ROS production and protein oxidation as a novel mechanism for seed dormancy alleviation

At harvest, sunflower (Helianthus annuus L.) seeds are dormant and unable to germinate at temperatures below 15 degrees C. Seed storage in the dry state, known as after-ripening, is associated with an alleviation of embryonic dormancy allowing subsequent germination at suboptimal temperatures. To identify the process by which dormancy is broken during after-ripening, we focused on the role of reactive oxygen species (ROS) in this phenomenon. After-ripening entailed a progressive accumulation of ROS, namely superoxide anions and hydrogen peroxide, in cells of embryonic axes. This accumulation, which was investigated at the cellular level by electron microscopy, occurred concomitantly with lipid peroxidation and oxidation (carbonylation) of specific embryo proteins. Incubation of dormant seeds for 3 h in the presence of hydrogen cyanide (a compound that breaks dormancy) or methylviologen (a ROS-generating compound) also released dormancy and caused the oxidation of a specific set of embryo proteins. From these observations, we propose a novel mechanism for seed dormancy alleviation. This mechanism involves ROS production and targeted changes in protein carbonylation patterns.     

NKp30-dependent cytolysis of filovirus-infected human dendritic cells

Understanding how protective innate immune responses are generated is crucial to defeating highly lethal emerging pathogens. Accumulating evidence suggests that potent innate immune responses are tightly linked to control of Ebola and Marburg filoviral infections. Here, we report that unlike authentic or inactivated Ebola and Marburg, filovirus-derived virus-like particles directly activated human natural killer (NK) cells in vitro, evidenced by pro-inflammatory cytokine production and enhanced cytolysis of permissive target cells. Further, we observed perforin- and CD95L-mediated cytolysis of filovirus-infected human dendritic cells (DCs), primary targets of filovirus infection, by autologous NK cells. Gene expression knock-down studies directly linked NK cell lysis of infected DCs to upregulation of the natural cytotoxicity receptor, NKp30. These results are the first to propose a role for NK cells in the clearance of infected DCs and the potential involvement of NKp30-mediated cytolysis in control of viral infection in vivo. Further elucidation of the biology of NK cell activation, specifically natural cytotoxicity receptors like NKp30 and NKp46, promises to aid our understanding of microbial pathology.     

Generation and characterization of soybean and marker-free tobacco plastid transformants over-expressing a bacterial 4-hydroxyphenylpyruvate dioxygenase which provides strong herbicide tolerance

Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is part of the biosynthetic pathway leading to plastoquinone and vitamin E. This enzyme is also the molecular target of various new bleaching herbicides for which genetically engineered tolerant crops are being developed. We have expressed a sensitive bacterial hppd gene from Pseudomonas fluorescens in plastid transformants of tobacco and soybean and characterized in detail the recombinant lines. HPPD accumulates to approximately 5% of total soluble protein in transgenic chloroplasts of both species. As a result, the soybean and tobacco plastid transformants acquire a strong herbicide tolerance, performing better than nuclear transformants. In contrast, the over-expression of HPPD has no significant impact on the vitamin E content of leaves or seeds, quantitatively or qualitatively. A new strategy is presented and exemplified in tobacco which allows the rapid generation of antibiotic marker-free plastid transformants containing the herbicide tolerance gene only. This work reports, for the first time, the plastome engineering for herbicide tolerance in a major agronomic crop, and a technology leading to marker-free lines for this trait.     

Dcp2 Decaps m2,2,7GpppN-capped RNAs, and its activity is sequence and context dependent

Hydrolysis of the mRNA cap plays a pivotal role in initiating and completing mRNA turnover. In nematodes, mRNA metabolism and cap-interacting proteins must deal with two populations of mRNAs, spliced leader trans-spliced mRNAs with a trimethylguanosine cap and non-trans-spliced mRNAs with a monomethylguanosine cap. We describe here the characterization of nematode Dcp1 and Dcp2 proteins. Dcp1 was inactive in vitro on both free cap and capped RNA and did not significantly enhance Dcp2 activity. Nematode Dcp2 is an RNA-decapping protein that does not bind cap and is not inhibited by cap analogs but is effectively inhibited by competing RNA irrespective of RNA sequence and cap. Nematode Dcp2 activity is influenced by both 5' end sequence and its context. The trans-spliced leader sequence on mRNAs reduces Dcp2 activity approximately 10-fold, suggesting that 5'-to-3' turnover of trans-spliced RNAs may be regulated. Nematode Dcp2 decaps both m(7)GpppG- and m(2,2,7)GpppG-capped RNAs. Surprisingly, both budding yeast and human Dcp2 are also active on m(2,2,7)GpppG-capped RNAs. Overall, the data suggest that Dcp2 activity can be influenced by both sequence and context and that Dcp2 may contribute to gene regulation in multiple RNA pathways, including monomethyl- and trimethylguanosine-capped RNAs.     

Patterns of protein oxidation in Arabidopsis seeds and during germination

Increased cellular levels of reactive oxygen species are known to occur during seed development and germination, but the consequences in terms of protein degradation are poorly characterized. In this work, protein carbonylation, which is an irreversible oxidation process leading to a loss of function of the modified proteins, has been analyzed by a proteomic approach during the first stages of Arabidopsis (Arabidopsis thaliana) seed germination. In the dry mature seeds, the legumin-type globulins (12S cruciferins) were the major targets. However, the acidic alpha-cruciferin subunits were carbonylated to a much higher extent than the basic (beta) ones, consistent with a model in which the beta-subunits are buried within the cruciferin molecules and the alpha-subunits are more exposed to the outside. During imbibition, various carbonylated proteins accumulated. This oxidation damage was not evenly distributed among seed proteins and targeted specific proteins as glycolytic enzymes, mitochondrial ATP synthase, chloroplastic ribulose bisphosphate carboxylase large chain, aldose reductase, methionine synthase, translation factors, and several molecular chaperones. Although accumulation of carbonylated proteins is usually considered in the context of aging in a variety of model systems, this was clearly not the case for the Arabidopsis seeds since they germinated at a high rate and yielded vigorous plantlets. The results indicate that the observed specific changes in protein carbonylation patterns are probably required for counteracting and/or utilizing the production of reactive oxygen species caused by recovery of metabolic activity in the germinating seeds.     

Iodinated vancomycin and mucopeptide biosynthesis by cell-free preparations from Micrococcus lysodeikticus

A particulate preparation from Micrococcus lysodeikticus was used to synthesize cell-wall mucopeptide. Radioactive iodinated vancomycin became attached to the preparation simultaneously with a complete inhibition of mucopeptide synthesis. After mucopeptide synthesis had occurred in the absence of antibiotic, the preparation took up more vancomycin, suggesting that new binding sites terminating in acyl-d-alanyl-d-alanine had been produced. The mucopeptide product was divided into a soluble and an insoluble portion, both sensitive to lysozyme. The soluble portion did not combine with vancomycin and hence had presumably lost its terminal d-alanine residues, either by transpeptidation or because of carboxy-peptidase action. The synthesis of both portions was unaffected by the presence of penicillin, but the insoluble part showed increased affinity for vancomycin, thus indicating that penicillin had caused conservation of d-alanyl-d-alanine termini.