Directed Evolution and In Silico Analysis of Reaction Centre Proteins Reveal Molecular Signatures of Photosynthesis Adaptation to Radiation Pressure

Evolutionary mechanisms adopted by the photosynthetic apparatus to modifications in the Earth's atmosphere on a geological time-scale remain a focus of intense research. The photosynthetic machinery has had to cope with continuously changing environmental conditions and particularly with the complex ionizing radiation emitted by solar flares. The photosynthetic D1 protein, being the site of electron tunneling-mediated charge separation and solar energy transduction, is a hot spot for the generation of radiation-induced radical injuries. We explored the possibility to produce D1 variants tolerant to ionizing radiation in Chlamydomonas reinhardtii and clarified the effect of radiation-induced oxidative damage on the photosynthetic proteins evolution. In vitro directed evolution strategies targeted at the D1 protein were adopted to create libraries of chlamydomonas random mutants, subsequently selected by exposures to radical-generating proton or neutron sources. The common trend observed in the D1 aminoacidic substitutions was the replacement of less polar by more polar amino acids. The applied selection pressure forced replacement of residues more sensitive to oxidative damage with less sensitive ones, suggesting that ionizing radiation may have been one of the driving forces in the evolution of the eukaryotic photosynthetic apparatus. A set of the identified aminoacidic substitutions, close to the secondary plastoquinone binding niche and oxygen evolving complex, were introduced by site-directed mutagenesis in un-transformed strains, and their sensitivity to free radicals attack analyzed. Mutants displayed reduced electron transport efficiency in physiological conditions, and increased photosynthetic performance stability and oxygen evolution capacity in stressful high-light conditions. Finally, comparative in silico analyses of D1 aminoacidic sequences of organisms differently located in the evolution chain, revealed a higher ratio of residues more sensitive to oxidative damage in the eukaryotic/cyanobacterial proteins compared to their bacterial orthologs. These results led us to hypothesize an archaean atmosphere less challenging in terms of ionizing radiation than the present one

Atomic Force Microscope nanolithography on chromosomes to genrate single-cell genetic probes

Abstract Background Chromosomal dissection provides a direct advance for isolating DNA from cytogenetically recognizable region to generate genetic probes for fluorescence in situ hybridization, a technique that became very common in cyto and molecular genetics research and diagnostics. Several reports describing microdissection methods (glass needle or a laser beam) to obtain specific probes from metaphase chromosomes are available. Several limitations are imposed by the traditional methods of dissection as the need for a large number of chromosomes for the production of a probe. In addition, the conventional methods are not suitable for single chromosome analysis, because of the relatively big size of the microneedles. Consequently new dissection techniques are essential for advanced research on chromosomes at the nanoscale level. Results We report the use of Atomic Force Microscope (AFM) as a tool for nanomanipulation of single chromosomes to generate individual cell specific genetic probes. Besides new methods towards a better nanodissection, this work is focused on the combination of molecular and nanomanipulation techniques which enable both nanodissection and amplification of chromosomal and chromatidic DNA. Cross-sectional analysis of the dissected chromosomes reveals 20 nm and 40 nm deep cuts. Isolated single chromosomal regions can be directly amplified and labeled by the Degenerate Oligonucleotide-Primed Polymerase Chain Reaction (DOP-PCR) and subsequently hybridized to chromosomes and interphasic nuclei. Conclusions Atomic force microscope can be easily used to visualize and to manipulate biological material with high resolution and accuracy. The fluorescence in situ hybridization (FISH) performed with the DOP-PCR products as test probes has been tested succesfully in avian microchromosomes and interphasic nuclei. Chromosome nanolithography, with a resolution beyond the resolution limit of light microscopy, could be useful to the construction of chromosome band libraries and to the molecular cytogenetic mapping related to the investigation of genetic diseases.</p

Size segregated ionic species collected in a harbour area

Water-soluble ions were analysed in size segregated aerosol samples collected in the port of Alicante (Southeastern Spain) during summer and winter using a multistage cascade impactor. Seasonal variations in the size distributions of the analysed components and the influence of bulk materials handling (loading/unloading and stockpiling) at the docks were investigated. The size distributions of SO42−, NH4+ and K+ were characterized by prominent peaks in the condensation and droplet modes, both in summer and winter, while those of Ca2+, Na+, Mg2+ and Cl− had a main peak centred at ∼4 μm. Although oxalate size distributions were similar during both seasons, the fraction of coarse-mode oxalate increased in summer most likely as a result of volatilization and repartition processes or reactions of oxalic acid with coarse alkaline particles. Nitrate size distributions were dominated by a coarse mode; however, during winter, modal peaks in the submicron size range were also observed due to favourable conditions for the formation of fine-mode NH4NO3. Harbour activities had a significant impact only on the concentrations of calcium, particularly in the coarse fraction, during both summer and winter.This work was supported by the Spanish Ministry of Science, Innovation and Universities (COSMOS Project, ref. RTI2018-098639-B-I00). The authors would also like to thank ACTRIS-Spain network (CGL2017-90884-REDT). A. Clemente thanks the Spanish Ministry of Education for a predoctoral grant (FPU18/00081)

Relationships linking primary production, sea ice melting, and biogenic aerosol in the Arctic

AbstractThis study examines the relationships linking methanesulfonic acid (MSA, arising from the atmospheric oxidation of the biogenic dimethylsulfide, DMS) in atmospheric aerosol, satellite-derived chlorophyll a (Chl-a), and oceanic primary production (PP), also as a function of sea ice melting (SIM) and extension of the ice free area in the marginal ice zone (IF-MIZ) in the Arctic. MSA was determined in PM10 samples collected over the period 2010–2012 at two Arctic sites, Ny Ålesund (78.9°N, 11.9°E), Svalbard islands, and Thule Air Base (76.5°N, 68.8°W), Greenland. PP is calculated by means of a bio-optical, physiologically based, semi-analytical model in the potential source areas located in the surrounding oceanic regions (Barents and Greenland Seas for Ny Ålesund, and Baffin Bay for Thule). Chl-a peaks in May in the Barents sea and in the Baffin Bay, and has maxima in June in the Greenland sea; PP follows the same seasonal pattern of Chl-a, although the differences in absolute values of PP in the three seas during the blooms are less marked than for Chl-a. MSA shows a better correlation with PP than with Chl-a, besides, the source intensity (expressed by PP) is able to explain more than 30% of the MSA variability at the two sites; the other factors explaining the MSA variability are taxonomic differences in the phytoplanktonic assemblages, and transport processes from the DMS source areas to the sampling sites. The taxonomic differences are also evident from the slopes of the correlation plots between MSA and PP: similar slopes (in the range 34.2–36.2 ng m−3of MSA/(gC m−2 d−1)) are found for the correlation between MSA at Ny Ålesund and PP in Barents Sea, and between MSA at Thule and PP in the Baffin Bay; conversely, the slope of the correlation between MSA at Ny Ålesund and PP in the Greenland Sea in summer is smaller (16.7 ng m−3of MSA/(gC m−2 d−1)). This is due to the fact that DMS emission from the Barents Sea and Baffin Bay is mainly related to the MIZ diatoms, which are prolific DMS producers, whereas in the Greenland Sea the DMS peak is related to an offshore pelagic bloom where low-DMS producer species are present. The sea ice dynamic plays a key role in determining MSA concentration in the Arctic, and a good correlation between MSA and SIM (slope = 39 ng m−3 of MSA/106 km2 SIM) and between MSA and IF-MIZ (slope = 56 ng m−3 of MSA/106 km2 IF-MIZ) is found for the cases attributable to bloomings of diatoms in the MIZ. Such relationships are calculated by combining the data sets from the two sites and suggest that PP is related to sea ice melting and to the extension of marginal sea ice areas, and that these factors are the main drivers for MSA concentrations at the considered Arctic sites