmodulin in various zones of Zea mays roots, a micro method of membrane preparation was developed. Most of the calmodulin was present in microsomes isolated from the root apex corresponding to the first 4 mm of a 15 +/- 2 mm root. An identical distribution was found by studying the steady state level of the protein by Northern blotting using a cDNA clone of Zea mays calmodulin.