Flavonoids in plant nuclei: detection by laser microdissection and pressure catapulting (LMPC), in vivo staining, and uv–visible spectroscopic titration

Previous studies in our laboratory have indicated that the nuclei of a number of trees are associated with flavonoids, especially flavan‐3‐ols. In the present study, three techniques were applied to verify that flavonoids are naturally incorporated into nuclei. These were histochemistry, UV–visible (UV‐VIS) titration and laser microdissection. Nuclei from intact seed wings of Tsuga canadensis were isolated from their cells using laser microdissection and pressure catapulting (LMPC). Thereafter, the excised nuclei were stained with p‐dimethylamino‐cinnamaldehyde (DMACA), which resulted in a blue coloration due to the presence of flavanols. Thus, there is no doubt that the nuclei were, prior to staining, associated with flavanols. The nuclei of the coniferous species Abies lasiocarpa, Cedrus deodara, Cedrus libani, Juniperus communis, Picea abies, Picea orientalis and Pseudotsuga menziessii(Douglas fir) showed a yellow fluorescence typical for flavonols from the beginning of bud break over the entire growing season. However, after the bud‐breaking period, the nuclei of all species, except for Cedrus deodara, showed additionally a blue reaction for flavanols. Rather late, in midsummer, blue‐stained flavanols in nuclei were found in Picea orientalis. Generally, zeatin intensified the flavanol association with the nuclei. The main components of nucleosomes are DNA and the histone proteins. The nature of their association with the flavonols quercetin and rutin was investigated by UV‐VIS spectroscopic titration. The data were evaluated by means of the Mauser (A and AD) diagrams. The results indicate that DNA shows largely no spectroscopically detectable association equilibria under the experimental conditions chosen. However, association (aggregation) equilibria can be observed with rutin or quercetin and histone sulphate in Tris buffer (pH 8.0, 7.4 and 7.0). In phosphate buffer, rutin shows spectroscopically no or only weak association with histone sulphate, in contrast to its behaviour towards quercetin.     

Regulation of carbohydrate transport activities in Salmonella typhimurium: use of the phosphoglycerate transport system to energize solute uptake.

The phosphoglycerate transport system was employed to supply energy-depleted, lysozyme-treated Salmonella typhimurium cells with a continuous intracellular source of phosphoenolpyruvate. When the cells had been induced to high levels of the phosphoglycerate transport system, a low extracellular concentration of phosphoenolpyruvate (0.1 mM) half maximally stimulated uptake of methyl alpha-glucoside via the phosphoenolpyruvate:sugar phosphotransferase system. If the phosphoglycerate transport system was not induced before energy depletion, 100 times this concentration of phosphoenolpyruvate was required for half-maximal stimulation. Phosphoenolpyruvate could not be replaced by other energy sources if potassium fluoride (an inhibitor of enolase) was present. Inhibition of [14C]-glycerol uptake into energy-depleted cells by methyl alpha-glucoside was demonstrated. A concentration of phosphoenolpyruvate which stimulated methyl alpha-glucoside accumulation counteracted the inhibitory effect of the glucoside. In the presence of potassium fluoride, phosphoenolpyruvate could not be replaced by other energy sources. Inhibition of glycerol uptake by methyl alpha-glucoside in intact untreated cells was also counteracted by phosphoenolpyruvate, but several energy sources were equally effective; potassium fluoride was without effect. These and other results were interpreted in terms of a mechanism in which the relative proportions of the phosphorylated and nonphosphorylated forms of a cell constituent influence the activity of the glycerol transport system.     

Loss of nuclear flavanols during drought periods in Taxus baccata

Normally, needles of Taxus baccata during the growth period prominently stain blue for nuclear flavanols with the histochemical DMACA procedure. However, under excess heat and drought conditions, nuclear flavanols of current‐year needles decline to zero. Nevertheless, greenish‐yellow‐coloured flavonols (quercetin derivatives) were still observed in nuclei. All of these yellow nuclei were in a silenced state and without mitosis. This link between drought and loss of nuclear flavanols was found in 3 years, 2003, 2007 and 2010. In 2007, exceptional drought occurred in early spring, interrupted by short rains. This, in turn, led to flushing of new sprouts, a characteristic feature in which nuclei were overloaded with flavanols. By the end of three drought periods, all nuclei developed blue‐coloured nuclear flavanols. The flavanols seem to be associated with the histone proteins of chromatin. The oxidative degradation of catechin in Tris buffer (pH 8.0) containing MgCl₂ was studied in the presence of the H4‐core fragment TYTEHAKRKTVTAMD, modified according to the epigenetic histone code. The results show that catechin degradation can be significantly inhibited by the non‐modified peptides and the methylated peptides (methylation at both lysine residues). The acetylated and formylated peptides do not show this behaviour. These observations indicate that flavanol association at chromosomes appears to be regulated by the epigenetic histone code.     

Evidence for regulation of gluconeogenesis by the fructose phosphotransferase system in Salmonella typhimurium.

A genetic locus designated fruR, previously mapped to min 3 on the Salmonella typhimurium chromosome, gave rise to constitutive expression of the fructose (fru) regulon and pleiotropically prevented growth on all Krebs cycle intermediates. Regulatory effects of fruR were independent of cyclic AMP and its receptor protein and did not prevent uptake of Krebs cycle intermediates. Instead, the phosphotransferase system appeared to regulate gluconeogenesis by controlling the activities of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate synthase.     

Coordinate regulation of adenylate cyclase and carbohydrate permeases by the phosphoenolpyruvate:sugar phosphotransferase system in Salmonella typhimurium.

Adenylate cyclase (EC 4.6.1.1) and several carbohydrate permeases are inhibited by D-glucose and other substrates of the phosphoenolpyruvate:sugar phosphotransferase system. These activities are coordinately altered by sugar substrates of the phosphotransferase system in a variety of bacterial strains which contain differing cellular levels of the protein components of the phosphotransferase system: Enzyme I, a small heat-stable protein, and Enzyme II. It is suggested that the activities of adenylate cyclase and the permease proteins are subject to allosteric regulation and that the allosteric effector is a regulatory protein which can be phosphorylated by the phosphotransferase system.     

Nuclei of tea flowers as targets for flavanols

The tea plant (Camellia sinensis L.) is famous for its flavanol-based constituents being valuable for human health. These flavanols associate with the nuclei of tea flowers, which is demonstrated histochemically by blue colouration using the selective staining reagent p-dimethylaminocinnamaldehyde (DMACA). Sepals, petals, stamens, pollen tubes, ovaries and ovules were studied. All these organs were shown to contain flavanols in vacuolar compartments, in nuclei and, exceptionally, also in the cytoplasm of pollen tubes. In all cells, even in those lacking vacuoles, the nuclei stained blue for flavanols. The extremely divergent development, shape and function of the diverse flower organs did not basically influence the nuclear flavanol association. Nevertheless, within the limits of this study, a few tissue-dependent differences in staining intensity were obvious. Interactions between epicatechin and nuclear histone proteins (histone sulphate) were studied by UV-VIS spectroscopic titration and by means of Mauser diagrams. The results show that the observed association equilibria are strongly dependent on pH (8.0 and 7.4) and on the buffer used (Tris, phosphate).     

Cytological and biochemical characterization of the FtsA cell division protein of Bacillus subtilis

The actin-like protein FtsA is present in many eubacteria, and genetic experiments have shown that it plays an important, sometimes essential, role in cell division. Here, we show that Bacillus subtilis FtsA is targeted to division sites in both vegetative and sporulating cells. As in other organisms FtsA is probably recruited immediately after FtsZ. In sporulating cells of B. subtilis FtsZ is recruited to potential division sites at both poles of the cell, but asymmetric division occurs at only one pole. We have now found that FtsA is recruited to only one cell pole, suggesting that it may play an important role in the generation of asymmetry in this system. FtsA is present in much higher quantities in B. subtilis than in Escherichia coli, with approximately one molecule of FtsA for five of FtsZ. This means that there is sufficient FtsA to form a complete circumferential ring at the division site. Therefore, FtsA may have a direct structural role in cell division. We have purified FtsA and shown that it behaves as a dimer and that it has both ATP-binding and ATP-hydrolysis activities. This suggests that ATP hydrolysis by FtsA is required, together with GTP hydrolysis by FtsZ, for cell division in B. subtilis (and possibly in most eubacteria).