Analysis of Chlorophyll Fluorescence Decays of Isolated Thylakoids in Various Stages of Greening

The decay of chlorophyll (Chl) fluorescence of etiochloroplasts isolated in various stage of greening of cucumber cotyledons was analysed in order to get structural information on a photosynthetic apparatus. Two model decays, multiexponential and stretched exponential, were applied in the analysis. The quality of fit in these two models was different in various stages of chloroplast greening. The two-exponent model did not provide a good fit at early greening stages. To improve the fit it was necessary to introduce an additional third component which became very low at later stages. However, chloroplasts in the early stage of greening could also be described by a stretched exponential with parameters indicating rather planar (two-dimensional) arrangement of donor and acceptor molecules. The chloroplasts treated by DCMU and/or photooxidized by strong irradiance exhibit a similar character of fractal decay as untreated samples but in the multiexponential model the exact values of lifetimes and amplitudes of components vary. This suggests that the structure of investigated system does not dramatically change as a result of these two types of treatment.     

The impact of brain imaging technology on our understanding of motor function and dysfunction

Brain imaging techniques have demonstrated functional specialisation of multiple areas within the motor system. They have also defined the patterns of interactions between these regions during normal motor function and in motor disorders. Functional imaging makes visible the changes in cortical activity that take place over time during motor functions, from the activations a fraction of a second before voluntary action to cortical neuronal plasticity several weeks after injury. Recently, the functional abnormalities underlying various acquired and developmental motor disorders have been described, as well as the effects of therapeutic intervention.     

Molecular marker systems for Oenothera genetics

The genus Oenothera has an outstanding scientific tradition. It has been a model for studying aspects of chromosome evolution and speciation, including the impact of plastid nuclear co-evolution. A large collection of strains analyzed during a century of experimental work and unique genetic possibilities allow the exchange of genetically definable plastids, individual or multiple chromosomes, and/or entire haploid genomes (Renner complexes) between species. However, molecular genetic approaches for the genus are largely lacking. In this study, we describe the development of efficient PCR-based marker systems for both the nuclear genome and the plastome. They allow distinguishing individual chromosomes, Renner complexes, plastomes, and subplastomes. We demonstrate their application by monitoring interspecific exchanges of genomes, chromosome pairs, and/or plastids during crossing programs, e.g., to produce plastome-genome incompatible hybrids. Using an appropriate partial permanent translocation heterozygous hybrid, linkage group 7 of the molecular map could be assigned to chromosome 9.8 of the classical Oenothera map. Finally, we provide the first direct molecular evidence that homologous recombination and free segregation of chromosomes in permanent translocation heterozygous strains is suppressed.     

Spectral properties of phthalocyanines incorporated into resting and stimulated human peripheral blood cells

Human peripheral blood cells stimulated by phytohemagglutinin (which serve as a model of cancerous cells) and resting cells were incubated in dimethyl sulfoxide solutions of various phthalocyanines. In order to diminish the influence of atmospheric oxygen the cells were embedded in a polymer (polyvinyl alcohol) film. Fluorescence spectra of the samples were measured over two regions of excitation wavelengths: at 405 nm (predominant absorption of the cell material) and in the regions of strong absorption of phthalocyanines (at about 605 nm and 337 nm). The intrinsic emission of cell material became changed as a result both of cells' stimulation and of incubation of cells in dye solution. In most cases the stimulated cells when stained by dye exhibited higher long wavelength fluorescence intensity than resting cells. This suggests higher efficiency of dye incorporation into cancerous cells than into healthy cells. The absorption spectra of samples were also measured. The spectra of various phthalocyanines in incubation solvent, in polymer and in the cells embedded in polymer, were compared. The comparison of properties of the cells stimulated for different time periods enabled to establish the conditions of stimulation creating a population of cells incorporating a large number of sensitizing molecules.     

Chloroplast DNA in Mature and Senescing Leaves: A Reappraisal

The fate of plastid DNA (ptDNA) during leaf development has become a matter of contention. Reports on little change in ptDNA copy number per cell contrast with claims of complete or nearly complete DNA loss already in mature leaves. We employed high-resolution fluorescence microscopy, transmission electron microscopy, semithin sectioning of leaf tissue, and real-time quantitative PCR to study structural and quantitative aspects of ptDNA during leaf development in four higher plant species (Arabidopsis thaliana, sugar beet [Beta vulgaris], tobacco [Nicotiana tabacum], and maize [Zea mays]) for which controversial findings have been reported. Our data demonstrate the retention of substantial amounts of ptDNA in mesophyll cells until leaf necrosis. In ageing and senescent leaves of Arabidopsis, tobacco, and maize, ptDNA amounts remain largely unchanged and nucleoids visible, in spite of marked structural changes during chloroplast-to-gerontoplast transition. This excludes the possibility that ptDNA degradation triggers senescence. In senescent sugar beet leaves, reduction of ptDNA per cell to ∼30% was observed reflecting primarily a decrease in plastid number per cell rather than a decline in DNA per organelle, as reported previously. Our findings are at variance with reports claiming loss of ptDNA at or after leaf maturation.In vascular plants, copy numbers of plastid genomes (plastomes) frequently range from <100 per cell in meristematic cells to several thousand per cell in fully developed diploid leaf parenchyma cells. Microscopy studies have shown that the multicopy organelle genomes are usually condensed in more or less distinct DNA regions (nucleoids) within the organelle matrix or stroma.During development, the ratio of nuclear to organelle genomes appears to be relatively stringently regulated (Herrmann and Possingham, 1980; Rauwolf et al., 2010). Disregarding greatly varying absolute values (summarized in Rauwolf et al., 2010; Liere and Börner, 2013), there is little dispute that the number of plastid genomes and nucleoids per organelle and cell increase during early leaf development in higher plants (Kowallik and Herrmann, 1972; Selldén and Leech, 1981; Baumgartner et al., 1989; Fujie et al., 1994; Li et al., 2006; Rauwolf et al., 2010). This increase is usually accompanied by an increase in both size and number of plastids per cell (Butterfass, 1979). By contrast, data about plastid DNA (ptDNA) amounts in chloroplasts and cells of mature, ageing, and senescent tissue differ and are highly controversial. Basically two patterns have been described: the maintenance of more or less constant amounts of ptDNA per cell and/or organelle (Li et al., 2006; Zoschke et al., 2007; Rauwolf et al., 2010; Udy et al., 2012) or a significant decrease in copy number brought about by either continued organelle and cell division without ptDNA replication (Lamppa and Bendich, 1979; Scott and Possingham, 1980; Tymms et al., 1983) or by ptDNA degradation (Baumgartner et al., 1989; Sodmergen et al., 1991). In a series of communications, Bendich and coworkers recently reported that ptDNA levels decline drastically before leaf maturation in several plant species. In Arabidopsis thaliana and maize (Zea mays), ptDNA levels were reported to decrease early and precipitously as leaves mature. It was concluded that, in fully expanded leaves, most chloroplasts contain no or only insignificant amounts of DNA long before the onset of leaf senescence (Oldenburg and Bendich, 2004; Rowan et al., 2004; Oldenburg et al., 2006; Shaver et al., 2006; Rowan et al., 2009). Retention of ptDNA was proposed to be dispensable after the photosynthetic machinery was established in that the plastome-encoded photosynthesis genes were no longer needed in adult leaves. Degradation or even entire loss of ptDNA was considered as an event during plastid and leaf development, common to all plants (Rowan et al., 2009). ptDNA degradation was also suggested to act as a signal inducing senescence (Sodmergen et al., 1991).A priori, there is no reason why different ptDNA patterns should not occur, and there is indeed evidence that organelle DNA can behave differently in different materials, both quantitatively and structurally (e.g., Selldén and Leech, 1981; Baumgartner et al., 1989). However, since contradictory data were reported for the same species that were grown under comparable, if not identical, conditions (Rowan et al., 2004, 2009; Li et al., 2006; Oldenburg et al., 2006; Shaver et al., 2006; Zoschke et al., 2007; Evans et al., 2010; Udy et al., 2012), it is apparent that some of them must reflect methodological insufficiencies of the experimental approaches employed.From a physiological point of view, the existence of DNA-deficient plastids in photosynthetically competent tissue seems unlikely. For instance, due to its susceptibility to photooxidative damage, the D1 protein (PsbA), a plastome-encoded core subunit of photosystem II, must be replaced continuously by a complex repair system to maintain photosynthesis (Prasil et al., 1992). This replacement requires de novo synthesis of the short-lived D1. There are no data available supporting an extreme mRNA stability, protein stability, or for another compensating biochemistry, preserving organelle functions for weeks or even months. The maximum mRNA half-life reported for psbA is in the range of 40 h (Kim et al., 1993).Resolving this controversy is of considerable scientific interest, both from a theoretical and an applied perspective. We therefore analyzed the fate of ptDNA in mature, ageing, and senescent leaves of four commonly studied higher plant species (Arabidopsis, sugar beet [Beta vulgaris], tobacco [Nicotiana tabacum], and maize; Figure 1) for which conflicting data have been reported. Four complementary methods were used for assessing the presence of ptDNA as well as its quantitative and morphological changes during leaf development: an improved 4′,6-diamidino-2-phenylindole (DAPI)–based fluorescence microscopy approach including deconvolution of fluorescence images, electron microscopy, semithin sectioning across leaf laminas, and real-time quantitative PCR (see Methods).Open in a separate windowFigure 1.Developmental Leaf Series of Sugar Beet, Tobacco, and Arabidopsis.(A) Sugar beet leaves, developmental stages II to VI (left to right; see text). Inset: leaf stages y1 and y3. Arrows indicate necrotic areas. Bar = 5 cm.(B) Tobacco leaves, developmental stages II and IV to VI. Inset: leaf stages y1 and y2. Bar = 5 cm; bar in inset = 1 cm.(C) Arabidopsis plants (left) from which leaves of developmental stages I to VI were taken. Bar = 4 cm.Figure 2, Supplemental Methods, and Supplemental Data Sets 1 to 4 present representative micrographs of developmental series of DAPI-stained chloroplasts in leaf spongy parenchyma cells of late ontogenetic stages from sugar beet, Arabidopsis, tobacco, and maize displaying clearly discernible nucleoid patterns. Figures 1A to 1C document some of the leaves from which samples were taken. Mesophyll cells of juvenile leaves investigated in our previous work (Li et al., 2006; Zoschke et al., 2007; Rauwolf et al., 2010) were included for comparison (Supplemental Data Sets 1 to 4, panels 1 to 37, 84 to 94, 112 to 117, and 123 to 128). The staining specificity of the fluorochrome was confirmed enzymatically. Treatment with DNase, but not DNase-free RNase or Proteinase K, either before or after staining with the fluorochrome, abolished the fluorescence but did not significantly affect chloroplast structure (compare with Rauwolf et al., 2010; see Methods).Open in a separate windowFigure 2.DAPI-DNA Fluorescence of Mature, Senescent, and Prenecrotic Leaf Mesophyll Cells or Cell Segments.Representative DAPI-stained squashed mesophyll cells of sugar beet ([A] to [C]), Arabidopsis ([D] to [F]), tobacco ([G] and [H]), and maize ([I] and [J]) leaflets or leaves (cell detail in [C], [E], [F], and [H]) of the developmental stages III/IV (I), IV ([A] and [D]), V ([B], [E], and [G]), and VI ([C], [F], [H], and [J]). Note that (E) represents a cell fragment of Supplemental Data Set 2, panel 102. Bar = 5 μm in (A), also for (B) to (J).     

Changes of the testes parameters,carcasses and antlers mass of harvested roe deer (Capreolus capreolus) bucks

The research was conducted during the hunting season in Poland from May to September 2012 in which materials of 58 roe deer bucks carcass, antler and testis masses were compared. In addition, the size of testes with epididymides was assessed. Significantly enlarged testes masses were observed in May till July (one-way ANOVA and LSD test). In August and September, the testes underwent abrupt involution. According to the literature and the results of this study, the enlargement of the testes which starts from the end of February, indicates that spring time is a preparatory period for the rut. On the other hand, monthly carcass mass fluctuations were not observed. There is, however, a trend suggesting that heavier roe deer individuals can display larger antler mass.