Light-independent accumulation of essential chlorophyll biosynthesis- and photosynthesis-related proteins in <Emphasis Type="Italic">Pinus mugo</Emphasis> and <Emphasis Type="Italic">Pinus sylvestris</Emphasis> seedlings

Dark-grown seedlings of Pinus mugo Turra and Pinus sylvestris L. accumulate chlorophyll (Chl) and its precursor protochlorophyllide (Pchlide). Pchlide reduction is a key regulatory step in Chl biosynthesis. In the dark, Pchlide is reduced by light-independent Pchlide oxidoreductase (DPOR) encoded by three plastid genes chlL, chlN, and chlB (chlLNB). To investigate the differences in chlLNB gene expressions, we compared the dark-grown and 24-h illuminated seedlings of P. mugo and P. sylvestris. Expression of these genes was found constitutive in all analyzed samples. We report light-independent accumulation of important proteins involved in Chl biosynthesis (glutamyl-tRNA reductase) and photosystem formation (D1 and LHCI). Chl and Pchlide content and plastid ultrastructure studies were also performed.     

A unified model for the origin of DNA sequence-directed curvature

The fine structure of the DNA double helix and a number of its physical properties depend upon nucleotide sequence. This includes minor groove width, the propensity to undergo the B-form to A-form transition, sequence-directed curvature, and cation localization. Despite the multitude of studies conducted on DNA, it is still difficult to appreciate how these fundamental properties are linked to each other at the level of nucleotide sequence. We demonstrate that several sequence-dependent properties of DNA can be attributed, at least in part, to the sequence-specific localization of cations in the major and minor grooves. We also show that effects of cation localization on DNA structure are easier to understand if we divide all DNA sequences into three principal groups: A-tracts, G-tracts, and generic DNA. The A-tract group of sequences has a peculiar helical structure (i.e., B*-form) with an unusually narrow minor groove and high base-pair propeller twist. Both experimental and theoretical studies have provided evidence that the B*-form helical structure of A-tracts requires cations to be localized in the minor groove. G-tracts, on the other hand, have a propensity to undergo the B-form to A-form transition with increasing ionic strength. This property of G-tracts is directly connected to the observation that cations are preferentially localized in the major groove of G-tract sequences. Generic DNA, which represents the vast majority of DNA sequences, has a more balanced occupation of the major and minor grooves by cations than A-tracts or G-tracts and is thereby stabilized in the canonical B-form helix. Thus, DNA secondary structure can be viewed as a tug of war between the major and minor grooves for cations, with A-tracts and G-tracts each having one groove that dominates the other for cation localization. Finally, the sequence-directed curvature caused by A-tracts and G-tracts can, in both cases, be explained by the cation-dependent mismatch of A-tract and G-tract helical structures with the canonical B-form helix of generic DNA (i.e., a cation-dependent junction model).     

The solution structure of d(G(4)T(4)G(3))(2): a bimolecular G-quadruplex with a novel fold

The G-rich 11-mer oligonucleotide d(G(4)T(4)G(3)) forms a bimolecular G-quadruplex in the presence of sodium ions with a topology that is distinct from the folds of the closely related and well-characterized sequences d(G(4)T(4)G(4)) and d(G(3)T(4)G(3)). The solution structure of d(G(4)T(4)G(3))(2) has been determined using a combination of NMR spectroscopy and restrained molecular dynamics calculations. d(G(4)T(4)G(3))(2) forms an asymmetric dimeric fold-back structure consisting of three stacked G-quartets. The two T(4) loops that span diagonally across the outer faces of the G-quartets assume different conformations. The glycosidic torsion angle conformations of the guanine bases are 5'-syn-anti-syn-anti-(T(4) loop)-anti-syn-anti in one strand and 5'-syn-anti-syn-anti-(T(4) loop)-syn-anti-syn in the other strand. The guanine bases of the two outer G-quartets exhibit a clockwise donor-acceptor hydrogen-bonding directionality, while those of the middle G-quartet exhibit the anti-clockwise directionality. The topology of this G-quadruplex, like other bimolecular fold-back structures with diagonal loops, places each strand of the G-quartet region next to a neighboring parallel and an anti-parallel strand. The two guanine residues not involved in G-quartet formation, G4 and G12 (i.e. the fourth guanine base of one strand and the first guanine base of the other strand), adopt distinct conformations. G4 is stacked on top of an adjacent G-quartet, and this base-stacking continues along with the bases of the loop residues T5 and T6. G12 is orientated away from the core of G-quartets; stacked on the T7 base and apparently involved in hydrogen-bonding interactions with the phosphodiester group of this same residue. The cation-dependent folding of the d(G(4)T(4)G(3))(2) quadruplex structure is distinct from that observed for similar sequences. While both d(G(4)T(4)G(4)) and d(G(3)T(4)G(3)) form bimolecular, diagonally looped G-quadruplex structures in the presence of Na(+), K(+) and NH(4)(+), we have observed this folding to be favored for d(G(4)T(4)G(3)) in the presence of Na(+), but not in the presence of K(+) or NH(4)(+). The structure of d(G(4)T(4)G(3))(2) exhibits a "slipped-loop" element that is similar to what has been proposed for structural intermediates in the folding pathway of some G-quadruplexes, and therefore provides support for the feasibility of these proposed transient structures in G-quadruplex formation.     

Bacterial Community Response to Petroleum Hydrocarbon Amendments in Freshwater,Marine, and Hypersaline Water-Containing Microcosms

Hydrocarbon-degrading bacterial communities from freshwater, marine, and hypersaline Brazilian aquatic ecosystems (with water salinities corresponding to 0.2%, 4%, and 5%, respectively) were enriched with different hydrocarbons (heptadecane, naphthalene, or crude oil). Changes within the different microcosms of bacterial communities were analyzed using cultivation approaches and molecular methods (DNA and RNA extraction, followed by genetic fingerprinting and analyses of clone libraries based on the 16S rRNA-coding gene). A redundancy analysis (RDA) of the genetic fingerprint data and a principal component analysis (PCA) of the clone libraries revealed hydrocarbon-enriched bacterial communities specific for each ecosystem studied. However, within the same ecosystem, different bacterial communities were selected according to the petroleum hydrocarbon used. In general, the results demonstrated that Acinetobacter and Cloacibacterium were the dominant genera in freshwater microcosms; the Oceanospirillales order and the Marinobacter, Pseudomonas, and Cycloclasticus genera predominated in marine microcosms; and the Oceanospirillales order and the Marinobacter genus were selected in the different hydrocarbon-containing microcosms in hypersaline water. Determination of total petroleum hydrocarbons (TPHs) in all microcosms after 32 days of incubation showed a decrease in the hydrocarbon concentration compared to that for the controls. A total of 50 (41.3%) isolates from the different hydrocarbon-contaminated microcosms were associated with the dominant operational taxonomic units (OTUs) obtained from the clone libraries, and their growth in the hydrocarbon contaminating the microcosm from which they were isolated as the sole carbon source was observed. These data provide insight into the general response of bacterial communities from freshwater, marine, and hypersaline aquatic ecosystems to petroleum hydrocarbon contamination.     

Condensation of oligonucleotides assembled into nicked and gapped duplexes: potential structures for oligonucleotide delivery

The condensation of nucleic acids into well-defined particles is an integral part of several approaches to artificial cellular delivery. Improvements in the efficiency of nucleic acid delivery in vivo are important for the development of DNA- and RNA-based therapeutics. Presently, most efforts to improve the condensation and delivery of nucleic acids have focused on the synthesis of novel condensing agents. However, short oligonucleotides are not as easy to condense into well-defined particles as gene-length DNA polymers and present particular challenges for discrete particle formation. We describe a novel strategy for improving the condensation and packaging of oligonucleotides that is based on the self-organization of half-sliding complementary oligonucleotides into long duplexes (ca. 2 kb). These non-covalent assemblies possess single-stranded nicks or single-stranded gaps at regular intervals along the duplex backbones. The condensation behavior of nicked- and gapped-DNA duplexes was investigated using several cationic condensing agents. Transmission electron microscopy and light-scattering studies reveal that these DNA duplexes condense much more readily than short duplex oligonucleotides (i.e. 21 bp), and more easily than a 3 kb plasmid DNA. The polymeric condensing agents, poly-l-lysine and polyethylenimine, form condensates with nicked- and gapped-DNA that are significantly smaller than condensates formed by the 3 kb plasmid DNA. These results demonstrate the ability for DNA structure and topology to alter nucleic acid condensation and suggest the potential for the use of this form of DNA in the design of vectors for oligonucleotide and gene delivery. The results presented here also provide new insights into the role of DNA flexibility in condensate formation.     

Chlorophyll biosynthesis and chloroplast development in etiolated seedlings of <Emphasis Type="Italic">Ginkgo biloba</Emphasis> L.

Ginkgo biloba L. is a large tree native in China with evolutionary affinities to the conifers and cycads. However unlike conifers, the gymnosperm G. biloba is not able to synthesize chlorophyll (Chl) in the dark, in spite of the presence of genes encoding subunits of light-independent protochlorophyllide oxidoreductase (DPOR) in the plastid genome. The principal aims of the present study were to investigate the presence of DPOR protein subunits (ChlL, ChlN, ChlB) as well as the key regulatory step in Chl formation: aminolevulinic acid (ALA) synthesis and abundance of the key regulatory enzyme in its synthesis: glutamyl-tRNA reductase (GluTR). In addition, functional stage of photosynthetic apparatus and assembly of pigment-protein complexes were investigated. Dark-grown, illuminated and circadian-grown G. biloba seedlings were used in our experiments. Our results clearly showed that no protein subunits of DPOR were detected irrespective of light conditions, what is consistent with the absence of Chl and Chl-binding proteins (D1, LHCI, LHCIIb) in the dark. This correlates with low ALA-synthesizing capacity and low amount of GluTR. The concentration of protochlorophyllide (Pchlide) in the dark is low and non-photoactive form (Pchlide₆₃₃) was predominant. Plastids were developed as typical etioplasts with prollamelar body and few prothylakoid membranes. Continual illumination (24 h) only slightly stimulated ALA and Chl synthesis, although Pchlide content was reduced. Prollamelar bodies disappeared, but no grana were formed, what was consistent with the absence of D1, LHCI, LHCIIb proteins. Lightinduced development of photosynthetic apparatus is extremely slow, as indicated by Chl fluorescence and gas exchange measurements. Even after 72 h of continuous illumination, the values of maximum (F_v/F_m) and effective quantum yield (Φ_PSII) and rate of net photosynthesis (P _N) did not reach the values comparable with circadian-grown plants.     

Changing the Chevreul illusion by a background luminance ramp: lateral inhibition fails at its traditional stronghold--a psychophysical refutation

The Chevreul illusion is a well-known 19^th century brightness illusion, comprising adjacent homogeneous grey bands of different luminance, which are perceived as inhomogeneous. It is generally explained by lateral inhibition, according to which brighter areas projected to the retina inhibit the sensitivity of neighbouring retinal areas. Lateral inhibition has been considered the foundation-stone of early vision for a century, upon which several computational models of brightness perception are built. One of the last strongholds of lateral inhibition is the Chevreul illusion, which is often illustrated even in current textbooks. Here we prove that lateral inhibition is insufficient to explain the Chevreul illusion. For this aim, we placed the Chevreul staircase in a luminance ramp background, which noticeably changed the illusion. In our psychophysical experiments, all 23 observers reported a strong illusion, when the direction of the ramp was identical to that of the staircase, and all reported homogeneous steps (no illusion) when its direction was the opposite. When the background of the staircase was uniform, 14 saw the illusion, and 9 saw no illusion. To see whether the change of the entire background area or that of the staircase boundary edges were more important, we placed another ramp around the staircase, whose direction was opposite to that of the original, larger ramp. The result is that though the inner ramp is rather narrow (mean = 0.51 deg, SD = 0.48 deg, N = 23), it still dominates perception. Since all conditions of the lateral inhibition account were untouched within the staircase, lateral inhibition fails to model these perceptual changes. Area ratios seem insignificant; the role of boundary edges seems crucial. We suggest that long range interactions between boundary edges and areas enclosed by them, such that diffusion-based models describe, provide a much more plausible account for these brightness phenomena, and local models are insufficient.     

20 Supramolecular polymerization of nucleobase-like monomers in water

Elucidating the physiochemical principles that govern molecular self-assembly is of great importance for understanding biological systems and may provide insight into the emergence of the earliest macromolecules of life, an important challenge facing the RNA World hypothesis. Self-assembly results from a delicate balance between multiple noncovalent interactions and solvent effects, but achieving efficient self-assembly in aqueous solution with synthetic molecules has proven particularly challenging. Here, we demonstrate how two physical properties – monomer solubility and large hydrophobic surfaces of intermediate structures – are key elements to achieving supramolecular polymers in aqueous solution (Cafferty et al., 2013). Applying these two principles, we report the highly cooperative self-assembly of two weakly interacting, low molecular weight monomers [cyanuric acid and a modified triaminopyrimidine] into a water-soluble supramolecular assembly (see scheme below). The observed equilibrium between only two appreciably populated states – free monomers and supramolecular assemblies – is in excellent agreement with the values previously determined for the free energy of hydrogen bonding (Klostermeier & Millar, 2002), π???π stacking (Frier et al., 1985), and the calculated free energy penalty for the solvation of hydrophobic structures in water (Chandler, 2005). The similarity of the molecules used in this study for the nucleobases found in contemporary nucleic acids and the demonstration that these monomers assemble while the natural nucleobases do not, suggests that the first informational polymers may have emerged from a similar self-assembly process, if the nucleobases were different then they are today (Hud et al., 2013).     

Influence of irradiance on chlorophyll synthesis in Picea abies calli cultures

Dark-grown seedlings of Picea abies (L) Karst. are able to accumulate the highest amounts of chlorophyll (Chl) and its precursor protochlorophyllide (Pchlide) in all Pinaceae, but calli derived from 14-d-old green cotyledons of P. abies are completely white during the cultivation in the dark. Pchlide reduction is catalysed in the dark by light-independent protochlorophyllide oxidoreductase (DPOR). This enzyme complex consists of three protein subunits ChlL, ChlN and ChlB, encoded by three plastid genes chlL, chlN and chlB. Using semiquantitative RT-PCR, we observed very low expression of chlLNB genes in dark-grown calli. It seems, that chlLNB expression and thus Chl accumulation could be modulated by light in P. abies calli cultures. This hypothesis is supported by the fact, that we observed low contents of glutamyl-tRNA reductase and Flu-like protein, which probably affected Chl biosynthetic pathway at the step of 5-aminolevulinic acid formation. ChlB subunit was not detected in dark-grown P. abies calli cultures. Our results indicated limited ability to synthesize Chl in callus during cultivation in the dark.     

A short overview of chlorophyll biosynthesis in algae

Chlorophylls are the most abundant classes of natural pigments and their biosynthesis is therefore a major metabolic activity in the ecosphere. Two pathways exist for chlorophyll biosynthesis, one taking place in darkness and the other requiring continuous light as a precondition. The key process for Chl synthesis is the reduction of protochlorophyllide (Pchlide). This enzymatic reaction is catalysed by two different enzymes — DPOR (dark-operative Pchlide oxidoreductase) or the structurally distinct LPOR (light-dependent Pchlide oxidoreductase). DPOR which consists of three subunits encoded by three plastid genes in eukaryotes was subject of our study. A short overview of our present knowledge of chlorophyll biosynthesis in Chlamydomonas reinhardtii in comparison with other plants is presented. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.