Luc Roef

Effect of indomethacin on cell cycle dependent cyclic AMP fluxes in tobacco BY-2 cells

The evolution of adenosine 3′,5′‐cyclic monophosphate (cAMP) levels was investigated in synchronised tobacco BY‐2 cells by virtue of a method based on immunoaffinity purification and analysis on electrospray tandem mass spectrometry. A transient peak in cAMP content was observed during the S and G1 phases of the cell cycle. Application of the prostaglandin inhibiting drug indomethacin at early S phase resulted in the loss of the cAMP peak in S phase and inhibited mitotic division. This inhibition of cAMP accumulation suggests the presence of a prostaglandin‐dependent adenylyl cyclase activity, analogous to animal cyclases. A potential role for cAMP during the plant cell cycle is postulated.

Benchmark study on algae harvesting with backwashable submerged flat panel membranes.

The feasibility of algae harvesting with submerged flat panel membranes was investigated as pre-concentration step prior to centrifugation. Polishing of the supernatant coming from the centrifuge was evaluated as well. The effect of membrane polymer (polyvinyl chloride [PVC], polyethersulfone polyvinyl-pyrollidone [PES-PVP], poly vinylidene fluoride [PVDF]), pore size (microfiltration [MF], ultrafiltration [UF]), algae cell concentrations and species were investigated at lab-scale. In addition, backwashing as fouling control was compared to standard relaxation. PVDF was the superior polymer, and UF showed better fouling resistance. Backwashing outperformed relaxation in fouling control. The backwashable membranes allowed up to 300% higher fluxes compared to commercial flat panel benchmark (PVC) membranes. Estimations on energy consumption for membrane filtration followed by centrifugation revealed relatively low values of 0.169 kW h/kg of dry weight of algae compared to 0.5 kW h/kg for algae harvesting via classical centrifuge alone.

Identification of Four Adenosine Kinase Isoforms in Tobacco By-2 Cells and Their Putative Role in the Cell Cycle-regulated Cytokinin Metabolism

Adenosine kinase (ADK), a key enzyme in the regulation of the intracellular level of adenosine is also speculated to be responsible for the conversion of cytokinin ribosides to their respective nucleotides. To elucidate the role of ADK in the cytokinin metabolism of tobacco BY-2 cells (Nicotiana tabacum cv. “Bright Yellow-2”; TBY-2), we have identified and characterized the full-length cDNAs encoding four ADK isoforms of N. tabacum and determined their catalytic properties. The four TBY-2 ADK isoforms (designated 1S, 2S, 1T, and 2T) display a high affinity for both adenosine (Km 1.88–7.30 μm) and three distinct types of cytokinin ribosides: isopentenyladenosine; zeatin riboside; and dihydrozeatin riboside (Km 0.30–8.71 μm). The Vmax/Km values suggest that ADK2S exhibits in vitro an overall higher efficiency in the metabolism of cytokinin ribosides than the other three isoforms. The expression pattern of NtADK genes is modulated significantly during the cell cycle. We suggest that the increased transcript accumulation of NtADK coupled to an increased ADK activity just prior to mitosis is associated with a very active cytokinin metabolism at that phase of the cell cycle of synchronized TBY-2 cells.

Quantitation of Cyclic Nucleotides in Biological Samples by Negative Electrospray Tandem Mass Spectrometry Coupled to Ion Suppression Liquid Chromatography

Measurement of cyclic nucleotides by electrospray ionization mass spectrometry constitutes an unequivocal quantitative and qualitative technique superior to the established estimation methods of UV spectroscopy, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and other mass spectrometric ionization techniques such as electron impact, thermospray and fast-atom bombardment. Optimal conditions for negative-ion electrospray tandem mass spectrometry showed a linear detection range using solutions with a concentration ranging from 10−6 and 5×10−9 M with an absolute detection limit of 100 fmol injected. Measurement of diagnostic product ions derived from low-energy collision-activated dissociation of the [M−H]− ions provided unambiguous identification. Quantitation of cyclic nucleotides present in biological samples in the femtomole region, needed further purification using on-line ion suppression high-performance liquid chromatography in order to avoid reduced sensitivity caused by matrix quenching.

The environmental sustainability of microalgae as feed for aquaculture: a life cycle perspective

The environmental sustainability of microalgae production for aquaculture purposes was analyzed using exergy analysis (EA) and life cycle assessment (LCA). A production process (pilot 2012, 240 m(2)) was assessed and compared with two upscaling scenarios (pilot 2013, 1320 m(2) and first production scale 2015, 2.5 ha). The EA at process level revealed that drying and cultivation had the lowest efficiencies. The LCA showed an improvement in resource efficiency after upscaling: 55.5 MJ(ex,CEENE)/MJ(ex) DW biomass was extracted from nature in 2012, which was reduced to 21.6 and 2.46 MJ(ex,CEENE)/MJ(ex) DW in the hypothetical 2013 and 2015 scenarios, respectively. Upscaling caused the carbon footprint to decline by factor 20 (0.09 kg CO2,eq/MJ(ex) DW in 2015). In the upscaling scenarios, microalgae production for aquaculture purposes appeared to be more sustainable in resource use than a reference fish feed (7.70 MJ(ex,CEENE) and 0.05 kg CO2,eq per MJ(ex) DW).

Plant Cyclic Nucleotide Signalling: Facts and Fiction

The presence of the cyclic nucleotides 3’,5’-cyclic adenyl monophosphate (cAMP) and 3’,5’-cyclic guanyl monophosphate (cGMP) in plants is now generally accepted. In addition, cAMP and cGMP have been implicated in the regulation of important plant processes such as stomatal functioning, monovalent and divalent cation fluxes, chloroplast development, gibberellic acid signalling, pathogen response and gene transcription. However, very little is known regarding the components of cyclic nucleotide signalling in plants. In this addendum, the evidence for specific mechanisms of plant cyclic nucleotide signalling is evaluated and discussed.

Cyclic AMP affinity purification and ESI-QTOF MS-MS identification of cytosolic glyceraldehyde 3-phosphate dehydrogenase and two nucleoside diphosphate kinase isoforms from tobacco BY-2 cells.

The soluble protein fraction of tobacco bright yellow 2 cells contained adenosine 3′,5′‐cyclic monophosphate (cAMP)‐binding activity, detected with both a conventional binding assay and a surface plasmon resonance biosensor. A cAMP‐agarose‐based affinity purification procedure yielded three proteins which were identified by mass spectrometry as glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) and two nucleoside diphosphate kinases (NDPKs). This is the first report describing an interaction between cAMP and these proteins in higher plants. Our findings are discussed in view of the reported role of the interaction of cAMP with GAPDH and NDPK in animals and yeast. In addition, we provide a rapid method to isolate both proteins from higher plants.

Indomethacin-induced G1/S phase arrest of the plant cell cycle.

In animal systems, indomethacin inhibits cAMP production via a prostaglandin‐adenylyl cyclase pathway. To examine the possibility that a similar mechanism occurs in plants, the effect of indomethacin on the cell cycle of a tobacco bright yellow 2 (TBY‐2) cell suspension was studied. Application of indomethacin during mitosis did not interfere with the M/G1 progression in synchronized BY‐2 cells but it inhibited cAMP production at the beginning of the G1 phase and arrested the cell cycle progression at G1/S. These observations are discussed in relation to the putative involvement of cAMP biosynthesis in the cell cycle progression in TBY‐2 cells.

A bioanalytical method for the proteome wide display and analysis of protein complexes from whole plant cell lysates

While protein interaction studies and protein network modeling come to the forefront, the isolation and identification of protein complexes in a cellular context remains a major challenge for plant science. To this end, a nondenaturing extraction procedure was optimized for plant whole cell matrices and the combined use of gel filtration and BN‐PAGE for the separation of protein complexes was studied. Hyphenation to denaturing electrophoresis and mass spectrometric analysis allows for the simultaneous identification of multiple (previously unidentified) protein interactions in single samples. The reliability and efficacy of the technique was confirmed (i) by the identification of well‐studied plant protein complexes, (ii) by the presence of nonplant interologs for several of the novel complexes (iii) by presenting physical evidence of previously hypothetical plant protein interactions and (iv) by the confirmation of found interactions using co‐IP. Furthermore practical issues concerning the use of this 2‐D BN/SDS‐PAGE display method for the analysis of protein–protein interactions are discussed.

Cytokinin Regulation of the Cell Division Cycle

Contrary to the situation in animal systems, plant shape and size are predominantly determined by developmental programs that govern the timely initiation and outgrowth of meristems. In plants, meristematic zones almost exclusively constitute the regions of dividing cells. The role of mitotic cell division in morphogenetic and developmental processes has been the subject of intense debate, but the present view seems to acknowledge the importance of proper cell division regulation for the correct elaboration and execution of developmental programs (29)