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Complex chromosomal rearrangements (CCRs), a class of structural variants (SVs) involving more than two chromosome breaks, were classically thought to be extremely rare. As advanced technologies become more available, it has become apparent that CCRs are more common than formerly thought, and are a substantial cause of genetic disorders. We attempted a novel approach for solving the mechanism of challenging CCRs, which involve repetitive sequences, by precisely identifying sequence-level changes and their order. Chromosomal microarray (CMA) and FISH analyses were used for interpretation of SVs detected by whole exome sequencing (WES). Breakpoint junctions were analyzed by Nanopore sequencing, a novel long-read whole genome sequencing tool. A large deletion identified by WES, encompassing the FOXF1 enhancer, was the cause of alveolar capillary dysplasia and respiratory insufficiency, resulting in perinatal death. CMA analysis of the newborn's mother revealed two duplications encompassing the deleted region in the proband, raising our hypothesis that the deletion resulted from the mother's CCR. Breakpoint junctions of complex SVs were determined at the nucleotide level using Nanopore long-read sequencing. According to sequencing results of breakpoint junctions, the CCR in the newborn was considered the consequence of at least one double-strand break during meiosis, and reassembly of DNA fragments by intra-chromosomal homologous recombination. Our comprehensive approach, combining cytogenetics and long-read sequencing, enabled delineation of the exact breakpoints in a challenging CCR, and proposal of a mechanism in which it arises. We suggest applying our integrative approach combining technologies for deciphering future challenging CCRs, enabling risk assessment in families.

Photosynthesis in deserts is challenging since it requires fast adaptation to rapid night-to-day changes, that is, from dawn’s low light (LL) to extreme high light (HL) intensities during the daytime. To understand these adaptation mechanisms, we purified photosystem I (PSI) from Chlorella ohadii, a green alga that was isolated from a desert soil crust, and identified the essential functional and structural changes that enable the photosystem to perform photosynthesis under extreme high light conditions. The cryo-electron microscopy structures of PSI from cells grown under low light (PSILL) and high light (PSIHL), obtained at 2.70 and 2.71 A, respectively, show that part of light-harvesting antenna complex I (LHCI) and the core complex subunit (PsaO) are eliminated from PSIHL to minimize the photodamage. An additional change is in the pigment composition and their number in LHCIHL; about 50% of chlorophyll b is replaced by chlorophyll a. This leads to higher electron transfer rates in PSIHL and might enable C. ohadii PSI to act as a natural photosynthesiser in photobiocatalytic systems. PSIHL or PSILL were attached to an electrode and their induced photocurrent was determined. To obtain photocurrents comparable with PSIHL, 25 times the amount of PSILL was required, demonstrating the high efficiency of PSIHL. Hence, we suggest that C. ohadii PSIHL is an ideal candidate for the design of desert artificial photobiocatalytic systems. Cryo-EM photosystem I structure reveals adaptation mechanisms to extreme high light in Chlorella ohadii.

The green alga Chlorella sp. and the filamentous cyanobacterium Leptolyngbya sp., regularly cope with various stressors including frequent hydration–desiccation cycles. When grown in axenic culture...

The unparalleled performance of Chlorella ohadii under irradiances of twice full sunlight underlines the gaps in our understanding of how the photosynthetic machinery operates, and what sets its upper functional limit. Rather than succumbing to photodamage under extreme irradiance, unique features of photosystem II function allow C. ohadii to maintain high rates of photosynthesis and growth, accompanied by major changes in composition and cellular structure. This remarkable resilience allowed us to investigate the systems response of photosynthesis and growth to extreme illumination in a metabolically active cell. Using redox proteomics, transcriptomics, metabolomics and lipidomics, we explored the cellular mechanisms that promote dissipation of excess redox energy, protein S-glutathionylation, inorganic carbon concentration, lipid and starch accumulation, and thylakoid stacking. C. ohadii possesses a readily available capacity to utilize a sudden excess of reducing power and carbon for growth and reserve formation, and post-translational redox regulation plays a pivotal role in this rapid response. Frequently the response in C. ohadii deviated from that of model species, reflecting its life history in desert sand crusts. Comparative global and case-specific analyses provided insights into the potential evolutionary role of effective reductant utilization in this extreme resistance of C. ohadii to extreme irradiation. The green alga, Chlorella ohadii, can survive under desert conditions of extreme irradiance. Redox proteomics, transcriptomics, metabolomics and lipidomics uncover how C. ohadii can absorb and use the sudden excesses of reducing power and carbon.

Mitochondria serve as major sites of ATP production and play key roles in many other metabolic processes that are critical to the cell. As relicts of an ancient bacterial endosymbiont, mitochondria contain their own hereditary material (i.e. mtDNA, or mitogenome) and a machinery for protein biosynthesis. The expression of the mtDNA in plants is complex, particularly at the post-transcriptional level. Following transcription, the polycistronic pre-RNAs undergo extensive modifications, including trimming, splicing and editing, before being translated by organellar ribosomes. Our study focuses on N6 -methylation of adenosine ribonucleotides (m6 A-RNA) in plant mitochondria. m6 A is a prevalent modification in nuclear-encoded mRNAs. The biological significance of this dynamic modification is under investigation, but it is widely accepted that m6 A mediates structural switches that affect RNA stability and/or activity. Using m6 A-pulldown/RNA-seq (m6 A-RIP-seq) assays of Arabidopsis and cauliflower mitochondria, we provide information on the m6 A-RNA landscapes in Arabidopsis thaliana and Brassica oleracea mitochondria. The results show that m6 A targets different types of mitochondrial transcripts, including known genes, mtORFs, as well as non-coding (transcribed intergenic) RNA species. While ncRNAs undergo multiple m6 A modifications, N6 -methylation of adenosine residues with mRNAs seem preferably positioned near start codons and may modulate their translatability.

Variants involving TBX4 are associated with a wide variety of disorders, including pulmonary arterial hypertension, ischiocoxopodopatellar syndrome (ICPPS)/small patella syndrome (SPS), lethal lung developmental disorders (LLDDs) in neonates, heart defects, and prenatally lethal posterior amelia with pelvic and pulmonary hypoplasia syndrome. The objective of our study was to elucidate the wide variable phenotypic expressivity and incomplete penetrance in a three-generation family with a truncating variant in TBX4. In addition to exome and genome sequencing analyses, a candidate noncoding regulatory single nucleotide variant (SNV) within the lung-specific TBX4 enhancer was functionally tested using an in vitro luciferase reporter assay. A heterozygous frameshift variant c.1112dup (p.Pro372Serfs*14) in TBX4 was identified in patients with mild interstitial lung disease (1), bronchiolitis obliterans (1), recurrent pneumothorax (1), ICPPS/SPS (1), LLDD (2), and in unaffected individuals (4). In two deceased neonates with LLDD, we identified a noncoding SNV rs62069651-C located in trans to the mutated TBX4 allele that reduced the TBX4 promoter activity by 63% in the reporter assay. Our findings provide a functional evidence for the recently reported model of complex compound inheritance in which both TBX4 coding and in trans noncoding hypomorphic variants in the lung-specific enhancer of TBX4 contribute to LLDD.

Red/far-red light signals regulate the cyanobacterial CO2-concentrating mechanism by activating/inactivating bicarbonate uptake., Desiccation-tolerant cyanobacteria can survive frequent hydration/dehydration cycles likely affecting inorganic carbon (Ci) levels. It was recently shown that red/far-red light serves as signal-preparing cells toward dehydration. Here, the effects of desiccation on Ci assimilation by Leptolyngbya ohadii isolated from Israel’s Negev desert were investigated. Metabolomic investigations indicated a decline in ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation activity, and this was accelerated by far-red light. Far-red light negatively affected the Ci affinity of L. ohadii during desiccation and in liquid cultures. Similar effects were evident in the non–desiccation-tolerant cyanobacterium Synechocystis. The Synechocystis Δcph1 mutant lacking the major phytochrome exhibited reduced photosynthetic Ci affinity when exposed to far-red light, whereas the mutant ΔsbtB lacking a Ci uptake inhibitory protein lost the far-red light inhibition. Collectively, these results suggest that red/far-red light perception likely via phytochromes regulates Ci uptake by cyanobacteria and that this mechanism contributes to desiccation tolerance in strains such as L. ohadii.

During meiosis, gene expression is silenced in aberrantly unsynapsed chromatin and in heterogametic sex chromosomes. Initiation of sex chromosome silencing is disrupted in meiocytes with sex chromosome-autosome translocations. To determine whether this is due to aberrant synapsis or loss of continuity of sex chromosomes, we engineered Caenorhabditis elegans nematodes with non-translocated, bisected X chromosomes. In early meiocytes of mutant males and hermaphrodites, X segments are enriched with euchromatin assembly markers and active RNA polymerase II staining, indicating active transcription. Analysis of RNA-seq data showed that genes from the X chromosome are upregulated in gonads of mutant worms. Contrary to previous models, which predicted that any unsynapsed chromatin is silenced during meiosis, our data indicate that unsynapsed X segments are transcribed. Therefore, our results suggest that sex chromosome chromatin has a unique character that facilitates its meiotic expression when its continuity is lost, regardless of whether or not it is synapsed., During meiosis, sex chromosomes show silenced gene expression that is disrupted when translocated to autosomes. Here, the authors engineer C. elegans with bisected X chromosomes and see active transcription in gonads, indicating continuity of sex chromosomes promotes silencing.

Introduction: More than one thousand variants have been described in the GLA gene. Some intronic variants and copy number variants in GLA can cause Fabry disease but will not be detected by classical Sanger sequence.Aims: We aimed to design and validate a method for sequencing the GLA gene using long read Oxford Nanopore sequencing technology.Methods: Twelve Fabry patients were blindly analyzed, both by conventional Sanger sequence and by long read sequencing of a 13kb PCR amplicon. We used minimap2 to align the long read data and Nanopolish and Sniffles to call variants.Results: All the variants detected by Sanger (including a deep intronic variant) were also detected by long read sequencing. One patient had a deletion that was not detected by Sanger sequencing but was detected by the new technology.Conclusions: Our long read sequencing-based method was able to detect missense variants and an exonic deletion, with the added advantage of intronic analysis. It can be used as an efficient and cost-effective tool for screening and diagnosing Fabry disease.

Various approaches have been proposed to control/eliminate toxic Microcystis sp. blooms including H2 O2 treatments. Earlier studies showed that pre-exposure of various algae to oxidative stress induced massive cell death when cultures were exposed to an additional H2 O2 treatment. We examined the vulnerability of exponential and stationary-phase Microcystis sp. strain MGK cultures to single and double H2 O2 applications. Stationary cultures show a much higher ability to decompose H2 O2 than younger cultures. Nevertheless, they are more sensitive to an additional H2 O2 dose given 1-6 h after the first one. Transcript analyses following H2 O2 application showed a fast rise in glutathione peroxidase abundance (227-fold within an hour) followed by a steep decline thereafter. Other genes potentially engaged in oxidative stress were far less affected. Metabolic-related genes were downregulated after H2 O2 treatments. Among those examined, the transcript level of prk (encoding phosphoribulose kinase) was the slowest to recover in agreement with the decline in photosynthetic rate revealed by fluorescence measurements. Our findings shed light on the response of Microcystis MGK to oxidative stress suggesting that two consecutive H2 O2 applications of low concentrations are far more effective in controlling Microcystis sp. population than a single dose of a higher concentration.

Cyanobacteria inhabiting desert biological soil crusts must prepare towards dehydration, or their revival after rewetting is severely impaired. The mechanisms involved are unknown but signalling of forthcoming dehydration by dawn illumination was demonstrated. Accurate and reproducible simulation of desert conditions enabled examination of physiological activities and transcript profiles in a model organism, Leptolyngbya ohadii, in response to specific conditions. Exposure to far red light or lack of ground warming during dawn severely reduced revival after rewetting and altered the network of gene expression. The data implicated phytochromes in light and temperature sensing. Many genes were up- or down-regulated before water content decline, while others were strongly affected by the progression of dehydration and desiccation. Transcription continues during the desiccated phase but only barely during early rewetting, although photosynthetic activity was regained. Application of rifampicin with or without a preceding dehydration phase demonstrated that RNA is stabilized/protected during desiccation, possibly by intrinsically disordered proteins. We conclude that increasing light and temperature at dawn activates a network of genes that prepare the cells towards dehydration. Quick resumption of photosynthesis upon rewetting in contrast to the slow change in the transcript profile suggested that in addition to preparing towards dehydration the cells also prepare for forthcoming rewetting, during dehydration. Unravelling the presently unknown function of many responding genes will help to clarify the networks involved.

The sparsely distributed Limodorum abortivum is a European-Mediterranean orchid species, which grows on decomposing plant material. Although some chlorophyll-pigmentation is observed in the degenerated scales-shaped leaf and stems regions of the plant, its photosynthetic capacity is assumed to be insufficient to support the full energy requirements of an adult plant. In Israel, L. abortivum shows a patchy distribution patterns in the Galilee, Golan, Carmel and Judean regions. To gain more insights into the physiology and photosynthetic activity of L. abortivum, we analyzed the organellar morphologies, photosynthetic activities the chloroplast-DNA sequence by Illumina-HTS. Microscopic analyses indicated to the presence of mature chloroplasts with well-organized grana-thylakoids in the leaves and stems of L. abortivum. However, the numbers of chloroplasts per cell and the grana ultrastructure density within the organelles were notably lower than those of model plant species and fully photosynthetically-active orchids. The cpDNA of L. abortivum (154,954 bp) encodes 60 proteins, 34 tRNAs and 4 rRNAs. The coding-regions of 24 genes are interrupted by 26 group-II intron-sequences. While many genes related to photosynthesis (RuBisCo, PSI, PSII and cytochrome b 6 /f subunits) have remained intact in the cpDNA, the majority of the NADH-dehydrogenase (ndh) subunits were either lost or became nonfunctional (i.e. pseudogenized). In agreement with previous reports, the photosynthetic-rates of adult Limodorum plants were found to be very low, further indicating that carbon-assimilation activity is insufficient to support the energy requirements of an adult plant, and may suggest that L. abortivum have adopted nutritional strategies similar to that of mycoheterotrophic orchid species.

Diatom dominance in contemporary aquatic environments indicates that they have developed unique and effective mechanisms to cope with the rapid and considerable fluctuations that characterize these environments. In view of their evolutionary history from a secondary endosymbiosis, inter-organellar regulation of biochemical activities may be of particular relevance. Diatom mitochondrial alternative oxidase (AOX) is believed to play a significant role in supplying chloroplasts with ATP produced in the mitochondria. Using the model diatom Phaeodactylum tricornutum we generated AOX knockdown lines, and followed sensitivity to stressors, photosynthesis and transcriptome and metabolome profiles of wild-type and knockdown lines. We show here that expression of the AOX gene is upregulated by various stresses including H2 O2 , heat, high light illumination, and iron or nitrogen limitation. AOX knockdown results in hypersensitivity to stress. Knockdown lines also show significantly reduced photosynthetic rates and their chloroplasts are more oxidized. Comparisons of transcriptome and metabolome profiles suggest a strong impact of AOX activity on gene expression, which is carried through to the level of the metabolome. Our data provide evidence for the involvement of mitochondrial AOX in processes central to the cell biology of diatoms, revealing that cross-talk between mitochondria and chloroplasts is crucial for maintaining sensitivity to changing environments.

Patients with type 1 diabetes mellitus (T1DM) exhibit reduced BMD and significant increases in fracture risk. Changes in BMD are attributed to blunted osteoblast activity and inhibited bone remodeling, but these cannot fully explain the impaired bone integrity in T1DM. The goal of this study was to determine the cellular mechanisms that contribute to impaired bone morphology and composition in T1DM. Nonobese diabetic (NOD) mice were used, along with μCT, histomorphometry, histology, Raman spectroscopy, and RNAseq analyses of several skeletal sites in response to naturally occurring hyperglycemia and insulin treatment. The bone volume in the axial skeleton was found to be severely reduced in diabetic NOD mice and was not completely resolved with insulin treatment. Decreased bone volume in diabetic mice was associated with increased sclerostin expression in osteocytes and attenuation of bone formation indices without changes in bone resorption. In the face of blunted bone remodeling, decreases in the mineral:matrix ratio were found in cortical bones of diabetic mice by Raman microspectroscopy, suggesting that T1DM did not affect the bone mineralization process per se, but rather resulted in microenvironmental alterations that favored mineral loss. Bone transcriptome analysis indicated metabolic shifts in response to T1DM. Dysregulation of genes involved in fatty acid oxidation, transport, and synthesis was found in diabetic NOD mice. Specifically, pyruvate dehydrogenase kinase isoenzyme 4 and glucose transporter 1 levels were increased, whereas phosphorylated‐AKT levels were significantly reduced in diabetic NOD mice. In conclusion, in addition to the blunted bone formation, osteoblasts and osteocytes undergo metabolic shifts in response to T1DM that may alter the microenvironment and contribute to mineral loss from the bone matrix. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Biological soil crusts (BSCs) are found in drylands, cover ∼12% of the Earth's surface in arid and semi-arid lands and their destruction is considered an important promoter of desertification. These crusts are formed by the adhesion of soil particles to polysaccharides excreted mostly by filamentous cyanobacteria, which are the pioneers and main primary producers in BSCs. Desert BSCs survive in one of the harshest environments on Earth, and are exposed to daily fluctuations of extreme conditions. The cyanobacteria inhabiting these habitats must precisely read the changing conditions and predict, for example, the forthcoming desiccation. Moreover, they evolved a comprehensive regulation of multiple adaptation strategies to enhance their stress tolerance. Here, we focus on what distinguishes cyanobacteria able to revive after dehydration from those that cannot. While important progress has been made in our understanding of physiological, biochemical and omics aspects, clarification of the sensing, signal transduction and responses enabling desiccation tolerance are just emerging. We plot the trajectory of current research and open questions ranging from general strategies and regulatory adaptations in the hydration/desiccation cycle, to recent advances in our understanding of photosynthetic adaptation. The acquired knowledge provides new insights to mitigate desertification and improve plant productivity under drought conditions.

Photosynthesis in deserts is challenging since it requires fast adaptation to rapid night-to-day changes, that is, from dawn's low light (LL) to extreme high light (HL) intensities during the daytime. To understand these adaptation mechanisms, we purified photosystem I (PSI) from Chlorella ohadii, a green alga that was isolated from a desert soil crust, and identified the essential functional and structural changes that enable the photosystem to perform photosynthesis under extreme high light conditions. The cryo-electron microscopy structures of PSI from cells grown under low light (PSI

SummaryIn multicellular organisms, Polycomb Repressive Complex2 (PRC2) is known to deposit H3K27me3 to establish and maintain gene silencing, critical for developmentally regulated processes. PRC2 complex is absent in both widely studied model yeasts which initially suggested that PRC2 arose with the emergence of multicellularity. However, its discovery in several unicellular species including microalgae questions its role in unicellular eukaryotes.Here, we use Phaeodactylum tricornutum enhancer of zeste E(z) knockouts and show that P. tricornutum E(z) is responsible for di and tri-methylation of lysine 27 of histone H3.H3K27me3 depletion abolishes cell morphology in P. tricornutum providing evidence for its role in cell differentiation. Genome wide profiling of H3K27me3 in fusiform and triradiate cells further revealed genes that may specify cell identity.These results suggest a role for PRC2 and its associated mark in cell differentiation in unicellular species and highlight their ancestral function in a broader evolutionary context than is currently appreciated.

During meiosis of heterogametic cells, such as XY meiocytes, sex chromosomes of many species undergo transcriptional silencing known as meiotic sex chromosome inactivation (MSCI). Silencing also occurs in aberrantly unsynapsed autosomal chromatin. The silencing of unsynapsed chromatin, is assumed to be the underline mechanism for MSCI. Initiation of MSCI is disrupted in meiocytes with sex chromosome-autosome translocations. Whether this is due to aberrant synapsis or the lack of sex chromosome integrity has never been determined. To address this, we used CRISPR to engineerCaenorhabditis elegansstable strains with broken X chromosomes that didn’t undergo translocations with autosomes. In early meiotic nuclei of these mutants, the X fragments lack silent chromatin modifications and instead the fragments are enriched with transcribing chromatin modifications. Moreover, the level of active RNA polymerase II staining on the X fragments in mutant nuclei is similar to that on autosomes, indicating active transcription on the X. Contrary to previous models, which predicted that any unsynapsed chromatin is silenced during meiosis, X fragments that did not synapse were robustly stained with RNA polymerase II and gene expression levels were high throughout the broken X. Therefore, lack of synapsis does not trigger MSCI if sex chromosome integrity is lost. Moreover, our results suggest that a unique character of the chromatin of sex chromosomes underlies their lack of meiotic silencing due to both unsynapsed chromatin and sex chromosome mechanisms when their integrity is lost.

Mitochondria play key roles in cellular energy metabolism within eukaryotic cells. As relics of endosymbiotic bacteria, most (but not all) mitochondria contain their own genome (mitogenome, mtDNA), as well as intrinsic biosynthetic machinery for making organelle RNAs and proteins. The expression of the mtDNA requires regulated metabolism of its transcriptome by nuclear-encoded factors. Post-transcriptional mtRNA modifications play a central role in the expression of the plant mitogenomes, and hence in organellar biogenesis and plant physiology. Despite extensive investigations, a full map of angiosperm mitochondrial transcriptomes, a prerequisite for the elucidation of the basic RNA biology of mitochondria, has not been reported yet. Using RNA-seq data, RT-PCR and bioinformatics, we sought to explore the gene expression profiles of land plant mitochondria. Here, we present the mitochondrial transcriptomic maps of two key Brassicaceae species, Arabidopsis thaliana (var Col-0) and cauliflower (Brassica oleracea var. botrytis). The revised transcriptome landscapes of Arabidopsis and cauliflower mitogenomes provide with more detail into mtRNA biology and processing in angiosperm mitochondria, and we expect that they would serve as a valuable resource for the plant organellar community.

Pgr5 proteins play a major direct role in cyclic electron flow paths in plants and eukaryotic phytoplankton. The genomes of many cyanobacterial species code for Pgr5-like proteins but their function is still uncertain. Here, we present evidence that supports a link between the Synechocystis sp. PCC6803 Pgr5-like protein and the regulation of intracellular redox balance. The knockout strain, pgr5KO, did not display substantial phenotypic response under our experimental conditions, confirming results obtained in earlier studies. However, the overexpression strain, pgr5OE, accumulated 2.5-fold more chlorophyll than the wild type and displayed increased content of photosystems matching the chlorophyll increase. As a result, electron transfer rates through the photosynthetic apparatus of pgr5OE increased, as did the amount of energy stored as glycogen. While, under photoautotrophic conditions, this metabolic difference had only minor effects, under mixotrophic conditions, pgr5OE cultures collapsed. Interestingly, this specific phenotype of pgr5OE mutants displayed a tendency for reverting, and cultures which previously collapsed in the presence of glucose were now able to survive. DNA sequencing of a pgr5OE strain revealed a second site suppression mutation in slr1916, a putative esterase associated with redox regulation. The phenotype of the slr1916 knockout is very similar to that of the strain reported here and to that of the pmgA regulator knockout. These data demonstrate that, in Synechocystis 6803, there is strong selection against overexpression of the Pgr5-like protein. The pseudoreversion event in a gene involved in redox regulation suggests a connection of the Pgr5-like protein to this network.

SummaryMitochondria are the main source of ATP production and also contribute to many other processes central to cellular function. Mitochondrial activities have been linked with growth, differentiation and aging. As relicts of bacterial endosymbionts, these organelles contain their own genetic system (i.e., mitogenome or mtDNA). The expression of the mtDNA in plants is complex, particularly at the posttranscriptional level. Following transcription, the polycistronic pre-RNAs in plant mitochondria are processed into individual RNAs, which then undergo extensive modifications, as trimming, splicing and C→U editing, before being translated by organellar ribosomes. Our study focuses on N6-methylation of Adenosine ribonucleotides (m6A-RNA) in plant mitochondria. m6A is the most common modification in eukaryotic mRNAs. The biological significance of this highly dynamic modification is under investigation, but it’s widely accepted that m6A mediates structural switches that affect RNA stability and activity. By performing m6A-pulldown/RNA-seq (m6A-RIP-seq) analyses of Arabidopsis and cauliflower mitochondrial transcripts (mtRNAs), we provide with detail information on the m6A landscapes in angiosperms mitochondria. The results indicate that m6A targets different types of mtRNAs, including coding sequences, UTRs, introns and non-coding RNA species. While introns and noncoding-RNAs undergo multiple m6A modifications along the transcript, in mRNAs m6A-modifications are preferably positioned near start-codons, and may modulate the translatability of the m6A-modified transcripts.

Various approaches have been proposed to control/eliminate toxic Microcystis sp. blooms including H

Mitochondria serve as major sites of ATP production and play key roles in many other metabolic processes that are critical to the cell. As relicts of an ancient bacterial endosymbiont, mitochondria contain their own hereditary material (i.e. mtDNA, or mitogenome) and a machinery for protein biosynthesis. The expression of the mtDNA in plants is complex, particularly at the post-transcriptional level. Following transcription, the polycistronic pre-RNAs undergo extensive modifications, including trimming, splicing and editing, before being translated by organellar ribosomes. Our study focuses on N

Toxic Microcystis spp. blooms constitute a serious threat to water quality worldwide. Aeromonas veronii was isolated from Microcystis sp. colonies collected in Lake Kinneret. Spent Aeromonas media inhibits the growth of Microcystis aeruginosa MGK isolated from Lake Kinneret. The inhibition was much stronger when Aeromonas growth medium contained spent media from MGK suggesting that Aeromonas recognized its presence and produced secondary metabolites that inhibit Microcystis growth. Fractionations of the crude extract and analyses of the active fractions identified several secondary metabolites including lumichrome in Aeromonas media. Application of lumichrome at concentrations as low as 4 nM severely inhibited Microcystis growth. Inactivation of aviH in the lumichrome biosynthetic pathway altered the lumichrome level in Aeromonas and the extent of MGK growth inhibition. Conversely, the initial lag in Aeromonas growth was significantly longer when provided with Microcystis spent media but Aeromonas was able to resume normal growth. The longer was pre-exposure to Microcystis spent media the shorter was the lag phase in Aeromonas growth indicating the presence of, and acclimation to, secondary MGK metabolite(s) the nature of which was not revealed. Our study may help to control toxic Microcystis blooms taking advantage of chemical languages used in the interspecies communication.

Filamentous cyanobacteria are the main founders and primary producers in biological desert soil crusts (BSCs) and are likely equipped to cope with one of the harshest environmental conditions on earth including daily hydration/dehydration cycles, high irradiance and extreme temperatures. Here, we resolved and report on the genome sequence of Leptolyngbya ohadii, an important constituent of the BSC. Comparative genomics identified a set of genes present in desiccation-tolerant but not in dehydration-sensitive cyanobacteria. RT qPCR analyses showed that the transcript abundance of many of them is upregulated during desiccation in L. ohadii. In addition, we identified genes where the orthologs detected in desiccation-tolerant cyanobacteria differs substantially from that found in desiccation-sensitive cells. We present two examples, treS and fbpA (encoding trehalose synthase and fructose 1,6-bisphosphate aldolase respectively) where, in addition to the orthologs present in the desiccation-sensitive strains, the resistant cyanobacteria also possess genes with different predicted structures. We show that in both cases the two orthologs are transcribed during controlled dehydration of L. ohadii and discuss the genetic basis for the acclimation of cyanobacteria to the desiccation conditions in desert BSC.

The factors rate-limiting growth of photosynthetic organisms under optimal conditions are controversial [1-8]. Adaptation to extreme environments is usually accompanied by reduced performance under optimal conditions [9, 10]. However, the green alga Chlorella ohadii, isolated from a harsh desert biological soil crust [11-17], does not obey this rule. In addition to resistance to photodamage [17, 18], it performs the fastest growth ever reported for photosynthetic eukaryotes. A multiphasic growth pattern (very fast growth [phase I], followed by growth retardation [phase II] and additional fast growth [phase III]) observed under constant illumination and temperature indicates synchronization of the algal population. Large physiological changes at transitions between growth phases suggest metabolic shifts. Indeed, metabolome analyses at points along the growth phases revealed large changes in the levels of many metabolites during growth with an overall rise during phase I and decline in phase II. Multivariate analysis of the metabolome data highlighted growth phase as the main factor contributing to observed metabolite variance. The analyses identified putrescine as the strongest predictive metabolite for growth phase and a putative growth regulator. Indeed, extracellular additions of polyamines strongly affected the growth rate in phase I and the growth arrest in phase II, with a marked effect on O2 exchange. Our data implicate polyamines as the signals harmonizing metabolic shifts and suggest that metabolic flexibility enables the immense growth capabilities of C. ohadii. The data provide a new dimension to current models focusing on growth-limiting processes in photosynthetic organisms where the anabolic and catabolic metabolisms must be strictly regulated.

Background With the emerging interest in phytoplankton research, the need to establish genetic tools for the functional characterization of genes is indispensable. The CRISPR/Cas9 system is now well recognized as an efficient and accurate reverse genetic tool for genome editing. Several computational tools have been published allowing researchers to find candidate target sequences for the engineering of the CRISPR vectors, while searching possible off-targets for the predicted candidates. These tools provide built-in genome databases of common model organisms that are used for CRISPR target prediction. Although their predictions are highly sensitive, the applicability to non-model genomes, most notably protists, makes their design inadequate. This motivated us to design a new CRISPR target finding tool, PhytoCRISP-Ex. Our software offers CRIPSR target predictions using an extended list of phytoplankton genomes and also delivers a user-friendly standalone application that can be used for any genome. Results The software attempts to integrate, for the first time, most available phytoplankton genomes information and provide a web-based platform for Cas9 target prediction within them with high sensitivity. By offering a standalone version, PhytoCRISP-Ex maintains an independence to be used with any organism and widens its applicability in high throughput pipelines. PhytoCRISP-Ex out pars all the existing tools by computing the availability of restriction sites over the most probable Cas9 cleavage sites, which can be ideal for mutant screens. Conclusions PhytoCRISP-Ex is a simple, fast and accurate web interface with 13 pre-indexed and presently updating phytoplankton genomes. The software was also designed as a UNIX-based standalone application that allows the user to search for target sequences in the genomes of a variety of other species. Electronic supplementary material The online version of this article (doi:10.1186/s12859-016-1143-1) contains supplementary material, which is available to authorized users.

The reasons for large multiannual fluctuations in phytoplankton biomass and composition in freshwater lakes are complex and involve many biotic and abiotic parameters. Here we studied the 2009 and 2010 summer-autumn blooms of the toxic, cylindrospermopsin producer, Aphanizomenon ovalisporum (hereafter Aphanizomenon) in Lake Kinneret (Sea of Galilee), Israel. During the summer the total dissolved phosphate concentration in the lake is very low, close to the detection level, limiting the development of phytoplankton. Earlier we showed that Aphanizomenon blooms are associated with a large rise in alkaline phosphatase (Apase) activity in the water body and that cylindrospermopsin produced by Aphanizomenon induces the PHO regulon, including secretion of Apase, in other alga thereby improving its own phosphate supply. Aphanizomenon transcripts of PHO and AOA (involved in cylindrospermopsin biosynthesis) genes in Lake Kinneret appeared much earlier in 2010 than in 2009 suggesting that the phytoplankton became phosphate-limited already at the beginning of its summer bloom in 2010 but much later in 2009. Water inflow and lake water temperatures were significantly higher in 2010 but the incoming nutrients were consumed by the much larger phytoplankton biomass early in 2010 before the beginning of the Aphanizomenon bloom. An analysis of abiotic and biological parameters provides an explanation for the very different development of Aphanizomenon populations during 2009 and 2010.

Desert biological soil crusts (BSC), among the harshest environments on Earth, are formed by the adhesion of soil particles to polysaccharides excreted mainly by filamentous cyanobacteria (see [1] and references therein). These species are the main primary producers in this habitat where they cope with various stressors including frequent hydration-dehydration cycles. Water is mainly provided as early-morning dew, followed by dehydration with rising temperatures and declining relative humidity. Earlier studies focused on community structure and cyanobacterial activities in various BSCs [1,2]. They identified genes present in dehydration-tolerant, but not -sensitive cyanobacteria [3], and suggested that abiotic conditions during natural dehydration (Figure 1A) are critical for the recovery upon rewetting. Inability of Leptolyngbya ohadii, which is abundant in the BSC examined here, to recover after rapid desiccation (Figure 1B) [4] suggested that the cells must prepare themselves toward forthcoming dehydration, but the nature of the signal involved was unknown. We show here that the rising dawn illumination, perceived by photo-sensors, serves as the signal inciting BSC-inhabiting cyanobacteria to prepare for forthcoming dehydration. L. ohadii filaments were exposed to simulated natural conditions from the morning of October 14th 2009, using our environmental chamber that enables accurate reproduction of BSC environment [4] (Supplemental Figure S1A). Samples were withdrawn at specific time points (Figure 1A), followed by RNA extraction and global transcript profiling (accession PRJNA391854). Four hours of dehydration led to up-regulation of 567 genes and down-regulation of 1597 (by more than 2-fold). Since BSC-inhabiting organisms have not been used as genetic models, the functions of 3258 (43.5% of the 7487 L. ohadii genes [3]) are unknown. Nevertheless, a pronounced rise in transcript levels of genes involved in carbon metabolism, transport, osmolyte production, energy dissipation and other cellular activities was observed. On the other hand, a declining transcript abundance for genes involved in light harvesting, photosynthetic metabolism, protein biosynthesis, cell division and other pathways was detected. The analysis unraveled clear distinctions between early- and late-responding genes. Supplemental Table S1 lists the 40 strongest differentially expressed genes verified by RT-qPCR and used in further analyses.

Oxidative stress has been implicated in the induction of programmed cell death in a wide variety of organisms. Acquiring antioxidant capacity is thought to enhance the viability of cells challenged by a subsequent oxidative stress. Counter-intuitively, we show that in two phytoplankton species, Chlamydomonas reinhardtii and Peridinium gatunense, representing the green and red plastid lineages, oxidative stress induced cell death in cultures that already possessed high antioxidant activity but not in cells that exhibited low activity. Cell death of low antioxidant possessing cultures was markedly enhanced by the addition of dehydroascorbate, a product of ascorbate peroxidase (APX), but not of ascorbate or reduced glutathione, and was preceded by increased metacaspase expression and activity. These data suggested that the level of APX and its products, strongly upregulated by oxidative stress, serves as a possible surveillance signal, reporting that the cells already experienced an earlier oxidative stress. Our data presents a novel role of APX in antioxidant activity and response to oxidative stress in photosynthetic microorganisms. Elimination of cysts production by phytoplankton cells that were already damaged by oxidative stress (indicated by the rise in oxidized proteins) as the inoculum for the following year's population may be the evolutionary trigger for this phenomenon.

International audience; Diatoms are one of the most ecologically successful classes of photosynthetic marine eukaryotes in the contemporary oceans. Over the past 30 million years, they have helped to moderate Earth's climate by absorbing carbon dioxide from the atmosphere, sequestering it via the biological carbon pump and ultimately burying organic carbon in the lithosphere. The proportion of planetary primary production by diatoms in the modern oceans is roughly equivalent to that of terrestrial rainforests. In photosynthesis, the efficient conversion of carbon dioxide into organic matter requires a tight control of the ATP/NADPH ratio which, in other photosynthetic organisms, relies principally on a range of plastid-localized ATP generating processes. Here we show that diatoms regulate ATP/NADPH through extensive energetic exchanges between plastids and mitochondria. This interaction comprises the re-routing of reducing power generated in the plastid towards mitochondria and the import of mitochondrial ATP into the plastid, and is mandatory for optimized carbon fixation and growth. We propose that the process may have contributed to the ecological success of diatoms in the ocean.

Background: Nucleosomes are the building blocks of chromatin where gene regulation takes place. Chromatin landscapes have been profiled for several species, providing insights into the fundamental mechanisms of chromatin-mediated transcriptional regulation of gene expression. However, knowledge is missing for several major and deep-branching eukaryotic groups, such as the Stramenopiles, which include the diatoms. Diatoms are highly diverse and ubiquitous species of phytoplankton that play a key role in global biogeochemical cycles. Dissecting chromatin-mediated regulation of genes in diatoms will help understand the ecological success of these organisms in contemporary oceans. Results: Here, we use high resolution mass spectrometry to identify a full repertoire of post-translational modifications on histones of the marine diatom Phaeodactylum tricornutum, including eight novel modifications. We map five histone marks coupled with expression data and show that P. tricornutum displays both unique and broadly conserved chromatin features, reflecting the chimeric nature of its genome. Combinatorial analysis of histone marks and DNA methylation demonstrates the presence of an epigenetic code defining activating or repressive chromatin states. We further profile three specific histone marks under conditions of nitrate depletion and show that the histone code is dynamic and targets specific sets of genes. Conclusions: This study is the first genome-wide characterization of the histone code from a stramenopile and a marine phytoplankton. The work represents an important initial step for understanding the evolutionary history of chromatin and how epigenetic modifications affect gene expression in response to environmental cues in marine environments. Instituto de Biotecnología Fil: Veluchamy, Alaguraj. Centre National de la Recherche Scientifique. Institut de Biologie de l’École Normale Supérieure. Ecology and Evolutionary Biology Section; Francia. King Abdullah University of Science and Technology. BESE Division. Center for Desert Agriculture; Arabia Saudita Fil: Rastogi, Achal. Centre National de la Recherche Scientifique. Institut de Biologie de l’École Normale Supérieure. Ecology and Evolutionary Biology Section; Francia Fil: Lin, Xin. Centre National de la Recherche Scientifique. Institut de Biologie de l’École Normale Supérieure. Ecology and Evolutionary Biology Section; Francia. Xiamen University. State key lab of Marine Environmental Science; China Fil: Lombard, Bérangère. Institut Curie PSL Research University. Centre de Recherche. Laboratoire de Spectrométrie de Masse Protéomique; Francia Fil: Murik, Omer. Centre National de la Recherche Scientifique. Institut de Biologie de l’École Normale Supérieure. Ecology and Evolutionary Biology Section; Francia Fil: Thomas, Yann. Centre National de la Recherche Scientifique. Institut de Biologie de l’École Normale Supérieure. Ecology and Evolutionary Biology Section; Francia Fil: Dingli, Florent. Institut Curie PSL Research University. Centre de Recherche. Laboratoire de Spectrométrie de Masse Protéomique; Francia Fil: Rivarola, Maximo Lisandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. University of Maryland School of Medicine. Institute for Genome Sciences; Estados Unidos Fil: Ott, Sandra. University of Maryland School of Medicine. Institute for Genome Sciences; Estados Unidos Fil: Liu, Xinyue. University of Maryland School of Medicine. Institute for Genome Sciences; Estados Unidos Fil: Sun, Yezhou. University of Maryland School of Medicine. Institute for Genome Sciences; Estados Unidos Fil: Rabinowicz, Pablo D. University of Maryland School of Medicine. Institute for Genome Sciences; Estados Unidos Fil: McCarthy, James. J. Craig Venter Institute; Estados Unidos Fil: Allen, Andrew E. J. Craig Venter Institute; Estados Unidos. University of California. Integrative Oceanography Division. Scripps Institution of Oceanography; Estados Unidos Fil: Loew, Damarys. Institut Curie PSL Research University. Centre de Recherche. Laboratoire de Spectrométrie de Masse Protéomique; Francia Fil: Bowler, Chris. Centre National de la Recherche Scientifique. Institut de Biologie de l’École Normale Supérieure. Ecology and Evolutionary Biology Section; Francia Fil: Tirichine, Leïla. Centre National de la Recherche Scientifique. Institut de Biologie de l’École Normale Supérieure. Ecology and Evolutionary Biology Section; Francia

The reasons for large multiannual fluctuations in phytoplankton biomass and composition in freshwater lakes are complex and involve many biotic and abiotic parameters. Here we studied the 2009 and 2010 summer-autumn blooms of the toxic, cylindrospermopsin producer, Aphanizomenon ovalisporum (hereafter Aphanizomenon) in Lake Kinneret (Sea of Galilee), Israel. During the summer the total dissolved phosphate concentration in the lake is very low, close to the detection level, limiting the development of phytoplankton. Earlier we showed that Aphanizomenon blooms are associated with a large rise in alkaline phosphatase (Apase) activity in the water body and that cylindrospermopsin produced by Aphanizomenon induces the PHO regulon, including secretion of Apase, in other alga thereby improving its own phosphate supply. Aphanizomenon transcripts of PHO and AOA (involved in cylindrospermopsin biosynthesis) genes in Lake Kinneret appeared much earlier in 2010 than in 2009 suggesting that the phytoplankton became phosphate-limited already at the beginning of its summer bloom in 2010 but much later in 2009. Water inflow and lake water temperatures were significantly higher in 2010 but the incoming nutrients were consumed by the much larger phytoplankton biomass early in 2010 before the beginning of the Aphanizomenon bloom. An analysis of abiotic and biological parameters provides an explanation for the very different development of Aphanizomenon populations during 2009 and 2010.

Summary Regulated programmed cell death (PCD) processes have been documented in several phytoplankton species and are hypothesized to play a role in popu- lation dynamics. However, the mechanisms leading to the coordinated collapse of phytoplankton blooms are poorly understood. We showed that the collapse of the annual bloom of Peridinium gatunense ,a n abundant dinoflagellate in Lake Kinneret, Israel, is initiated by CO2 limitation followed by oxidative stress that triggers a PCD-like cascade. We provide evidences that a protease excreted by senescing P. gatunense cells sensitizes younger cells to oxidative stress and may consequently trigger syn- chronized cell death of the population. Ageing of the P. gatunense cultures was characterized by a remark- able rise in DNA fragmentation and enhanced sensi- tivity to H2O2. Exposure of logarithmic phase (young) cultures to conditioning media from stationary phase (old) cells sensitized them to H2O2 and led to prema- ture massive cell death. We detected the induction of specific extracellular protease activity, leupeptin- sensitive, in ageing cultures and in lake waters during the succession of the P. gatunense bloom. Partial purification of the conditioned media revealed that this protease activity is responsible for the higher susceptibility of young cells to oxidative stress. Inhi- bition of the protease activity lowered the sensitivity to oxidative stress, whereas application of papain to logarithmic phase P. gatunense cultures mimicked the effect of the spent media and enhanced cell death. We propose a novel mechanistic framework by which a population of unicellular phytoplankton orches- trates a coordinated response to stress, thereby determine the fate of its individuals.

The reason(s) for glucose sensitivity in certain cyanobacterial strains is poorly understood. Inactivation of genes encoding the putative sensor kinase Hik31 inSynechocystissp. strain PCC 6803 resulted in a mutant unable to grow in the presence ofd-glucose. Sensitivities tod-glucose, its analogue 2-deoxy-d-glucose, and fructose, were alleviated in mutants in whichglcP, encoding the glucose transporter, was inactivated. These data indicate that permeation of these substrates is required to inflict cell death. The mutant Δhik31, and the glucose-sensitive strain ofSynechocystis, do not possess glucokinase activity, although a transcript originating fromglk, encoding glucokinase, is present. Inactivation ofglkled to severe sensitivity to glucose, indicating that the presence of glucose itself, within the cells, inflicted this sensitivity. On the other hand, sensitivity to 2-deoxy-d-glucose was lower in Δglk, thus distinguishing between the effect of glucose itself and that of its analogue, which, in the absence of glucokinase activity, may not be phosphorylated. Addition of glucose led to a small rise in glucose-6-phosphate dehydrogenase activity in the wild type, but constitutive activity was observed in the Δhik31mutant regardless of the presence of glucose. Microarray analyses showed only small changes in the abundance of global transcripts inSynechocystisfollowing glucose addition, but the transcription levels of several genes, includingicfG, but notglk, were strongly affected by inactivation ofhik31. The mechanism(s) whereby Hik31 is involved in glucose sensing and response is discussed.