Plastids of diatoms and related algae with complex plastids of red algal origin are surrounded by four membranes, which also define the periplastidic compartment (PPC), the space between the second and third membranes. Metabolic reactions as well as cell biological processes take place in the PPC; however, genome-wide predictions of the proteins targeted to this compartment were so far based on manual annotation work. Using published experimental protein localizations as reference data, we developed the first automatic prediction method for PPC proteins, which we included as a new feature in an updated version of the plastid protein predictor ASAFind. With our method, at least a subset of the PPC proteins can be predicted with high specificity, with an estimate of at least 81 proteins (0.7% of the predicted proteome) targeted to the PPC in the model diatom Phaeodactylum tricornutum. The proportion of PPC proteins varies, since 180 PPC proteins (1.3% of the predicted proteome) were predicted in the genome of the diatom Thalassiosira pseudonana. The new ASAFind version can also generate a newly designed graphical output that visualizes the contribution of each position in the sequence to the score and accepts the output of the recent versions of SignalP (5.0) and TargetP (2.0) as input data. Furthermore, we release a script to calculate custom scoring matrices that can be used for predictions in a simplified score cut-off mode. This allows for adjustments of the method to other groups of algae.

• Klíčová slova
• chloroplast, diatoms, evolution, gene transfer, genome annotation, mitochondria, organelle, periplastidic compartment, protein transport, secretory pathway, technical advance,
• MeSH
• bílkoviny řas * metabolismus   MeSH
• plastidy * metabolismus   MeSH
• proteom MeSH
• Rhodophyta metabolismus   MeSH
• rozsivky * metabolismus   genetika   MeSH
• software * MeSH
• výpočetní biologie * metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bílkoviny řas * MeSH
• proteom MeSH

The plant-signaling molecule auxin triggers fast and slow cellular responses across land plants and algae. The nuclear auxin pathway mediates gene expression and controls growth and development in land plants, but this pathway is absent from algal sister groups. Several components of rapid responses have been identified in Arabidopsis, but it is unknown if these are part of a conserved mechanism. We recently identified a fast, proteome-wide phosphorylation response to auxin. Here, we show that this response occurs across 5 land plant and algal species and converges on a core group of shared targets. We found conserved rapid physiological responses to auxin in the same species and identified rapidly accelerated fibrosarcoma (RAF)-like protein kinases as central mediators of auxin-triggered phosphorylation across species. Genetic analysis connects this kinase to both auxin-triggered protein phosphorylation and rapid cellular response, thus identifying an ancient mechanism for fast auxin responses in the green lineage.

• Klíčová slova
• RAF kinase, auxin, plant evolution, protein phosphorylation,
• MeSH
• Arabidopsis genetika   metabolismus   MeSH
• bílkoviny řas metabolismus   MeSH
• fosforylace MeSH
• kyseliny indoloctové metabolismus   MeSH
• proteinkinasy metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné proteiny metabolismus   MeSH
• rostliny metabolismus   MeSH
• signální transdukce * MeSH
• vyšší rostliny * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bílkoviny řas MeSH
• kyseliny indoloctové MeSH
• proteinkinasy MeSH
• rostlinné proteiny MeSH

Elicitins are proteinaceous elicitors that induce the hypersensitive response and plant resistance against diverse phytopathogens. Elicitin recognition by membrane receptors or high-affinity sites activates a variety of fast responses including the production of reactive oxygen species (ROS) and nitric oxide (NO), leading to induction of plant defense genes. Beta-cryptogein (CRY) is a basic β-elicitin secreted by the oomycete Phytophthora cryptogea that shows high necrotic activity in some plant species, whereas infestin 1 (INF1) secreted by the oomycete P. infestans belongs to acidic α-elicitins with a significantly weaker capacity to induce necrosis. We compared several mutated forms of β-CRY and INF1 with a modulated capacity to trigger ROS and NO production, bind plant sterols and induce cell death responses in cell cultures of Nicotiana tabacum L. cv. Xanthi. We evidenced a key role of the lysine residue in position 13 in basic elicitins for their biological activity and enhancement of necrotic effects of acidic INF1 by the replacement of the valine residue in position 84 by larger phenylalanine. Studied elicitins activated in differing intensity signaling pathways of ROS, NO and phytohormones jasmonic acid, ethylene and salicylic acid, known to be involved in triggering of hypersensitive response and establishment of systemic resistance.

• Klíčová slova
• Cryptogein, Elicitins, Infestin, Nitric oxide, Plant defense responses, Reactive oxygen species,
• MeSH
• bílkoviny řas genetika   MeSH
• dusík * MeSH
• fungální proteiny metabolismus   MeSH
• kyslík MeSH
• Phytophthora * MeSH
• reaktivní formy kyslíku MeSH
• rostliny metabolismus   MeSH
• sekvence aminokyselin MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bílkoviny řas MeSH
• dusík * MeSH
• fungální proteiny MeSH
• kyslík MeSH
• reaktivní formy kyslíku MeSH

Antenna proteins play a major role in the regulation of light-harvesting in photosynthesis. However, less is known about a possible link between their sizes (oligomerization state) and fluorescence intensity (number of photons emitted). Here, we used a microscopy-based method, Fluorescence Correlation Spectroscopy (FCS), to analyze different antenna proteins at the particle level. The direct comparison indicated that Chromera Light Harvesting (CLH) antenna particles (isolated from Chromera velia) behaved as the monomeric Light Harvesting Complex II (LHCII) (from higher plants), in terms of their radius (based on the diffusion time) and fluorescence yields. FCS data thus indicated a monomeric oligomerization state of algal CLH antenna (at our experimental conditions) that was later confirmed also by biochemical experiments. Additionally, our data provide a proof of concept that the FCS method is well suited to measure proteins sizes (oligomerization state) and fluorescence intensities (photon counts) of antenna proteins per single particle (monomers and oligomers). We proved that antenna monomers (CLH and LHCIIm) are more "quenched" than the corresponding trimers. The FCS measurement thus represents a useful experimental approach that allows studying the role of antenna oligomerization in the mechanism of photoprotection.

• Klíčová slova
• Chromera velia, antenna proteins, fluorescence correlation spectroscopy, light-harvesting, microscopy, photosynthesis, protein diffusion, protein oligomerization,
• MeSH
• bílkoviny řas chemie   metabolismus   MeSH
• fluorescence * MeSH
• fluorescenční spektrometrie MeSH
• fotosyntéza * MeSH
• kinetika MeSH
• multimerizace proteinu MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bílkoviny řas MeSH

Programmable site-specific nucleases, such as the clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas (Chlamydomonas reinhardtii). However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous double-stranded DNA (dsDNA) encoding a selectable marker gene. Repair of the dsDNA breaks induced by the RNPs is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. Here, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker)-in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precise edits in the homolog of bacterial ftsY or the WD and TetratriCopeptide repeats protein 1 genes in ∼1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonas acetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways, or structures.

• MeSH
• bílkoviny řas genetika   MeSH
• Chlamydomonas genetika   MeSH
• CRISPR-Cas systémy * MeSH
• editace genu metody   MeSH
• ribonukleoproteiny genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• bílkoviny řas MeSH
• ribonukleoproteiny MeSH

Temperature is one of the key factors affecting growth and division of algal cells. High temperature inhibits the cell cycle in Chlamydomonas reinhardtii. At 39 °C, nuclear and cellular divisions in synchronized cultures were blocked completely, while DNA replication was partly affected. In contrast, growth (cell volume, dry matter, total protein, and RNA) remained unaffected, and starch accumulated at very high levels. The cell cycle arrest could be removed by transfer to 30 °C, but a full recovery occurred only in cultures cultivated up to 14 h at 39 °C. Thereafter, individual cell cycle processes began to be affected in sequence; daughter cell release, cell division, and DNA replication. Cell cycle arrest was accompanied by high mitotic cyclindependent kinase activity that decreased after completion of nuclear and cellular division following transfer to 30 °C. Cell cycle arrest was, therefore, not caused by a lack of cyclin-dependent kinase activity but rather a blockage in downstream processes.

• Klíčová slova
• Chlamydomonas reinhardtii, DNA replication, cell cycle arrest, cell size, cyclin-dependent kinase, starch accumulation, supraoptimal temperature, synchronized cultures,
• MeSH
• bílkoviny řas metabolismus   MeSH
• buněčné kultury metody   MeSH
• Chlamydomonas reinhardtii cytologie   fyziologie   MeSH
• cyklin-dependentní kinasy metabolismus   MeSH
• down regulace MeSH
• fyziologický stres MeSH
• kontrolní body buněčného cyklu * MeSH
• regulace genové exprese u rostlin MeSH
• vysoká teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bílkoviny řas MeSH
• cyklin-dependentní kinasy MeSH

Mitogen-activated protein kinase (MAPK) pathways are central cellular signalling mechanisms in all eukaryotes. They are key regulators of the cell cycle and stress responses, yet evolution of MAPK families took markedly different paths in the animal and plant kingdoms. Instead of the characteristic divergence of MAPK types in animals, in plants an expanded network of ERK-like MAPKs has emerged. To gain insight into the early evolution of the plant MAPK family we identified and analysed MAPKs in 13 representative species across green algae, a large and diverse early-diverging lineage within the plant kingdom. Our results reveal that the plant MAPK gene family emerged from three types of progenitor kinases, which are ubiquitously present in algae, implying their formation in an early ancestor. Low number of MAPKs is characteristic across algae, the few losses or duplications are associated with genome complexity rather than habitat ecology, despite the importance of MAPKs in environmental signalling in flowering plants. ERK-type MAPKs are associated with cell cycle regulation in opisthokont models, yet in plants their stress-signalling function is more prevalent. Unicellular microalgae offer an excellent experimental system to study the cell cycle, and MAPK gene expression profiles show CDKB-like peaks around S/M phase in synchronised Chlamydomonas reinhardtii cultures, suggesting their participation in cell cycle regulation, in line with the notion that the ancestral eukaryotic MAPK was a cell cycle regulator ERK-like kinase. Our work also highlights the scarcity of signalling knowledge in microalgae, in spite of their enormous ecological impact and emerging economic importance.

• MeSH
• bílkoviny řas metabolismus   MeSH
• buněčný cyklus genetika   MeSH
• Chlorophyta enzymologie   MeSH
• duplikace genu MeSH
• fylogeneze MeSH
• genetická variace MeSH
• mitogenem aktivované proteinkinasy chemie   genetika   metabolismus   MeSH
• molekulární evoluce * MeSH
• multigenová rodina MeSH
• proteom metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• rostliny enzymologie   MeSH
• sekvence aminokyselin MeSH
• selekce (genetika) MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bílkoviny řas MeSH
• mitogenem aktivované proteinkinasy MeSH
• proteom MeSH

The progression of the cell cycle in green algae dividing by multiple fission is, under otherwise unlimited conditions, affected by the growth rate, set by a combination of light intensity and temperature. In this study, we compared the cell cycle characteristics of Desmodesmus quadricauda at 20 °C or 30 °C and upon shifts between these two temperatures. The duration of the cell cycle in cells grown under continuous illumination at 20 °C was more than double that at 30 °C, suggesting that it was set directly by the growth rate. Similarly, the amounts of DNA, RNA, and bulk protein content per cell at 20 °C were approximately double those of cells grown at the higher temperature. For the shift experiments, cells grown at either 20 °C or 30 °C were transferred to darkness to prevent further growth, and then cultivated at the same or the other temperature. Upon transfer to the lower temperature, fewer nuclei and daughter cells were produced, and not all cells were able to finish the cell cycle by division, remaining multinuclear. Correspondingly, cells placed in the dark at the higher temperature divided faster into more daughter cells than the control cells. These differences correlated with shifts in the preceding cyclin-dependent kinase activity, suggesting that cell cycle progression was not related to growth rate or cell biomass but correlated with cyclin-dependent kinase activity.

• MeSH
• bílkoviny řas genetika   metabolismus   MeSH
• buněčný cyklus * MeSH
• Chlorophyta cytologie   růst a vývoj   fyziologie   MeSH
• cyklin-dependentní kinasy genetika   metabolismus   MeSH
• nízká teplota MeSH
• tma MeSH
• vysoká teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bílkoviny řas MeSH
• cyklin-dependentní kinasy MeSH

Two untapered, heterocytous species were observed and collected from the intertidal and supratidal zones of the Mexican coastline of the Pacific Ocean near Oaxaca and from the Gulf of Mexico. These populations were highly similar in morphology to the freshwater taxon Petalonema incrustans in the Scytonemataceae. However, 16S rRNA sequence data and phylogenetic analysis indicated that they were sister taxa to the epiphyllic, Brazilian species Phyllonema aveceniicola in the Rivulariaceae, described from culture material. While genetic identity between the two new species was high, they differed significantly in morphology, 16S rRNA gene sequence identity, and sequence and structure of the 16S-23S ITS region. Their morphology differed markedly from the generitype of the previously monotypic Phyllonema, which has tapered, heteropolar, single-false branched trichomes with very thin or absent sheath. The two new species, Phyllonema ansata and Phyllonema tangolundensis, described from both culture and environmental material, have untapered, isopolar, geminately false branched trichomes with thick, lamellated sheaths, differences so significant that the species would not be placed in Phyllonema without molecular corroboration. The morphological differences are so significant that a formal emendation of the genus is required. These taxa provide a challenge to algal taxonomy because the morphological differences are such that one would logically conclude that they represent different genera, but the phylogenetic evidence for including them all in the same genus is conclusive. This conclusion is counter to the current trend in algal taxonomy in which taxa with minor morphological differences have been repeatedly placed in separate genera based primarily upon DNA sequence evidence.

• Klíčová slova
• Phyllonema, 16S-23S ITS, ITS secondary structure, Mexico, Oaxaca, Rivulariaceae, Scytonemataceae, cyanobacteria,
• MeSH
• bílkoviny řas analýza   MeSH
• fylogeneze MeSH
• mezerníky ribozomální DNA analýza   MeSH
• RNA řas analýza   MeSH
• RNA ribozomální 16S analýza   MeSH
• sekundární struktura proteinů MeSH
• sekvenční analýza RNA MeSH
• sinice klasifikace   cytologie   genetika   ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Mexiko MeSH
• Názvy látek
• bílkoviny řas MeSH
• mezerníky ribozomální DNA MeSH
• RNA řas MeSH
• RNA ribozomální 16S MeSH

Photoprotective non-photochemical quenching (NPQ) represents an effective way to dissipate the light energy absorbed in excess by most phototrophs. It is often claimed that NPQ formation/relaxation kinetics are determined by xanthophyll composition. We, however, found that, for the alveolate alga Chromera velia, this is not the case. In the present paper, we investigated the reasons for the constitutive high rate of quenching displayed by the alga by comparing its light harvesting strategies with those of a model phototroph, the land plant Spinacia oleracea. Experimental results and in silico studies support the idea that fast quenching is due not to xanthophylls, but to intrinsic properties of the Chromera light harvesting complex (CLH) protein, related to amino acid composition and protein folding. The pKa for CLH quenching was shifted by 0.5 units to a higher pH compared with higher plant antennas (light harvesting complex II; LHCII). We conclude that, whilst higher plant LHCIIs are better suited for light harvesting, CLHs are 'natural quenchers' ready to switch into a dissipative state. We propose that organisms with antenna proteins intrinsically more sensitive to protons, such as C. velia, carry a relatively high concentration of violaxanthin to improve their light harvesting. In contrast, higher plants need less violaxanthin per chlorophyll because LHCII proteins are more efficient light harvesters and instead require co-factors such as zeaxanthin and PsbS to accelerate and enhance quenching.

• MeSH
• Alveolata fyziologie   MeSH
• bílkoviny řas metabolismus   MeSH
• fotosyntéza * MeSH
• protony * MeSH
• protozoální proteiny metabolismus   MeSH
• rostlinné proteiny metabolismus   MeSH
• Spinacia oleracea fyziologie   MeSH
• světlosběrné proteinové komplexy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• bílkoviny řas MeSH
• protony * MeSH
• protozoální proteiny MeSH
• rostlinné proteiny MeSH
• světlosběrné proteinové komplexy MeSH