Sexual reproduction in higher plants I: fertilization and the initiation of zygotic program

Sexual plant reproduction is a critical developmental step in the life cycle of higher plants, to allow maternal and paternal genes to be transmitted in a highly regulated manner to the next generation. During evolution, a whole set of signal transduction machinery is developed by plants to ensure an error-free recognition between male and female gametes and initiation of zygotic program. In the past few years, the molecular machineries underlying this biological process have been elucidated, particularly on the importance of synergid cells in pollen tube guidance, the Ca⁺⁺ spike as the immediate response of fertilization and the epigenetic regulation of parental gene expressions in early zygotic embryogenesis. This review outlines the most recent development in this area.     

A pathogen- and salicylic acid-induced WRKY DNA-binding activity recognizes the elicitor response element of the tobacco class I chitinase gene promoter

Using a simple oligo selection procedure, we have previously identified a tobacco sequence-specific DNA-binding activity, TDBA12, that increases markedly during the tobacco mosaic virus (TMV)-induced hypersensitive response (HR). Based on the binding specificity and the two cDNA clones isolated, TDBA12 is related to a novel class of DNA-binding factors containing WRKY domains. In the present study, we report that TDBA12 could be induced not only by TMV infection but also by treatment with salicylic acid (SA) or its biologically active analogs capable of inducing pathogenesis-related (PR) genes and enhanced resistance. TDBA12 was sensitive to temperature and the protein dissociating agent sodium deoxycholate, suggesting that it may be a multimeric factor in which protein–protein interaction is important for the enhanced DNA-binding activity. Pre-treatment of nuclear extracts with alkaline phosphatase abolished TDBA12, suggesting that protein phosphorylation is important for its high DNA-binding activity. TDBA12 specifically recognized the elicitor response element of the tobacco class I basic chitinase gene promoter. The increase in the levels of TDBA12 following TMV infection or SA treatment preceded the induced expression of the tobacco chitinase gene. These results strongly suggest that certain WRKY DNA-binding proteins may be activated by enhanced protein phosphorylation and regulate inducible expression of defense-related genes during pathogen- and SA-induced plant defense responses.     

Visualization of the uptake of high-density lipoprotein by rat aortic endothelial cells and smooth muscle cells in vitro

High-density lipoproteins (HDL) were conjugated to Fluorescein 1,1-dioctadecyl 3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI) or colloidal gold for the investigation of ultrastructural aspects of binding and uptake of HDL by cholesterol-loaded cultured endothelial and smooth muscle cells from rat aorta. When cells were incubated for 2h at 4°C, HDL–DiI and HDL–gold conjugates were seen only on the cell surface. When cells were returned to incubation at 37°C for 5min, HDL–DiI appeared in the cytoplasm and colocalized with the fluorescent cholesteryl ester tag BODIPY-FL-C₁₂. HDL–gold conjugates appeared in the plasmalemmal invaginations and plasmalemmal vesicles. After incubation for 15min, most of the HDL–gold conjugates reappeared on the cell surface. After incubation for 30min, only a few conjugates were observed and they localized in lysosomal-like bodies. Quantitative data indicated that when the cholesterol-loaded cells were incubated at 4°C for 2h, the numbers of HDL–gold associated in clusters on the endothelial cell surface was 1.18 clusters/m. When cells were returned to incubation at 37°C for 5min, this value decreased to 0.7, increased again to 1.13 at 15min, and decreased to 0.29 at 30min. The numbers of clusters in the plasmalemmal invaginations were 0.06 clusters/m at 4°C for 2h, increased to 0.34 at 37°C for 5min and decreased gradually to 0.19 and 0.04 at 15 and 30min, respectively. The incidence of clusters in the plasmalemmal vesicles per non-nuclear cytoplasm was 0.01 clusters/m² at 4°C for 2h, increased significantly to 1.08 at 37°C for 5min, and decreased to 0.43 and 0.14 at 15 and 30min, respectively. This work supports that the plasmalemmal invaginations and plasmalemmal vesicles are linked to the HDL uptake in cholesterol-loaded aortic endothelial cells and smooth muscle cells.     

Mutagenesis of D80-82 and G83 residues in West Nile Virus NS2B: Effects on NS2B-NS3 activity and viral replication

Flaviviral NS2B is a required cofactor for NS3 serine protease activity and plays an important role in promoting functional NS2B-NS3 protease configuration and maintaining critical interactions with protease catalysis substrates. The residues D⁸⁰DDG in West Nile virus (WNV) NS2B are important for protease activity. To investigate the effects of D⁸⁰DDG in NS2B on protease activity and viral replication, the negatively charged region D⁸⁰DD and the conserved residue G83 of NS2B were mutated (D⁸⁰DD/E⁸⁰EE, D⁸⁰DD/K⁸⁰KK, D⁸⁰DD/A⁸⁰AA, G83F, G83S, G83D, G83K, and G83A), and NS3 D75A was designated as the negative control. The effects of the mutations on NS2B-NS3 activity, viral translation, and viral RNA replication were analyzed using kinetic analysis of site-directed enzymes and a transient replicon assay. All substitutions resulted in significantly decreased enzyme activity and blocked RNA replication. The negative charge of D⁸⁰DD is not important for maintaining NS2B function, but side chain changes in G83 have dramatic effects on protease activity and RNA replication. These results demonstrate that NS2B is important for viral replication and that D⁸⁰DD and G83 substitutions prevent replication; they will be useful for understanding the relationship between NS2B and NS3.     

Factors influencing parasitism of Trichogramma dendrolimi on eggs of the Asian corn borer, Ostrinia furnacalis

Laboratory studies were made to determine the capacity of Trichogramma dendrolimi to parasitize eggs of Ostrinia furnacalis, as affected by the rearing host species, substrate of host eggs, host age, original locality of host populations, and cold storage of host eggs. Wasps reared from eggs of Antheraea pernyi showed parasitic capacity on eggs of O. furnacalis on average twice as high as that of the wasps reared from eggs of Corcyra cephalonica. When the age of O. furnacalis eggs at 26 °C increased from 0–6 h to 18–24 h, the proportion of wasps that successfully parasitized host eggs, the number of host eggs parasitized, and the rate of parasitization all decreased by >50%. The number of O. furnacalis eggs parasitized per female T. dendrolimi increased with the number of host eggs available, and reached 22.9 in a 24 h period. However, the parasitic capacity of female T. dendrolimi on eggs of O. furnacalis laid on plant leaves was similar to that of O. furnacalis eggs laid on wax paper. Levels of parasitism of O. furnacalis eggs from two widely separated localities, i.e. Changchun (43.50° N, 125.20° E) and Hangzhou (30.18° N, 120.07° E), were similar. Cold storage of O. furnacalis eggs at 4 °C for 5 days did not affect parasitization. Results obtained in this study indicate that although O. furnacalis is a less preferred and less suitable host than many other hosts, such as Dendrolimus punctatus, Actias selene ningpoane, Philosamia cynthia, A. pernyi, C. cephalonica, within the host-species range of T. dendrolimi, the parasitoid has the potential to achieve 50–60% or even higher rates of parasitization of O. furnacalis eggs in corn fields under suitable conditions, and could be used in the biological control of the pest.     

Comparative studies of products obtained from solvolysis liquefaction of oil palm empty fruit bunch fibres using different solvents

Solvolysis of oil palm empty fruit bunches (EFB) fibres using different solvents (acetone, ethylene glycol (EG), ethanol, water and toluene) were carried out using an autoclave at 275°C for 60 min. The solvent efficiency in term of conversion yield was found to be: EG>water>ethanol>acetone>toluene. The liquid products and residue obtained were analyzed using Fourier transform infrared spectroscopy (FTIR) and gas chromatography/mass selectivity. The obtained results showed that the chemical properties of the oil product were significantly affected by the type of solvent used for the solvolysis process. The higher heating value (HHV) of oil products obtained using ethanol is ～29.42 MJ/kg, which is the highest among the oil products produced using different solvents. Water, ethanol and toluene yield major phenolic compounds. While EG favors the formation of alcohol compounds and acetone yields ketone and aldehyde compounds.     

Cytoplasmic targeting of mutant poly(A)-binding protein nuclear 1 suppresses protein aggregation and toxicity in oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. The autosomal dominant form of this disease is caused by a polyalanine expansion from 10 to 12-17 residues, located at the N-terminus of the poly(A)-binding protein nuclear 1 (PABPN1). A distinct pathological hallmark of OPMD is the presence of filamentous intranuclear aggregates in patients' skeletal muscle cells. Wildtype PABPN1 protein is expressed ubiquitously and was shown to be mostly concentrated in discrete nuclear domains called 'speckles'. Using an established cell- culture model, we show that most mutant PABPN1- positive (alanine expanded form) intranuclear aggregates are structures distinct from intranuclear speckles. In contrast, the promyelocytic leukaemia protein, a major component of nuclear bodies, strongly colocalized to intranuclear aggregates of mutant PABPN1. Wildtype PABPN1 can freely shuttle between the nucleus and cytoplasm. We determined whether the nuclear environment is necessary for mutant PABPN1 inclusion formation and cellular toxicity. This was achieved by inactivating the mutant PABPN1 nuclear localization signal and by generating full-length mutant PABPN1 fused to a strong nuclear export sequence. A green fluorescence protein tag inserted at the N-terminus of both wildtype PABPN1 (ala10) and mutant PABPN1 (ala17) proteins allowed us to visualize their subcellular localization. Targeting mutant PABPN1 to the cytoplasm resulted in a significant suppression of both intranuclear aggregates formation and cellular toxicity, two histological consequences of OPMD. Our results indicate that the nuclear localization of mutant PABPN1 is crucial to OPMD pathogenesis.     

Effect of Bacillus thuringiensis on Habrobracon hebetor during combined biological control of Plodia interpunctella

Abstract  The suitability of combining microbial pesticides and an insect parasitoid for pest management of stored cereal in China was evaluated using laboratory assays. For this purpose, interactions between Bacillus thuringiensis ( Bt ), Bt -intoxicated host larvae and the parasitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae) were tested against Plodia interpunctella Hübner (Lepidoptera: Pyralidae) . Bt or H. hebetor alone caused 41.67% and 35.35% P. interpunctella larval mortality respectively. The Bt –parasitoid combined treatment significantly increased mortality of P. interpunctella (86%). Progeny development of H. hebetor was dependent upon its susceptibility to Bt . Fewer parasitoids emerged from Bt –parasitoid combined treatment than in non- Bt treatments. However, since Bt did not prevent parasitoid development, a combined treatment with Bt and parasitoid release could produce better protection against P. interpunctella than either treatments when used singly, because their lethal effects were additive to each other.     

Epidermal growth factor receptor and notch pathways participate in the tumor suppressor function of gamma-secretase

Gamma-secretase, a unique aspartyl protease, is required for the regulated intramembrane proteolysis of Notch and APP, pathways that are implicated, respectively, in the pathogenesis of cancer and Alzheimer disease. However, the mechanism whereby reduction of gamma-secretase causes tumors such as squamous cell carcinoma (SCC) remains poorly understood. Here, we demonstrate that gamma-secretase functions in epithelia as a tumor suppressor in an enzyme activity-dependent manner. Notch signaling is down-regulated and epidermal growth factor receptor (EGFR) is activated in SCC caused by genetic reduction of gamma-secretase. Moreover, the level of EGFR is inversely correlated with the level of gamma-secretase in fibroblasts, suggesting that the up-regulation of EGFR stimulates hyperproliferation in epithelia of mice with genetic reduction of gamma-secretase. Supporting this notion is our finding that the proliferative response of fibroblasts lacking gamma-secretase activity is more sensitive when challenged by either EGF or an inhibitor of EGFR as ompared with wild type cells. Interestingly, the up-regulation of EGFR is independent of Notch signaling, suggesting that the EGFR pathway functions in parallel with Notch in the tumorigenesis of SCC. Collectively, our results establish a novel mechanism linking the EGFR pathway to the tumor suppressor role of gamma-secretase and that mice with genetic reduction of gamma-secretase represent an excellent rodent model for clarifying pathogenesis of SCC and for testing therapeutic strategy to ameliorate this type of human cancer.