Increased Glucosinolate Production in Brassica oleracea var. italica Cell Cultures Due to Coronatine Activated Genes Involved in Glucosinolate Biosynthesis.

In this work, the effect of different elicitors and culture conditions on the production of glucosinolates in broccoli cell cultures was studied. The results showed that 0.5 µM coronatine was the best elicitor for increasing glucosinolate production (205-fold increase over untreated cells after 72 h of treatment). Furthermore, the expression levels of some genes related to the biosynthetic pathway of glucosinolates as well as three Myb transcription factors also have been studied. The highest glucosinolate levels found in coronatine-treated cells were closely correlated with the highest gene expression levels of Cyp79b2, Cyp83b1, St5a, Myb51, and Myb122 after 6 h of treatment. The data shown in this study provide new insight into the key metabolic steps involved in the biosynthesis of glucosinolates, which will be of use for future applications of metabolic engineering techniques in broccoli.

Changes in the secretome of Vitis vinifera cv. Monastrell cell cultures treated with cyclodextrins and methyl jasmonate.

Elicitors induce defense responses that resemble those triggered by pathogen attack, including the synthesis of phytoalexins and pathogen-related proteins, which are accumulated in the extracellular space. In this work we analyze the changes in the secretome of Vitis vinifera cv. Monastrell cell cultures. This refers to the secreted proteome obtained from cell suspension cultures, in response to treatment with cyclodextrins and methyl jasmonate, separately or in combination using label-free quantitative approaches. Of the proteins found, thirty-three did not show significant differences in response to the different treatments carried out, indicating that these proteins were expressed in a constitutive way in both control and elicited grapevine cell cultures. These proteins included pathogenesis-related proteins 4 and 5, class III peroxidases, NtPRp-27, chitinases and class IV endochitinases, among others. Moreover, eleven proteins were differentially expressed in the presence of cyclodextrins and/or methyl jasmonate: three different peroxidases, two pathogenesis related protein 1, LysM domain-containing GPI-anchored protein 1, glycerophosphoryl diester phosphodiesterase, reticulin oxidase, heparanase, ß-1,3-glucanase and xyloglucan endotransglycosylase. Treatments with cyclodextrins reinforced the defensive arsenal and induced the accumulation of peroxidase V and xyloglucan endotransglycosylase. However, elicitation with methyl jasmonate decreased the levels of several proteins such as pathogenesis related protein 1, LysM domain-containing GPI-anchored protein 1, cationic peroxidase, and glycerophosphoryl diester phosphodiesterase, but increased the levels of new gene products such as heparanase, ß-1,3 glucanase, reticulin oxidase, and peroxidase IV, all of which could be used as potential biomarkers in the grapevine defense responses.

The endothelial transcription factor ERG mediates Angiopoietin-1-dependent control of Notch signalling and vascular stability.

Notch and Angiopoietin-1 (Ang1)/Tie2 pathways are crucial for vascular maturation and stability. Here we identify the transcription factor ERG as a key regulator of endothelial Notch signalling. We show that ERG controls the balance between Notch ligands by driving Delta-like ligand 4 (Dll4) while repressing Jagged1 (Jag1) expression. In vivo, this regulation occurs selectively in the maturing plexus of the mouse developing retina, where Ang1/Tie2 signalling is active. We find that ERG mediates Ang1-dependent regulation of Notch ligands and is required for the stabilizing effects of Ang1 in vivo. We show that Ang1 induces ERG phosphorylation in a phosphoinositide 3-kinase (PI3K)/Akt-dependent manner, resulting in ERG enrichment at Dll4 promoter and multiple enhancers. Finally, we demonstrate that ERG directly interacts with Notch intracellular domain (NICD) and ß-catenin and is required for Ang1-dependent ß-catenin recruitment at the Dll4 locus. We propose that ERG coordinates Ang1, ß-catenin and Notch signalling to promote vascular stability.

Long-term sampling of dioxin-like substances from a clinker kiln stack using alternative fuels.

The aim of this work is to characterize atmospheric emissions of polychlorinated dibenzo-p-dioxins (PCDDs)/polychlorinated dibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls (dl-PCBs) from a cement production plant where the existing clinker production line was completely replaced by a new state-of-the-art installation. The project started in April 2008 with the installation of a long-term sampling system in the stack of the clinker kiln that used petroleum coke as fuel; PCDD/PCDF and dl-PCB emissions were then evaluated for a two year period. To carry out the second part of the study, in 2010 the sampling system was moved to the new installation in which, apart from conventional fuel, recovered derived fuel (RDF) and WWTP sludge were used as alternative fuels. For both the old and new clinker kilns, PCDD/PCDF emission values were well below the limit established by the European Waste Incineration Directive 2000/76/CE (EWID) of 100 pg I-TEQ/Nm(3); values ranged from 0.43 to 2.02 and from 0.07 to 3.31 pg I-TEQ/Nm(3), respectively. dl-PCBs accounted for approximately 25% of the WHO-TEQ toxicity. These results prove that the installation is capable of reducing PCDD/PCDF and dl-PCB emissions when alternative fuels are integrated into the process. In the case of PCDDs/PCDFs, the major contributions to total TEQ were usually from 2,3,7,8-TCDD (owing to its relative abundance) and 2,3,4,7,8-PeCDF (due to its high I-TEF of 0.5); while for dl-PCBs, the major contribution was from PCB-126. The slight shift in the congener profile between the old and new installations was characterized and a regression model was proposed for dl-PCB emissions depending on the RDF flow rate in the clinker.

New method to enhance ajmalicine production in Catharanthus roseus cell cultures based on the use of cyclodextrins.

The joint use of cyclodextrins and methyljasmonate, when accompanied by a short exposure to UV, enhanced extracellular ajmalicine accumulation to 1040 ± 26.6 mg/l in suspension cultured cells of Catharanthus roseus. The success of this strategy is due to the use of cyclodextrins, which not only induce ajmalicine biosynthesis but also promote adduct formation. This removes ajmalicine from the medium, reduces feedback inhibition and ajmalicine degradation, and allows its accumulation in the culture medium at elevated concentrations.

Class III peroxidases in plant defence reactions.

When plants are attacked by pathogens, they defend themselves with an arsenal of defence mechanisms, both passive and active. The active defence responses, which require de novo protein synthesis, are regulated through a complex and interconnected network of signalling pathways that mainly involve three molecules, salicylic acid (SA), jasmonic acid (JA), and ethylene (ET), and which results in the synthesis of pathogenesis-related (PR) proteins. Microbe or elicitor-induced signal transduction pathways lead to (i) the reinforcement of cell walls and lignification, (ii) the production of antimicrobial metabolites (phytoalexins), and (iii) the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Among the proteins induced during the host plant defence, class III plant peroxidases (EC 1.11.1.7; hydrogen donor: H(2)O(2) oxidoreductase, Prxs) are well known. They belong to a large multigene family, and participate in a broad range of physiological processes, such as lignin and suberin formation, cross-linking of cell wall components, and synthesis of phytoalexins, or participate in the metabolism of ROS and RNS, both switching on the hypersensitive response (HR), a form of programmed host cell death at the infection site associated with limited pathogen development. The present review focuses on these plant defence reactions in which Prxs are directly or indirectly involved, and ends with the signalling pathways, which regulate Prx gene expression during plant defence. How they are integrated within the complex network of defence responses of any host plant cell will be the cornerstone of future research.