SARS-CoV-2 is a novel positive-sense single-stranded RNA virus from the Coronaviridae family (genus Betacoronavirus), which has been established as causing the COVID-19 pandemic. The genome of SARS-CoV-2 is one of the largest among known RNA viruses, comprising of at least 26 known protein-coding loci. Studies thus far have outlined the coding capacity of the positive-sense strand of the SARS-CoV-2 genome, which can be used directly for protein translation. However, it has been recently shown that transcribed negative-sense viral RNA intermediates that arise during viral genome replication from positive-sense viruses can also code for proteins. No studies have yet explored the potential for negative-sense SARS-CoV-2 RNA intermediates to contain protein-coding loci. Thus, using sequence and structure-based bioinformatics methodologies, we have investigated the presence and validity of putative negative-sense ORFs (nsORFs) in the SARS-CoV-2 genome. Nine nsORFs were discovered to contain strong eukaryotic translation initiation signals and high codon adaptability scores, and several of the nsORFs were predicted to interact with RNA-binding proteins. Evolutionary conservation analyses indicated that some of the nsORFs are deeply conserved among related coronaviruses. Three-dimensional protein modeling revealed the presence of higher order folding among all putative SARS-CoV-2 nsORFs, and subsequent structural mimicry analyses suggest similarity of the nsORFs to DNA/RNA-binding proteins and proteins involved in immune signaling pathways. Altogether, these results suggest the potential existence of still undescribed SARS-CoV-2 proteins, which may play an important role in the viral lifecycle and COVID-19 pathogenesis.
- MeSH
- COVID-19 * genetika MeSH
- genom virový MeSH
- lidé MeSH
- pandemie MeSH
- proteiny vázající RNA genetika MeSH
- RNA virová chemie genetika MeSH
- SARS-CoV-2 * genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In Part I, by using 31P-NMR spectroscopy, we have shown that isolated granum and stroma thylakoid membranes (TMs), in addition to the bilayer, display two isotropic phases and an inverted hexagonal (HII) phase; saturation transfer experiments and selective effects of lipase and thermal treatments have shown that these phases arise from distinct, yet interconnectable structural entities. To obtain information on the functional roles and origin of the different lipid phases, here we performed spectroscopic measurements and inspected the ultrastructure of these TM fragments. Circular dichroism, 77 K fluorescence emission spectroscopy, and variable chlorophyll-a fluorescence measurements revealed only minor lipase- or thermally induced changes in the photosynthetic machinery. Electrochromic absorbance transients showed that the TM fragments were re-sealed, and the vesicles largely retained their impermeabilities after lipase treatments-in line with the low susceptibility of the bilayer against the same treatment, as reflected by our 31P-NMR spectroscopy. Signatures of HII-phase could not be discerned with small-angle X-ray scattering-but traces of HII structures, without long-range order, were found by freeze-fracture electron microscopy (FF-EM) and cryo-electron tomography (CET). EM and CET images also revealed the presence of small vesicles and fusion of membrane particles, which might account for one of the isotropic phases. Interaction of VDE (violaxanthin de-epoxidase, detected by Western blot technique in both membrane fragments) with TM lipids might account for the other isotropic phase. In general, non-bilayer lipids are proposed to play role in the self-assembly of the highly organized yet dynamic TM network in chloroplasts.
Build-up of the energized state of thylakoid membranes and the synthesis of ATP are warranted by organizing their bulk lipids into a bilayer. However, the major lipid species of these membranes, monogalactosyldiacylglycerol, is a non-bilayer lipid. It has also been documented that fully functional thylakoid membranes, in addition to the bilayer, contain an inverted hexagonal (HII) phase and two isotropic phases. To shed light on the origin of these non-lamellar phases, we performed 31P-NMR spectroscopy experiments on sub-chloroplast particles of spinach: stacked, granum and unstacked, stroma thylakoid membranes. These membranes exhibited similar lipid polymorphism as the whole thylakoids. Saturation transfer experiments, applying saturating pulses at characteristic frequencies at 5 °C, provided evidence for distinct lipid phases-with component spectra very similar to those derived from mathematical deconvolution of the 31P-NMR spectra. Wheat-germ lipase treatment of samples selectively eliminated the phases exhibiting sharp isotropic peaks, suggesting easier accessibility of these lipids compared to the bilayer and the HII phases. Gradually increasing lipid exchanges were observed between the bilayer and the two isotropic phases upon gradually elevating the temperature from 5 to 35 °C, suggesting close connections between these lipid phases. Data concerning the identity and structural and functional roles of different lipid phases will be presented in the accompanying paper.
Light quality significantly influences plant metabolism, growth and development. Recently, we have demonstrated that leaves of barley and other plant species grown under monochromatic green light (500-590 nm) accumulated a large pool of chlorophyll a (Chl a) intermediates with incomplete hydrogenation of their phytyl chains. In this work, we studied accumulation of these geranylgeranylated Chls a and b in pigment-protein complexes (PPCs) of Arabidopsis plants acclimated to green light and their structural-functional consequences on the photosynthetic apparatus. We found that geranylgeranylated Chls are present in all major PPCs, although their presence was more pronounced in light-harvesting complex II (LHCII) and less prominent in supercomplexes of photosystem II (PSII). Accumulation of geranylgeranylated Chls hampered the formation of PSII and PSI super- and megacomplexes in the thylakoid membranes as well as their assembly into chiral macrodomains; it also lowered the temperature stability of the PPCs, especially that of LHCII trimers, which led to their monomerization and an anomaly in the photoprotective mechanism of non-photochemical quenching. Role of geranylgeranylated Chls in adverse effects on photosynthetic apparatus of plants acclimated to green light is discussed.
SARS-CoV-2 is an intensively investigated virus from the order Nidovirales (Coronaviridae family) that causes COVID-19 disease in humans. Through enormous scientific effort, thousands of viral strains have been sequenced to date, thereby creating a strong background for deep bioinformatics studies of the SARS-CoV-2 genome. In this study, we inspected high-frequency mutations of SARS-CoV-2 and carried out systematic analyses of their overlay with inverted repeat (IR) loci and CpG islands. The main conclusion of our study is that SARS-CoV-2 hot-spot mutations are significantly enriched within both IRs and CpG island loci. This points to their role in genomic instability and may predict further mutational drive of the SARS-CoV-2 genome. Moreover, CpG islands are strongly enriched upstream from viral ORFs and thus could play important roles in transcription and the viral life cycle. We hypothesize that hypermethylation of these loci will decrease the transcription of viral ORFs and could therefore limit the progression of the disease.
- MeSH
- COVID-19 virologie MeSH
- CpG ostrůvky * MeSH
- genom virový MeSH
- lidé MeSH
- metylace DNA MeSH
- mutace * MeSH
- SARS-CoV-2 genetika MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Barley (Hordeum vulgare) accumulates phenolic compounds (PhCs), which play a key role in plant defense against environmental stressors as antioxidants or UV screening compounds. The influence of light and atmospheric CO2 concentration ([CO2]) on the accumulation and localization of PhCs in barley leaves was examined for two varieties with different tolerances to oxidative stress. PhC localization was visualized in vivo using fluorescence microscopy. Close relationships were found between fluorescence-determined localization of PhCs in barley leaves and PhC content estimated using liquid chromatography coupled with mass spectroscopy detection. Light intensity had the strongest effect on the accumulation of PhCs, but the total PhC content was similar at elevated [CO2], minimizing the differences between high and low light. PhCs localized preferentially near the surfaces of leaves, but under low light, an increasing allocation of PhCs in deeper mesophyll layers was observed. The PhC profile was significantly different between barley varieties. The relatively tolerant variety accumulated significantly more hydroxycinnamic acids, indicating that these PhCs may play a more prominent role in oxidative stress prevention. Our research presents novel evidence that [CO2] modulates the accumulation of PhCs in barley leaves. Mesophyll cells, rather than epidermal cells, were most responsive to environmental stimuli in terms of PhC accumulation.
- Publikační typ
- časopisecké články MeSH
The role of non-bilayer lipids and non-lamellar lipid phases in biological membranes is an enigmatic problem of membrane biology. Non-bilayer lipids are present in large amounts in all membranes; in energy-converting membranes they constitute about half of their total lipid content-yet their functional state is a bilayer. In vitro experiments revealed that the functioning of the water-soluble violaxanthin de-epoxidase (VDE) enzyme of plant thylakoids requires the presence of a non-bilayer lipid phase. 31P-NMR spectroscopy has provided evidence on lipid polymorphism in functional thylakoid membranes. Here we reveal reversible pH- and temperature-dependent changes of the lipid-phase behaviour, particularly the flexibility of isotropic non-lamellar phases, of isolated spinach thylakoids. These reorganizations are accompanied by changes in the permeability and thermodynamic parameters of the membranes and appear to control the activity of VDE and the photoprotective mechanism of non-photochemical quenching of chlorophyll-a fluorescence. The data demonstrate, for the first time in native membranes, the modulation of the activity of a water-soluble enzyme by a non-bilayer lipid phase.
- MeSH
- diferenciální skenovací kalorimetrie MeSH
- epoxidové sloučeniny metabolismus MeSH
- kinetika MeSH
- koncentrace vodíkových iontů MeSH
- lipidové dvojvrstvy chemie MeSH
- lipidy chemie MeSH
- magnetická rezonanční spektroskopie MeSH
- oxidoreduktasy metabolismus MeSH
- rozpustnost MeSH
- Spinacia oleracea metabolismus MeSH
- světlo MeSH
- teplota MeSH
- tylakoidy chemie MeSH
- voda chemie MeSH
- xanthofyly metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Earlier experiments, using 31 P-NMR and time-resolved merocyanine fluorescence spectroscopy, have shown that isolated intact, fully functional plant thylakoid membranes, in addition to the bilayer phase, contain three non-bilayer (or non-lamellar) lipid phases. It has also been shown that the lipid polymorphism of thylakoid membranes can be characterized by remarkable plasticity, i.e. by significant variations in 31 P-NMR signatures. However, changes in the lipid-phase behaviour of thylakoids could not be assigned to changes in the overall membrane organization and the photosynthetic activity, as tested by circular dichroism and 77 K fluorescence emission spectroscopy and the magnitude of the variable fluorescence of photosystem II, which all showed only marginal variations. In this work, we investigated in more detail the temporal stability of the different lipid phases by recording 31 P-NMR spectra on isolated thylakoid membranes that were suspended in sorbitol- or NaCl-based media. We observed, at 5°C during 8 h in the dark, substantial gradual enhancement of the isotropic lipid phases and diminishment of the bilayer phase in the sorbitol-based medium. These changes compared well with the gradually increasing membrane permeability, as testified by the gradual acceleration of the decay of flash-induced electrochromic absorption changes and characteristic changes in the kinetics of fast chlorophyll a-fluorescence transients; all variations were much less pronounced in the NaCl-based medium. These observations suggest that non-bilayer lipids and non-lamellar lipid phases play significant roles in the structural dynamics and functional plasticity of thylakoid membranes.
Photosystem II (PSII) catalyses the photoinduced oxygen evolution and, by producing reducing equivalents drives, in concert with PSI, the conversion of carbon dioxide to sugars. Our knowledge about the architecture of the reaction centre (RC) complex and the mechanisms of charge separation and stabilisation is well advanced. However, our understanding of the processes associated with the functioning of RC is incomplete: the photochemical activity of PSII is routinely monitored by chlorophyll-a fluorescence induction but the presently available data are not free of controversy. In this work, we examined the nature of gradual fluorescence rise of PSII elicited by trains of single-turnover saturating flashes (STSFs) in the presence of a PSII inhibitor, permitting only one stable charge separation. We show that a substantial part of the fluorescence rise originates from light-induced processes that occur after the stabilisation of charge separation, induced by the first STSF; the temperature-dependent relaxation characteristics suggest the involvement of conformational changes in the additional rise. In experiments using double flashes with variable waiting times (∆τ) between them, we found that no rise could be induced with zero or short ∆τ, the value of which depended on the temperature - revealing a previously unknown rate-limiting step in PSII.
- MeSH
- chlorofyl a metabolismus MeSH
- fluorescence * MeSH
- fotosyntéza MeSH
- fotosystém II - proteinový komplex antagonisté a inhibitory metabolismus MeSH
- Spinacia oleracea metabolismus MeSH
- Synechococcus metabolismus MeSH
- Synechocystis metabolismus MeSH
- teplota MeSH
- tylakoidy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Chloroplast thylakoid membranes contain virtually all components of the energy-converting photosynthetic machinery. Their energized state, driving ATP synthesis, is enabled by the bilayer organization of the membrane. However, their most abundant lipid species is a non-bilayer-forming lipid, monogalactosyl-diacylglycerol; the role of lipid polymorphism in these membranes is poorly understood. Earlier 31P-NMR experiments revealed the coexistence of a bilayer and a non-bilayer, isotropic lipid phase in spinach thylakoids. Packing of lipid molecules, tested by fluorescence spectroscopy of the lipophilic dye, merocyanine-540 (MC540), also displayed heterogeneity. Now, our 31P-NMR experiments on spinach thylakoids uncover the presence of a bilayer and three non-bilayer lipid phases; time-resolved fluorescence spectroscopy of MC540 also reveals the presence of multiple lipidic environments. It is also shown by 31P-NMR that: (i) some lipid phases are sensitive to the osmolarity and ionic strength of the medium, (ii) a lipid phase can be modulated by catalytic hydrogenation of fatty acids and (iii) a marked increase of one of the non-bilayer phases upon lowering the pH of the medium is observed. These data provide additional experimental evidence for the polymorphism of lipid phases in thylakoids and suggest that non-bilayer phases play an active role in the structural dynamics of thylakoid membranes.
