Role of reactive oxygen species in the regulation of Arabidopsis seed dormancy

Freshly harvested seeds of Arabidopsis thaliana, Columbia (Col) accession were dormant when imbibed at 25°C in the dark. Their dormancy was alleviated by continuous light during imbibition or by 5 weeks of storage at 20°C (after-ripening). We investigated the possible role of reactive oxygen species (ROS) in the regulation of Col seed dormancy. After 24 h of imbibition at 25°C, non-dormant seeds produced more ROS than dormant seeds, and their catalase activity was lower. In situ ROS localization revealed that germination was associated with an accumulation of superoxide and hydrogen peroxide in the radicle. ROS production was temporally and spatially regulated: ROS were first localized within the cytoplasm upon imbibition of non-dormant seeds, then in the nucleus and finally in the cell wall, which suggests that ROS play different roles during germination. Imbibition of dormant and non-dormant seeds in the presence of ROS scavengers or donors, which inhibited or stimulated germination, respectively, confirmed the role of ROS in germination. Freshly harvested seeds of the mutants defective in catalase (cat2-1) and vitamin E (vte1-1) did not display dormancy; however, seeds of the NADPH oxidase mutants (rbohD) were deeply dormant. Expression of a set of genes related to dormancy upon imbibition in the cat2-1 and vet1-1 seeds revealed that their non-dormant phenotype was probably not related to ABA or gibberellin metabolism, but suggested that ROS could trigger germination through gibberellin signaling activation.     

The promoter of the wheat puroindoline-a gene (<Emphasis Type="Italic">PinA</Emphasis>) exhibits a more complex pattern of activity than that of the <Emphasis Type="Italic">PinB</Emphasis> gene and is induced by wounding and pathogen attack in rice

Puroindolines form the molecular basis of wheat grain hardness. However, little is known about puroindoline gene regulation. We previously reported that the Triticum aestivum puroindoline-b gene (PinB) promoter directs β-glucuronidase gene (uidA) seed-specific expression in transgenic rice. In this study, we isolated a puroindoline-a gene (PinA), analyzed PinA promoter activity by 5′ deletions and compared PinA and PinB promoters in transgenic rice. Seeds of PinA-1214 and PinB-1063 transgenic plants strongly expressed uidA in endosperm, in the aleurone layer and in epidermis cells in a developmentally regulated manner. The GUS activity was also observed in PinA-1214 embryos. Whereas the PinB promoter is seed specific, the PinA promoter also directed, but to a lower level, uidA expression in roots of seedlings and in the vascular tissues of palea and pollen grains of dehiscent anthers during flower development. In addition, the PinA promoter was induced by wounding and by Magnaporthe grisea. By deletion analysis, we showed that the “390-bp” PinA promoter drives the same expression pattern as the “1214-bp” promoter. Moreover, the “214-bp” PinA promoter drives uidA expression solely in pollen grains of dehiscent anthers. The presence of putative cis-regulatory elements that may be related to PinA expression is discussed from an evolutionary point of view. By electrophoretic mobility shift assay, we showed that putative cis-elements (WUN-box, TCA motifs and as-1-like binding sites) whose presence in the PinA promoter may be related to wounding and/or the pathogen response form complexes with nuclear extracts isolated from wounded wheat leaves.     

Mechanisms of degradation of the fungal ornithine decarboxylase

Ornithine decarboxylase (ODC) is the first enzyme in polyamine biosynthesis in numerous living organisms, from bacteria to mammalian cells. Its control is under negative feedback regulation by the end products of the pathway. In dimorphic fungi, ODC activity and therefore polyamine concentrations are related to the morphogenetic process. From the fission yeast Schizosaccharomyces pombe to human, polyamines induce antizyme synthesis which in turn inactivates ODC. This is hydrolyzed by the 26S proteasome without ubiquitination. The regulatory mechanism of antizyme on polyamines is conserved, although to date no antizyme homology has been identified in some fungal species. The components that are responsible for regulating polyamine levels in cells and the current knowledge of ODC regulation in dimorphic fungi are presented in this review. ODC degradation is of particular interest because inhibitors of this pathway may lead to the discovery of novel antifungal drugs.     

A PIF-dependent recombinogenic signal in the mitochondrial DNA of yeast

From their recombination properties, tandem rho^- mutants of the mitochondrial genome of Saccharomyces cerevisiae were divided into two categories. In crosses between PIF-independent rho^- and rho⁺ strains, the recombination frequency is low and similar in PIF/pif and pif/pif diploids. In crosses between PIF-dependent rho^- and rho⁺ strains, the recombination frequency is stimulated 10-50 times in PIF/pif diploids and is drastically decreased in pif/pif diploids. These results suggest that a recombinogenic signal is present in the mitochondrial (mt) DNA of PIF-dependent rho^- clones. This signal is not recognized in pif mutants. Sequence analysis of a series of small (<300 bp) overlapping tandem rho^- genomes located in the ery region of the 21S rRNA gene led us to identify an essential element of this signal within a 41-bp A+T sequence exhibiting over 26 bp a perfect dyad symmetry. However the recombinogenic signal is not sequence-specific since the sequence described above does not characterize PIF-dependent rho^- clones located in the oli1 region. Our results rather suggest that the recombinogenic signal is related to the topology of rho^- DNA. Denaturated sites in the double helix or cruciform structures elicited by local negative supercoiling might be preferred sites of the initiation of recombination.     

Endoglin (CD105) expression is regulated by the liver X receptor alpha (NR1H3) in human trophoblast cell line JAR

Human implantation involves invasion of the uterine wall and remodeling of uterine arteries by extravillous cytotrophoblasts. Defects in these early steps of placental development lead to poor placentation and are often associated with preeclampsia, a frequent complication of human pregnancy. One of the complex mechanisms controlling trophoblast invasion involves the activation of the liver X receptor beta (or NR1H2, more commonly known as LXRbeta) by oxysterols known as potent LXR activators. This activation of LXRbeta leads to a decrease of trophoblast invasion. The identification of new target genes of LXR in the placenta could aid in the understanding of their physiological roles in trophoblast invasion. In the present study, we show that the endoglin (ENG) gene is a direct target of the liver X receptor alpha (NR1H3, also known as LXRalpha). ENG, whose gene is highly expressed in syncytiotrophoblasts, is part of the transforming growth factor (TGF) receptor complex that binds several members of the TGFbeta superfamily. In the human placenta, ENG has been shown to be involved in the inhibition of trophoblast invasion. Treatment of human choriocarcinoma JAR cells with T0901317, a synthetic LXR-selective agonist, leads to a significant increase in ENG mRNA and protein levels. Using transfection and electrophoretic mobility shift assays, we demonstrate that LXR (as a heterodimer with the retinoid X receptor) is able to bind the ENG promoter on an LXR response element and mediates the activation of ENG gene expression by LXRalpha in JAR cells. This study suggests a novel mechanism by which LXR may regulate trophoblast invasion in pathological pregnancy such as preeclampsia.     

The N-terminal domain of annexin 2 serves as a secondary binding site during membrane bridging

Annexin A2 (AnxA2) is a Ca(2+)- and acidic phospholipid-binding protein involved in many cellular processes. It undergoes Ca(2+)-mediated membrane bridging at neutral pH and has been demonstrated to be involved in an H(+)-mediated mechanism leading to a novel AnxA2-membrane complex structure. We used fluorescence techniques to characterize this H(+)-dependent mechanism at the molecular level; in particular, the involvement of the AnxA2 N-terminal domain. This domain was labeled at Cys-8 either with acrylodan or pyrene-maleimide fluorescent probes. Steady-state and time-resolved fluorescence analysis for acrylodan and fluorescence quenching by doxyl-labeled phospholipids revealed direct interaction between the N-terminal domain and the membrane. The absence of pyrene excimer suggested that interactions between N termini are not involved in the H(+)-mediated mechanism. These findings differ from those previously observed for the Ca(2+)-mediated mechanism. Protein titration experiments showed that the protein concentration for half-maximal membrane aggregation was twice for Ca(2+)-mediated compared with H(+)-mediated aggregation, suggesting that AnxA2 was able to bridge membranes either as a dimer or as a monomer, respectively. An N-terminally deleted AnxA2 was 2-3 times less efficient than the wild-type protein for H(+)-mediated membrane aggregation. We propose a model of AnxA2-membrane assemblies, highlighting the different roles of the N-terminal domain in the H(+)- and Ca(2+)-mediated membrane bridging mechanisms.     

Role of ATM in the telomere response to the G-quadruplex ligand 360A

Telomeres are known to prevent chromosome ends from being recognized as DNA double-strand breaks. Conversely, many DNA damage response proteins, including ATM, are thought to participate to telomere maintenance. However, the precise roles of ATM at telomeres remain unclear due to its multiple functions in cell checkpoints and apoptosis. To gain more insights into the role of ATM in telomere maintenance, we determined the effects of the G-quadruplex ligand 360A in various cell lines lacking functional ATM. We showed, by using Fluorescence in situ hybridization (FISH) and Chromosome Orientation-FISH using telomere PNA probes, that 360A induced specific telomere aberrations occurring during or after replication, mainly consisting in sister telomere fusions and also recombinations that involved preferentially the lagging strand telomeres. We demonstrate that ATM reduced telomere instability independently of apoptosis induction. Our results suggest thus that ATM has a direct role in preventing inappropriate DNA repair at telomeres, which could be related to its possible participation to the formation of protected structures at telomeres.     

Role of histone tails in the conformation and interactions of nucleosome core particles

The goal of this work was to test the role of the histone tails in the emergence of attractive interactions between nucleosomes above a critical salt concentration that corresponds to the complete tail extension outside the nucleosome [Mangenot, S., et al (2002) Biophys. J. 82, 345-356; Mangenot, S., et al (2002) Eur. Phys. J. E 7, 221-231]. Small angle X-ray scattering experiments were performed in parallel with intact and trypsin tail-deleted nucleosomes with 146 +/- 3 bp DNA. We varied the monovalent salt concentration from 10 to 300 monovalent salt concentration and followed the evolution of (i) the second virial coefficient that characterizes the interactions between particles and (ii) the conformation of the particle. The attractive interactions do not emerge in the absence of the tails, which validates the proposed hypothesis.     

Bacillus subtilis alpha-amylase: interactions of a partially folded conformer with small unilamellar vesicles

We studied the interactions between conformers of exocellular alpha-amylase and small unilamellar vesicles (SUV) composed of the major membrane lipids of Bacillus subtilis under physiological conditions of pH, temperature and ionic strength. Using fluorescence spectroscopy, surface plasmon resonance (SPR) and phase separation, we show that the native alpha-amylase has no affinity for the SUV, whereas a partially folded form, displaying structural properties in common with the competent state for secretion, binds to the vesicles (KA approximately 10(5) M(-1)). This association prevented its subsequent folding. The complex was destabilized in the presence of PrsA, a major peripheric lipoprotein of B. subtilis which displays a strong affinity for SUV (KA approximately 1.5x10(8) M(-1)). Vesicles coated with PrsA lost their ability to bind the partially folded conformer. The approach in vitro, in which our aim was to mimic the last stage of alpha-amylase translocation, indicates that PrsA possibly helps, in vivo, the secreted protein to acquire its native conformation by modulating the interaction between the latter and the lipid polar heads on the trans side of the cytoplasmic membrane.