Maintenance of DNA methylation during the Arabidopsis life cycle is essential for parental imprinting

Imprinted genes are expressed predominantly from either their paternal or their maternal allele. To date, all imprinted genes identified in plants are expressed in the endosperm. In Arabidopsis thaliana, maternal imprinting has been clearly demonstrated for the Polycomb group gene MEDEA (MEA) and for FWA. Direct repeats upstream of FWA are subject to DNA methylation. However, it is still not clear to what extent similar cis-acting elements may be part of a conserved molecular mechanism controlling maternally imprinted genes. In this work, we show that the Polycomb group gene FERTILIZATION-INDEPENDENT SEED2 (FIS2) is imprinted. Maintenance of FIS2 imprinting depends on DNA methylation, whereas loss of DNA methylation does not affect MEA imprinting. DNA methylation targets a small region upstream of FIS2 distinct from the target of DNA methylation associated with FWA. We show that FWA and FIS2 imprinting requires the maintenance of DNA methylation throughout the plant life cycle, including male gametogenesis and endosperm development. Our data thus demonstrate that parental genomic imprinting in plants depends on diverse cis-elements and mechanisms dependent or independent of DNA methylation. We propose that imprinting has evolved under constraints linked to the evolution of plant reproduction and not by the selection of a specific molecular mechanism.     

GTP enhances the degradation of the photosystem II D1 protein irrespective of its conformational heterogeneity at the Q(B) site

The light exposure history and/or binding of different herbicides at the Q(B) site may induce heterogeneity of photosystem II acceptor side conformation that affects D1 protein degradation under photoinhibitory conditions. GTP was recently found to stimulate the D1 protein degradation of photoinactivated photosystem II (Spetea, C. , Hundal, T., Lohmann, F., and Andersson, B. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6547-6552). Here we report that GTP enhances the cleavage of the D1 protein D-E loop following exposure of thylakoid membranes to either high light, low light, or repetitive single turnover flashes but not to trypsin. GTP does not stimulate D1 protein degradation in the presence of herbicides known to affect the accessibility of the cleavage site to proteolysis. However, GTP stimulates degradation that can be induced even in darkness in some photosystem II conformers following binding of the PNO8 herbicide (Nakajima, Y., Yoshida, S., Inoue, Y., Yoneyama, K., and Ono, T. (1995) Biochim. Biophys. Acta 1230, 38-44). Both the PNO8- and the light-induced primary cleavage of the D1 protein occur in the grana membrane domains. The subsequent migration of photosytem II containing the D1 protein fragments to the stroma domains for secondary proteolysis is light-activated. We conclude that the GTP effect is not confined to a specific photoinactivation pathway nor to the conformational state of the photosystem II acceptor side. Consequently, GTP does not interact with the site of D1 protein cleavage but rather enhances the activity of the endogenous proteolytic system.     

Outcome of the first electron microscopy validation task force meeting

This Meeting Review describes the proceedings and conclusions from the inaugural meeting of the Electron Microscopy Validation Task Force organized by the Unified Data Resource for 3DEM (http://www.emdatabank.org) and held at Rutgers University in New Brunswick, NJ on September 28 and 29, 2010. At the workshop, a group of scientists involved in collecting electron microscopy data, using the data to determine three-dimensional electron microscopy (3DEM) density maps, and building molecular models into the maps explored how to assess maps, models, and other data that are deposited into the Electron Microscopy Data Bank and Protein Data Bank public data archives. The specific recommendations resulting from the workshop aim to increase the impact of 3DEM in biology and medicine.     

Meconium ileus caused by mutations in GUCY2C, encoding the CFTR-activating guanylate cyclase 2C

Meconium ileus, intestinal obstruction in the newborn, is caused in most cases by CFTR mutations modulated by yet-unidentified modifier genes. We now show that in two unrelated consanguineous Bedouin kindreds, an autosomal-recessive phenotype of meconium ileus that is not associated with cystic fibrosis (CF) is caused by different homozygous mutations in GUCY2C, leading to a dramatic reduction or fully abrogating the enzymatic activity of the encoded guanlyl cyclase 2C. GUCY2C is a transmembrane receptor whose extracellular domain is activated by either the endogenous ligands, guanylin and related peptide uroguanylin, or by an external ligand, Escherichia coli (E. coli) heat-stable enterotoxin STa. GUCY2C is expressed in the human intestine, and the encoded protein activates the CFTR protein through local generation of cGMP. Thus, GUCY2C is a likely candidate modifier of the meconium ileus phenotype in CF. Because GUCY2C heterozygous and homozygous mutant mice are resistant to E. coli STa enterotoxin-induced diarrhea, it is plausible that GUCY2C mutations in the desert-dwelling Bedouin kindred are of selective advantage.     

Deregulation of electron flow within photosystem II in the absence of the PsbJ protein

The photosystem II (PSII) complex of photosynthetic oxygen evolving membranes comprises a number of small proteins whose functions remain unknown. Here we report that the low molecular weight protein encoded by the psbJ gene is an intrinsic component of the PSII complex. Fluorescence kinetics, oxygen flash yield, and thermoluminescence measurements indicate that inactivation of the psbJ gene in Synechocystis 6803 cells and tobacco chloroplasts lowers PSII-mediated oxygen evolution activity and increases the lifetime of the reduced primary acceptor Q(A)(-) (more than a 100-fold in the tobacco DeltapsbJ mutant). The decay of the oxidized S(2,3) states of the oxygen-evolving complex is considerably accelerated, and the oscillations of the Q(B)(-)/S(2,3) recombination with the number of exciting flashes are damped. Thus, PSII can be assembled in the absence of PsbJ. However, the forward electron flow from Q(A)(-) to plastoquinone and back electron flow to the oxidized Mn cluster of the donor side are deregulated in the absence of PsbJ, thereby affecting the efficiency of PSII electron flow following the charge separation process.     

Mutations in FIE, a WD polycomb group gene, allow endosperm development without fertilization

A fundamental problem in biology is to understand how fertilization initiates reproductive development. Higher plant reproduction is unique because two fertilization events are required for sexual reproduction. First, a sperm must fuse with the egg to form an embryo. A second sperm must then fuse with the adjacent central cell nucleus that replicates to form an endosperm, which is the support tissue required for embryo and/or seedling development. Here, we report cloning of the Arabidopsis FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) gene. The FIE protein is a homolog of the WD motif-containing Polycomb proteins from Drosophila and mammals. These proteins function as repressors of homeotic genes. A female gametophyte with a loss-of-function allele of fie undergoes replication of the central cell nucleus and initiates endosperm development without fertilization. These results suggest that the FIE Polycomb protein functions to suppress a critical aspect of early plant reproduction, namely, endosperm development, until fertilization occurs.     

Detection of protein-protein interactions in plants using bimolecular fluorescence complementation

Protein function is often mediated via formation of stable or transient complexes. Here we report the determination of protein-protein interactions in plants using bimolecular fluorescence complementation (BiFC). The yellow fluorescent protein (YFP) was split into two non-overlapping N-terminal (YN) and C-terminal (YC) fragments. Each fragment was cloned in-frame to a gene of interest, enabling expression of fusion proteins. To demonstrate the feasibility of BiFC in plants, two pairs of interacting proteins were utilized: (i) the alpha and beta subunits of the Arabidopsis protein farnesyltransferase (PFT), and (ii) the polycomb proteins, FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA). Members of each protein pair were transiently co-expressed in leaf epidermal cells of Nicotiana benthamiana or Arabidopsis. Reconstitution of a fluorescing YFP chromophore occurred only when the inquest proteins interacted. No fluorescence was detected following co-expression of free non-fused YN and YC or non-interacting protein pairs. Yellow fluorescence was detected in the cytoplasm of cells that expressed PFT alpha and beta subunits, or in nuclei and cytoplasm of cells that expressed FIE and MEA. In vivo measurements of fluorescence spectra emitted from reconstituted YFPs were identical to that of a non-split YFP, confirming reconstitution of the chromophore. Expression of the inquest proteins was verified by immunoblot analysis using monoclonal antibodies directed against tags within the hybrid proteins. In addition, protein interactions were confirmed by immunoprecipitations. These results demonstrate that plant BiFC is a simple, reliable and relatively fast method for determining protein-protein interactions in plants.     

Protein phosphorylation and redox sensing in chloroplast thylakoids

Transduction of light dependent signals to redox sensitive kinases in photosynthetic membranes modulates energy transfer to the photochemical reaction centres and regulates biogenesis, stability and turnover of thylakoid protein complexes. The occupancy of the quinol-oxidation site of the cytochrome bf complex by plastoquinol and the redox state of protein thiol groups act as elements of the signal transducing chains.     

Functional Graded Germanium–Lead Chalcogenide‐Based Thermoelectric Module for Renewable Energy Applications

High thermoelectric conversion efficiencies can be achieved by making use of materials with, as high as possible, figure of merit, ZT, values. Moreover, even higher performance is possible with appropriate geometrical optimization including the use of functionally graded materials (FGM) technology. Here, an advanced n‐type functionally graded thermoelectric material based on a phase‐separated (PbSn_0.05Te)_0.92(PbS)_0.08 matrix is reported. For assessment of the thermoelectric potential of this material, combined with the previously reported p‐type Ge_0.87Pb_0.13Te showing a remarkable dimensionless figure of merit of 2.2, a finite‐element thermoelectric model is developed. The results predict, for the investigated thermoelectric couple, a very impressive thermoelectric efficiency of 14%, which is more than 20% higher than previously reported values for operating under cold and hot junction temperatures of 50 °C and 500 °C, respectively. Validation of the model prediction is done by a thermoelectric couple fabricated according to the model's geometrical optimization conditions, showing a good agreement to the theoretically calculated results, hence approaching a higher technology readiness level.