Melting of the Greenland Ice Sheet is a leading cause of land-ice massloss and cryosphere-attributed sea level rise. Blooms of pigmented glacier ice algaelower ice albedo and accelerate surface melting in the ice sheet’s southwest sector.Although glacier ice algae cause up to 13% of the surface melting in this region,the controls on bloom development remain poorly understood. Here we show a directlink between mineral phosphorus in surface ice and glacier ice algae biomass throughthe quantification of solid and fluid phase phosphorus reservoirs in surfacehabitats across the southwest ablation zone of the ice sheet. We demonstrate thatnutrients from mineral dust likely drive glacier ice algal growth, and therebyidentify mineral dust as a secondary control on ice sheet melting. Melting of the Greenland Ice Sheet—a threat for sea level rise—isaccelerated by ice algal blooms. Here the authors find a link between mineralphosphorus and glacier algae, indicating that dust-derived nutrients aid bloomdevelopment, thereby impacting ice sheet melting. Mass loss is predominantly determined by theincoming shortwave radiation flux4modulated by surface albedo5. There has been a ~40% increase in surfacemelting and runoff from the GrIS over the last quarter of acentury6 as a north−south oriented band of low-albedo ice,known as the Dark Zone, has developed along the western margin of the icesheet7. Albedo depends on the physical structure ofsurface ice8and the presence of light absorbing particulates (LAP), which include pigmentedglacier snow and ice algae, black carbon (BC), and mineraldust7. Glacier ice algae  producephotoprotective phenolic pigments14 that lower ice sheet albedo on thelandscape-scale, thereby contributing to melting16. Glacier algae were calculated to bedirectly responsible for 9−13% of the surface melting in the Dark Zone in201616, and there are comparable indirect effects dueto water retention and changes to ice crystal fabric as a result of algalgrowth8. While glacier algal blooms can cover up to 78%of the ice surface16, they exhibit a high degree of interannualvariability in intensity and spatial extent7 that is yet to be understood.Thus, there is a pressing need to better quantify the parameters that controlglacier algal growth and constrain the impact of these blooms on ice sheet albedo,melting, and contributions to sea-level rise. Here we demonstrate, through nutrientaddition experiments and spatially resolved mineralogical and geochemical data, thatphosphorus is a limiting nutrient for glacier algae in the Dark Zone. We identifyphosphorus-bearing minerals (hydroxylapatite) as the likely phosphorous nutrientsource fueling glacier algal blooms. We also disentangle the biogeochemical controlson ice sheet darkening by characterizing the source, composition, and nutrientdelivery capacity of mineral dust. These results, in combination with nutrientaddition experiments, demonstrate that phosphorus from mineral dust is a limitingnutrient for algal blooms in the Dark Zone.The Greenland Ice Sheet (GrIS) comprises only 11.2% of land ice onEarthbio) ice, Hbiosnow, dispersed cryoconite (DCC) ice, cryoconite holes (CCH), a floatingbiofilm, and supraglacial stream water  that was melted in the dark over24 h, and re-incubated for 120 h across five treatments , control). Concomitantly, the healthand productivity of glacier algae assemblages were monitored using rapid lightresponse curves17 performed with pulse amplitude modulation(PAM) fluorometery at 24, 72, 120 h. A significant response to phosphorusaddition was apparent after 120 h of incubation and maximum rates of electron transport (rETRmax: proxy for photosynthesis rate).Both parameters displayed positive responses to increased P availability(PO43- and +ALLtreatments) compared to other treatments  and revealed that organic phosphorus(Porg) accounted for up to 86% of the solid-phase Pin Hbio ice  and mineral phosphorus comprised the remaining solid-phase P.Molar concentrations of total organic carbon (TOC), total nitrogen (TN), andPorg all showed a positive correlation withparticulate Pmin concentrations ,which reflect the nutrient pools in the algal dominated biomass, indicate P asthe limiting nutrient, particularly when compared to Redfield ratios . In the presentstudy, we find that as the concentration of solid-phasePmin increases, the C:N:P ratio decreases andapproaches the ideal Redfield C:N:P ratio. This trend was observed forsolid-phase samples from all habitats and locations sampled in bothyears.To assess potential phosphorus sources, sequential P extractionswere conducted 3(OH); < 1.1 wt%],which we have identified in Dark Zone surface ice dust using Rietveldrefinement25 of powder X-ray diffraction (XRD) datacontained lower abundances of ferromagnesian phases than inland sites, with thecorresponding meltwater enriched in dissolved major cations mineral dustweathering, or through retention of cations via adsorption to negatively chargedcell exteriors.The hydroxylapatite presents an important link between mineral dustand glacier algal blooms because it contains bio-essentialphosphorusin Sites  and 5co2+)Fig. . ElevateAncylonema nordenskioeldii and Mesotaenium sp. comprised between 66 and 99% of thealgal community in all samples , bacterial (16 S), and fungal (ITS2)community compositions clustered according to sampling sites but exhibitedspatial variability across the Dark Zone Fig. –13. Allice Fig.  and aggrice Fig. .Microbiice Fig.  and trapice Fig. .Fig. 4Co26 and fungi27 can accelerate apatiteweathering beyond abiotic rates, thereby transferring solid-phase P to theorganic reservoir. This accounts for the lower Pminconcentration at sites hosting more prolific algal blooms (4 and 5), where ahigher proportion of Pmin has been transformed intoPorg through bioweathering and algal biomassaccumulation. Glacier algal productivity outstrips that of associatedheterotrophic assemblages23, and likely drives recycling ofsolubilized P. Site 4a Hbio and DCC ice contained threeto four times more dissolved P than clean ice or supraglacial stream waterthan DCC ice (36 ± 5 μg L−1) and clean ice(16 ± 13 μg L−1), respectively  as significant contributors signatures of the mineral fraction inour surface samples were used to assess dust source. The REE signatures werehomogenous and exhibited a positive Eu/Eu* anomaly analogous to local sources being intermixed with felsicphases (79−93 wt%), namely feldspars and quartz, which are adept at scatteringlight. The measured refractive index of the dust44, demonstrated that snowalgae respond to the addition of fertilizer45, and inferred thatglacier algal abundance correlate with mineral dustloading24. Here we demonstrate how glacier algaerespond in situ to the addition of specific nutrients and link this response tocollocated nutrient-bearing mineral dust. Our findings demonstrate that mineralnutrient availability is a second-order control on albedo by modulating glacieralgal bloom development. Comparable datasets spanning the GrIS are thereforerequired to incorporate mineral dust as a factor in the positive feedbackbetween algal growth and surface melting. Algae induced melting liberatesice-bound dust, from which heterotrophs can extract micronutrients. Recyclednutrients augment glacier algal blooms, thereby further reducing albedo andpromoting additional melting. Furthermore, trapping and binding of mineral dustby microbial exopolymers  indicate thatthese locations are geochemically primed to host future glacier algal blooms.These trends may be exacerbated by increased atmospheric delivery of mineraldust to the ice sheet through increased windblown dust from exposed forefieldlithologies48, and by projected increased snowfall overthe GrIS49.Nevertheless, factors controlling the timing, spatial extent, andintensity of algal blooms remain knowledge gaps limiting our ability to projectbiological albedo reduction and melt. Bloom initiation depends on bare iceexposure following snowpack retreat, as indicated by glacier algal cell countsfor the same locationsThe complexity of this rapidly changing Arctic system makes itdifficult to anticipate future changes to ice sheet albedo, melt rates, andcontributions to sea level. Our data provide a quantitative link betweenmineral-derived nutrients and glacier algae blooms, and demonstrate that mineraldust is an essential nutrient source for glacier algae. This biogeochemicalprocess must therefore be incorporated into predictive models thereby improvingour understanding of how glacier algal blooms will contribute to ice sheetmelting in the future.n = 1), cleansurface ice , high algal biomassice ,high algal biomass snow (n = 2), anddispersed cryoconite ice . Inaddition, supraglacial stream water (n = 2),a sample of cryoconite hole (CCH) material (n = 1), a cryoconite hole layer from an ice core (n = 1), and a floating algal biofilm (n = 1) were collected. Details of sample types andcollection locations are in Supplementary Table bio ice and snowconsisted of surface ice and snow containing visible glacier algal andparticulate material, and DCC consisted of ice surfaces covered in disseminatedparticulate material from melted out cryoconite holes. Cryoconite hole materialwas sampled to provide a biogeochemical reference for the DCC samples. Thebiofilm sample consisted of a semi-coherent slick of aggregated microbial andparticulate material floating on the surface of ponded meltwater. Ice and snowsamples were collected from the top 3–5 cm of surface into sterile plastic bags,melted at ambient temperatures (5–10 °C) (details in51). Aliquots of filtered melted sampleswere processed as described below for fluid chemistry analyses. While on theice, melted samples were filtered through glass fiber filters , from which the accumulated LAP were removed using a stainless steelspatula. The collected solid LAP were air-dried and stored in glassvials.Surface snow and ice samples were collected along a transect acrossthe ablation zone of the southwestern margin of the Greenland Ice Sheet duringthe 2016 (July 27–August 17) and 2017 (June 1–28) melt seasons. Thesesamples were used as the inoculum for the incubation experiments and had algalcounts of 8.0 ± 2.1 × 103cells mL−1 . Algal cells were counted using a modifiedFuchs-Rosenthal haemocytometer  on a Leica DM 2000 epifluorescencemicroscope with attached MC120 HD microscope camera . Theinoculum was incubated in 30 mL microalgal culturing flasks  inquadruplicates across five nutrient treatments: control (no nutrient addition),+NH4+ , +NO3− , +PO43−, and +ALL nutrients .Nutrient concentrations were designed to provide ~10-fold ambient dissolvedinorganic nitrogen (DIN) and dissolved inorganic phosphorus (DIP) concentrationspreviously reported for GrIS supraglacial ice53.A nutrient addition incubation experiment was performed during the2017 ablation season at our base camp location (Site 4) to determine thelimiting nutrients for glacier algal assemblages. Five, 20 × 20 × 2 cm depthsurface ice areas containing a conspicuous loading of glacier algae were sampledon June 2217, providing information on energy usefrom limiting through to saturating levels ofirradiance54. All samples were dark-adapted for20 minutes prior to RLC assessment, which was undertaken with a saturating pulseof ca. 8,600 μmol photons m−2s−1 for 600 ms duration and nine 20 s incrementallight steps ranging from 0 to4000 μmol photons m−2 s−1.Maximum quantum efficiency (Fv/Fm) was calculated from minimum(Fo) and maximum(Fm) fluorescenceyields in the dark-adapted state as Fv/Fm = (Fm − Fo)/Fm. Electron transport throughphotosystem II (PSII) was calculated from PSII quantum efficiency (YPSII) in relative units(rETR = YPSII × PAR × 0.5) assuming an equal division of photosyntheticallyactive radiation (PAR) between photosystem I and PSII. Analysis of rapid lightcurves (rETR ~ PAR)followed17 with iterative curve fitting in R (v.3.6.0)and calculation of the relative maximum electron transport rate (rETRmax), theoreticalmaximum light utilization coefficient (α),and light saturation coefficient (Ek) following Eilers andPeeters55. Statistical differences inphotophysiological parameters were assessed using two-way ANOVA with the fixedvariables of treatment (5 levels) and date (2 levels: 24 and 120 h) and theinteraction term, following tests of homogeneity of variance and normality ofdistribution. Tukey HSD tests were used to assess statistically significantdifferences in quantum efficiency and relative maximum electron transport ratebetween nutrient treatments. Simultaneous to photophysiological measurements, afurther 5 mL of homogenized sample was fixed using 25% glutaraldehyde at 2%final concentration and transported back to the University of Bristol, UK, toassess glacier algal cell abundance (cellsml−1)9, completed within 1 monthof sample return. Detailed statistical outputs and final cell count data can befound in Supplementary Tables After 24 h, 72 h, and 120 h, measurements of variable chlorophyllfluorescence were performed on 3 mL incubation sub-samples with a WaterPAMfluorometer and attached red-light emitter/detector cuvette system . During the experiment, the ambient air temperature ranged between −4and +4 °C. Rapid light response curves (RLCs) were performed to constrainglacier algae photophysiologybio ice, DCCice, Hbio snow, and CCH samples was determined using amodified version of the SEDEX sequential extractionprotocol20. Steps I, III, IV, and V were completed as ameans of quantifying loosely bound/exchangeable P(Pexch), mineral P (Pmin), andorganic P (Porg). The extracted P was measured in thefluid phase as described below for the melted ice samples. Detailed results canbe found in Supplementary Table The phosphorus content of the H3. ICP-MS wasconducted by Stephen Reid at the University of Leeds, UK. Phosphorus was eithermeasured using segmented flow-injection analysis , or for samples containing lower concentration of phosphorus using a100 cm WPI Liquid Waveguide Capillary Cell in conjunction with an Ocean OpticsUSB2000 + spectrophotometer with a precision of 1.6% and a LOD of2 nmol L−1. Aliquots of the non-acidified 0.22 μmfiltered samples were also analyzed in replicates by ion chromatography byAndrea Viet-Hillebrand at the German Research Centre for Geosciences, Potsdam,Germany. Analyses were carried out with a conductivity detector on a Dionex ICS3000 system, equipped with an AS 11 HC Dionex analytical column run at 35 °C forchromatographic separation of the anions. Standards containing all investigatedinorganic ions  were analyzed andall replicate samples had a standard deviation <10%. Detailed resultscan be found in Supplementary Table Melted surface samples and supraglacial stream water samples werefiltered using 0.22 μm single use syringe filters into acid-washed Nalgenebottles. Inductively-coupled plasma mass spectroscopy  was used to measure fluid phase cations in the filtered water samplesthat were acidified using Aristar HNObioice, DCC ice, Hbio snow, and cryoconite hole materialusing an elemental analyzer  and with the TOC concentrations subsequently measuredfollowing an in situ decalcification step. Note, the organic carbon fractionincludes a contribution from black carbon. TC/TN analyses were conducted byBirgit Plessen and Sylvia Pinkerneil at the German Research Centre forGeosciences, Potsdam, Germany. Detailed results found in SupplementaryTable r-values in Fig. Aliquots from the 0.7 µm GFF filtered, dried and hand-milledsamples were analyzed for their bulk total and organic carbon (TC and TOC) andtotal nitrogen (TN) content. This was done for the Hwp values. The 2016 COD and 1996 ICDD databases wereused to complete phase identification for each sample, in conjunction withDIFFRACplus Eva v.2 software56. Topas V4.256 and the fundamental parametersapproach57 were used to complete Rietveldrefinements59. No preferred orientation corrections wereused because refinements are typically more accurate for samples containing manyphases that are known to exhibit severe preferred orientation  when such corrections are excluded60. In some cases, the useof multiple K-feldspar, plagioclase feldspar, and orthopyroxene structures wereused in a single refinement because this approach provided substantiallyimproved fit statistics and visual fits to observed XRD patterns. This mayreflect the incorporation of dust from multiple source rocks of differingmineralogical composition. Mineral phases identified using XRD were grouped intothe following classes: quartz, plagioclase feldspars, amphiboles (refined using the structure ofactinolite), potassium feldspars (orthoclase/microcline), pyroxene(enstatite/augite/diopside), and micas (refined using the structure ofmuscovite). Detailed results are found in Supplementary Tables The mineralogy of the dust was determined using a Bruker D8 AdvanceEco X-ray diffractometer  with a Cu source, operated at40 kV and 40 mA at the University of Leeds, UK. Samples were hand-milled in anagate mortar and pestle prior to loading in 5 or 10 mm low-background siliconmounts. The small quantity of material per sample necessitated the use ofshallow sample mounts, thereby making the sample not infinitely thick withrespect to X-rays, and thus rendering this analysis semi-quantitative.Furthermore, because it was necessary to rely on hand grinding, XRD patternsexhibit the effects of non-ideal particle size statistics and preferredorientation on some phases, which can result in higherRbio ice), two high algal biomass snow (Hbio_snow),one biofilm, four dispersed cryoconite (DCC) , and four clean ice (CI) samples  were collected into sterile 50 mL centrifuge tubes or sterile sampling bags (CI). After gentle thawing at field-labtemperatures (~5–10 °C), and concentrating by gravimetric settling of particles or filtering (CI) through sterile Nalgene single-use filtration units(pore size 0.22 µm), up to 5 replicate from the concentrates or 1 filter persampling event were transferred to 5 ml cryo-tubes and immediately frozen inliquid nitrogen. Samples were returned to the German Research Centre forGeosciences in Potsdam, Germany in a cryo-shipper at liquid nitrogentemperatures and stored at −80 °C until processing.A total of 26 samples comprising 15 high algal biomass ice or PowerWater (CI) DNA Isolation kits (MoBio Laboratories). The 16 SrRNA, 18 S rRNA and ITS amplicons were prepared according to the Illumina “16 SMetagenomic Sequencing Library Preparation” guide. 16 S rRNA genes wereamplified using the bacterial primers 341 F (5′-CCTACGGGNGGCWGCAG) and 785 R(5′-GACTACHVGGGTATCTAATCC) spanning the V3-V4 hypervariable regions. 18 S rRNAgenes were amplified using the eukaryotic primers 528 F (5′ GCGGTAATTCCAGCTCCAA)and 706 R  spanning the V4-V5hypervariable regions. ITS amplicons were amplified using the primers 5.8SbF (5′CGATGAAGAACGCAGCG) and ITS4R (5′ TCCTCCGCTTATTGATATGC) spanning the ITS2 region.All primers were tagged with the Illumina adapter sequences. Polymerase chainreactions (PCR) were performed using KAPA HiFi HotStart ReadyMix. Initialdenaturation at 95 °C for 3 min was followed by 25 cycles of denaturation at95 °C for 30 s, annealing at 55 °C for 30 s, and elongation at 72 °C for 30 s.The final elongation was at 72 °C for 5 min. All PCRs were carried out inreaction volumes of 25 µL. All pre-amplification steps were done in a laminarflow hood with DNA-free certified plastic ware and filter tips. Amplicons werebarcoded using the Nextera XT Index kit. The pooled library was sequenced on theIllumina MiSeq using paired 300-bp reads at the University of Bristol GenomicsFacility, Bristol, UK.DNA was extracted from all samples using the PowerSoil. Theimported libraries were quality-filtered using the dada2 pipeline . The ampliconsequence variants (ASV) in the filtered libraries were classified usingclassify-sklearn and the respective databases Greengenes 62, Silva 63, and Unite 64. ASVs skewing the results were removedfrom each data set . Feature tables containing solely algal  or fungal (ITS2: --p-include Fungi)sequences were created. Subsequently, all feature tables were rarefied to thelowest yet sufficient sample size and low-coverage samples below this thresholdwere discarded . Further, only ASVs with aminimum frequency count of 10 were retained in the feature tables. Detailedresults can be found in Supplementary Tables The sequenced 16 S, 18 S, and ITS2 libraries were individuallyimported into Qiime2 v.2019.1).161. ItsThe filtered feature tables were imported into R (v.3.6.0) tocreate bar charts representing the respective community compositions based ontheir relative abundances. Non-metric multidimensional scaling (NMDS) analyseswere performed using the “metaMDS” function (Bray-Curtis distances) of the Rpackage “vegan” and plots were created using the package “ggplot2”. Analysis ofsimilarities (ANOSIM) was carried out using the “anosim” function of the “vegan”package and “sites” and “habitats” as treatment groups.bio ice, cryoconite, DCC ice, and biofilmsamples were fixed using 2.5 % v/v glutaraldehyde and stored at 4 °C. Sampleswere dehydrated via an ethanol dehydration series  for 15 min at each step, followed by 10 min in each of: 50:50 ethanol:hexamethyldisilazane (HMDS), and 2 × 100% HMDS. The HMDS was removed and thesamples were air-dried prior to being mounted on stainless steel stubs usingadhesive carbon tabs. Samples were coated with 5 nm of iridium using an AgarHigh Resolution sputter coater. SEM characterization of the samples wasconducted using a Hitatchi 8230 SEM at the Leeds Electron Microscopy andSpectroscopy Centre (LEMAS), University of Leeds, UK.Hbio ice, DCC ice, and cryoconite hole particulatesolid materials filtered onto 0.7 µm GFF and hand-milled in an agate mortar andpestle. To remove organic matter, samples were ashed in a slightly open ceramiccrucible in a muffle furnace at 450 °C for 4 h. Rock samples collected from site6 by Gilda Varliero and Gary Barker  (representinglithologies of the catchment area) were cut into centimeter-sized cubes prior tomilling in a tungsten ring mill. Acid dissolution of the mineral fraction wasachieved in Savillex® beakers using pro-analysis acids previously purified bydistillation and sub-boiling. Dissolution was first performed with 2 mL 14 MHNO3 and 1 mL 23 M HF on a hot plate at 120 °C for48 h and later, after evaporation to dryness, with 2 mL 6 M HCl on a hot plateat 120 °C for 24 h. REE concentrations were determined using HR-ICP-MS(ThermoFisher Element 2) at the Vrije Universiteit Brussel, Belgium. Traceelement concentrations were calibrated using elemental standard solutions andUSGS reference material (AGV-2). Precision for all elements is better than 2%RSD. Detailed results can be found in Supplementary Table Rare Earth Element (REE) analysis was conducted onH2O2 (w/w) (Honeywell Fluka™)was added in order to remove the organic content. The tubes were sonicated (VWRultrasonic cleaner) for 10 min to disaggregate the solids. The samples wereagitated in an orbital shaking incubator operating at 100 rpm at 35 °C. After72 h, the samples were centrifuged at 4000 rpm for 10 min (Eppendorf centrifuge5810). The supernatant was removed and replaced with newH2O2. This was repeated sixtimes until no more organic oxidation was observed. The mineral fraction waswashed three times in water (Sartorius arium pro ultrapure water) for 24 h, withcentrifugation succeeding each wash. The organic-free mineral fractions weredried at 35 °C prior to particle size analysis measured by Kerstin Jurkschatusing a DC24000 CPS disc centrifuge65 at Oxford Materials CharacterisationServices, Oxford, UK.Approximately 100 mg of each sample was transferred to a 50 mLcentrifuge tube, to which 35 mL of 30%HAliquots of melted snow and ice samples of known volumes werefiltered in the field successively through pre-weighed 5 μm and 0.2 μmpolycarbonate filters. The filters were returned to the University of Leeds, UKwhere they were dried and weighed to determine the mass of LAP per volume ofmelted sample. The sum of the total organic carbon and nitrogen was used as aproxy to indicate the biomass fraction of each sample, with the remaining samplemass allocated to mineral dust. These values were used to calculate the mineraldust mass loading per unit of melted ice.Further information on research design is available inthe Supplementary InformationReporting Summary


Ixodes ricinus instar abundance at 159 sites in southern Scandinavia in August-September, 2016, and collected 29,440 tick nymphs at 50 of these sites. We additionally measured abundance at 30 sites in August-September, 2017. We tested the 29,440 tick nymphs in pools of 10 in a Fluidigm real-time PCR chip to screen for 17 different tick-associated pathogens, 2 pathogen groups and 3 tick species. We present data on the geolocation, habitat type and instar abundance of the surveyed sites, as well as presence/absence of each pathogen in all analysed pools from the 50 collection sites and individual prevalence for each site. These data can be used alone or in combination with other data for predictive modelling and mapping of high-risk areas.Ticks carry pathogens that can cause disease in both animals and humans, and there is a need to monitor the distribution and abundance of ticks and the pathogens they carry to pinpoint potential high risk areas for tick-borne disease transmission. In a joint Scandinavian study, we measured  Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.12523133 Incidences of tick-borne diseases such as Lyme borreliosis have increased in many regions – including Scandinavia9. Left untreated, Lyme borreliosis can pose serious health threats to humans. Other tick-borne diseases, affecting humans and animals such as anaplasmosis (caused by Anaplasma phagocytophilumn), relapsing fever (caused by Borrelia miyamotoi), babesiosis (caused by Babesia spp.), rickettsiosis (caused by Rickettsia spp.) and infections by Neoehrlichia mikurensis, are also on the rise13. As climate has changed over the past decades, there is great concern that a warmer climate has an impact on increased incidence of vector-borne diseases as vectors might expand their distribution to more northern climates in Scandinavia14. Human behaviour evidently affects the risk of exposure to ticks and their pathogens, but multiple studies have shown how tick abundance affect not only human tick-exposure but also the prevalence of pathogens harboured in ticks21. More effective control and prevention of these diseases requires a better understanding of factors affecting vectors and their associated pathogens. Hence, there is a great need for data-based analyses and models that can identify the main drivers of vector abundance and distribution as well as drivers of disease transmission. Developing predictive models for larger areas requires underlying data, which can oftentimes be economically and logistically unfeasible. Free access to data from multiple studies would not only support any large-scale modelling attempt but also act as a reference to future repeated surveys, and thus sharing of data would be of great value to the scientific community.Tick-borne pathogens have become more prevalent over the last decades30, and some of these studies have also made data accessible through a free repository33. However, this is only a fraction of the published data, and finding data on tick abundance and tick-borne pathogen prevalence from a large number of sites for use in risk assessments and modelling, requires a thorough review of the immense amount of papers reporting these data, as for example was done by Estrada-Peña et al. both for European and South American tick data35. Common and free access to data on tick abundance and tick-borne pathogen prevalence would greatly benefit epidemiological research and sharing of data should always be encouraged. Therefore, we have created a public database for tick abundance and tick-borne pathogen prevalence in the figshare repository and describe the collected data in this manuscript.In Europe there are numerous studies on ticks and their pathogens that have been reported in scientific journalsIxodes ricinus, I. persulcatus, and Dermacentor reticulatus. The dataset of 50 georeferenced sites of approximately 60 pools of ten nymphs each tested for 17 pathogens, 2 pathogen groups and 3 tick species thus consist of approximately 66,000 individual PCR tests. The field study was conducted within a short time-frame, from August to September in both 2016 and 2017, thus any differences between measures of abundance and pathogen prevalence within the same year is likely due to geographical differences within the region.The data is part of a multi-national study on ticks in southern Scandinavia. The tick abundance and prevalence data described here, was obtained using a standardised stratified design, comparable between the participating countries. The data consist of tick instar abundance measured from a total of 159 sites in 2016 and 30 sites in 2017 in Denmark, Norway and Sweden and constitute 75.6 km’s of flagged transects. We furthermore collected tick nymphs at 50 of the 159 surveyed sites, counting a total of 24,667 ticks. We used a Fluidigm real-time PCR chip to screen for 17 different pathogens and 2 pathogen groups expected to be in the region , to further divide these habitat pixels into high NDVI  and low NDVI . An overview of the stratification scheme can be found in Fig. The stratification of the study region has been described in Kjær 40 (sampleStratified in the raster41 package). Our original aim was to both measure instar abundance and collect 600 tick nymphs from 30 1st priority sites consisting of 80% forest and 20% meadow in each country36. To account for potential difficulties collecting the required number of nymphs at some of the sites, we created 10 alternative sites with the same stratification score for each of the 30 first priority sites in each country, where these 10 alternative sites were ranked after smallest distance to the original site. To account for difficulties collecting enough tick nymphs in meadow sites, we also created 10 alternative forest sites for each meadow site with the same NDVI score. We also created 20 random sites  along the Oslo Fjord in Norway, as we were interested in investigating ticks in that region.We randomly selected sites in Denmark, Norway and Sweden using R 3.4.2For logistical reasons, we needed to ascertain whether it was possible to collect 600 tick nymphs within a reasonable time frame at a given site. Thus, we defined a measure of effort as the amount of nymphs collected by one person during 30 minutes. We determined that a site should allow collection of 600 nymphs in 10 man hours, and thus one person should be able to collect 30 nymphs in 30 minutes. If the number of nymphs collected was below 30, no attempt was made to collect 600 tick nymphs for pathogen testing and an alternative site was explored to obtain the 600 nymphs. Tick instar abundance was, however, measured at all sites, which could result in abundance measures from more than 30 sites per country.42 a white flannel cloth (1.05 × 1.15 m with lead weights in one end) along each transect in both directions  and counted and removed tick instar every 50 meters. We measured all tick instar abundance between 15 August and 30 September, 2016 within the hours of 11–16. After measuring abundance at a site, we used our preciously described effort measure to gauge whether it was feasible to collect 600 tick nymphs at the site. If the site was deemed feasible, we collected the tick nymphs, placed them in tubes and stored them on dry ice. When returning from the field, we stored the ticks at −80 °C until use. Within the allocated collection time period, we obtained abundance measures for 37 sites in Denmark, 47 sites in Norway and 75 sites in Sweden. Out of these sites, we collected approximately 600 tick nymphs from 30 sites in Denmark, 11 sites in Norway and 9 sites in Sweden  using a TissueLyser II  for 2 × 2.5 min at 25 Hz in a mixture of 75 µl Incubation buffer  and 75 µl Lysis buffer . We briefly centrifuged the samples at 10,000 × g for 60 s and added 30 µl Proteinase K. After an overnight incubation of the samples at 56 °C, we added 300 µl Lysis buffer  and vortexed the samples for a short time. We then extracted Genomic DNA, using the Maxwell 16 LEV Blood DNA kit  on a MaxwellE. coli control5 and one negative water control. We used primers for bacterial and parasitic tick-borne pathogens previously identified in the most common tick species, I. ricinus, from Scandinavia, and also included common tick-borne pathogens found throughout Europe5. To screen for different tick species, we used primers for I. ricinus, I. persulcatus, and D. reticulatus; tick species observed in various regions of Scandinavia44. All pathogens targeted in the PCR assays are listed in Table We performed high-throughput microfluidic real-time PCR, using the BioMark real-time PCR system . We applied the 192.24 dynamic arrays with 22 pathogen primers, as well as one positive E. coli controls were positive, amplification curves were accepted by Fluidigm’s algorithm for ideal curves and CP values were ≤28. Sensitivity and specificity of the test have been discussed previously45.We considered a PCR run to be valid (Pass/Fail) when all water controls were negative, all et al.46, assuming 100% test sensitivity and specificity. Exact confidence limits (CIs) were calculated based on binomial theory46.We estimated the individual-level pathogen prevalence at each site based on the number of positive pools and number of examined pools of ten nymphs each using method 3 from Cowling I. ricinus and negative for I. persulcatus and D. reticulatus47. We did not detect F. tularensis, C. burnetii, B. canis or B. henselae in any of the pools, and these data are omitted from the published database.All of our pools were positive for 48. The 3 files can be linked with each other through the SiteID variable, which is a unique identifier for each site.A.The tick instar abundance table has 12 columns: (1) the unique site ID, (2) the country where the site is situated, (3) the date of measurement, (4) the site selection criteria, (5) the longitude of the site, (6) the latitude of the site, (7) the habitat type of the site, (8) the total number of larva counted from both transects, (9) the total number of nymphs counted from both transects, (10) the total number of adult females counted from both transects, (11) the total number of adult males counted from both transects, (12) the total number of tick nymphs collected and used for pathogen screening.B.B. miyamotoi, (5) the Pass/Fail test result for B. burgdorferi sensu lato, (6) the Pass/Fail test result for B. afzelii, (7) the Pass/Fail test result for B. burgdorferi sensu strictu, (8) the Pass/Fail test result for B. garinii, (9) the Pass/Fail test result for B. lusitaniae, (10) the Pass/Fail test result for B. spielmanii, (11) the Pass/Fail test result for B. valaisiana, (12) the Pass/Fail test result for A. phagocytophilum, (13) the Pass/Fail test result for B. divergens, (14) the Pass/Fail test result for B. microti, (15) the Pass/Fail test result for B. venatorum, (16) the Pass/Fail test result for N. mikurensis, (17) the Pass/Fail test result for R. helvetica, (18) the Pass/Fail test result for SFG Rickettsiae, (19) the tick species found within the pool.The pathogen pool results table has 19 columns: (1) the country where the site is situated, (2) the unique site ID, (3) The pool number for the particular site, (4) the Pass/Fail test result for C.B. miyamotoi, (4) the individual prevalence in percent along with exact confidence limits for B. burgdorferi sensu lato, (5) the individual prevalence in percent along with exact confidence limits for B. afzelii, (6) the individual prevalence in percent along with exact confidence limits for B. burgdorferi sensu strictu, (7) the individual prevalence in percent along with exact confidence limits for B. garinii, (8) the individual prevalence in percent along with exact confidence limits for B. lusitaniae, (9) the individual prevalence in percent along with exact confidence limits for B. spielmanii, (10) the individual prevalence in percent along with exact confidence limits for B. valaisiana, (11) the individual prevalence in percent along with exact confidence limits or A. phagocytophilum, (12) the individual prevalence in percent along with exact confidence limits for B. divergens, (13) the individual prevalence in percent along with exact confidence limits for B. microti, (14) the individual prevalence in percent along with exact confidence limits for B. venatorum, (15) the individual prevalence in percent along with exact confidence limits for N. mikurensis, (16) the individual prevalence in percent along with exact confidence limits for R. helvetica, (17) the individual prevalence in percent along with exact confidence limits for SFG Rickettsiae.The pathogen individual nymph prevalence table has 17 columns: (1) the country where the site is situated, (2) the unique site ID, (3) the individual prevalence in percent along with exact confidence limits for The database consists of 3 csv filesWe held several meetings to discuss the standardisation of measuring tick abundance, and a video demonstrating the “dragging” method was shared with each researcher in the group to ensure that the collections were standardised as much as possible. We furthermore ensured that each country used the same fabric and same fabric size of the cloth used for “dragging”, by having these cloths produced and made by one partner for all 3 countries.45.The Fluidigm real-time PCR method has been validated through numerous studies, particularly studies pertaining to tick pathogens


TisAFP7 from the snow mold fungus Typhula ishikariensis. TisAFP7 exhibited intermediate activity, with the ability to bind the basal plane, compared with a hyperactive isoform TisAFP8 and a moderately active isoform TisAFP6. Analysis of the TisAFP7 crystal structure revealed a bound-water network arranged in a zigzag pattern on the surface of the protein’s ice-binding site (IBS). While the three AFP isoforms shared the water network pattern, the network on TisAFP7 IBS was not extensive, which was likely related to its intermediate activity. Analysis of the TisAFP7 crystal structure also revealed the presence of additional water molecules that form a ring-like network surrounding the hydrophobic side chain of a crucial IBS phenylalanine, which might be responsible for the increased adsorption of AFP molecule onto the basal plane. Based on these observations, we propose that the extended water network and hydrophobic hydration at IBS together determine the TisAFP activity.Antifreeze proteins (AFPs) inhibit ice growth by adsorbing onto specific ice planes. Microbial AFPs show diverse antifreeze activity and ice plane specificity, while sharing a common molecular scaffold. To probe the molecular mechanisms responsible for AFP activity, we here characterized the antifreeze activity and crystal structure of  Antifreeze proteins (AFP) are part of one such strategy. These proteins are structurally diverse and produced by various organisms, including fishTf) of a solution below the melting point (Tm)6 via Gibbs–Thomson effect7. The difference between Tm and non-equilibrium Tf is called thermal hysteresis (TH), which is a prime indicator of the AFP antifreeze activity9. Based on the TH value, AFPs are classified into two main groups, namely, moderately active and hyperactive AFPs. Hyperactive AFPs achieve up to 2–5 °C TH, which is 10–100 times that of the moderately active AFPs at identical protein concentrations10. Hyperactivity can be ascribed to the binding affinity to the basal plane of ice as well as prism and pyramidal planes, leading to rapid ice growth (burst) below Tf, and expansion perpendicular to the ice c-axis with a dendritic pattern with a sixfold symmetry11. By contrast, typical moderately active AFPs bind to prism and/or pyramidal planes to form a bipyramidal ice between Tm and the non-equilibrium Tf (defined as the TH gap). Below the non-equilibrium Tf, the ice crystal bursts at the two unbound bipyramid tips, which lie along the c-axis, forming a needle-shaped ice14. The specific affinity toward sets of water molecules constituting discrete ice surfaces is attributable to the ice-binding site (IBS), localized on the flat surface of an AFP molecule9. IBS in various AFPs show vast amino acid sequence and structure diversity, which implies that each AFP evolved from a different ancestor molecule to adapt to the cold environment by acquiring ice-binding ability9. Therefore, understanding the detailed molecular mechanism that defines ice-binding specificity is crucial for the elucidation of adaptation to cold environment associated with AFP evolution.AFPs have specific affinity for a single ice crystal, to arrest its growth in the direction normal to the bound surface. They noncolligatively depress the non-equilibrium freezing point  349418 in the Pfam database19, and currently categorized as “ice-binding–like family”. Characterization of DUF3494 AFPs  revealed their highly diversified antifreeze activities. For example, AFPs from Colwellia sp. (ColAFP)20, Flavobacterium frigoris PS1 (FfIBP)21, Typhula ishikariensis isoform 8 (TisAFP8)22, and Shewanella frigidimarina (SfIBP_1)23 are hyperactive, whereas those from Leucosporidium sp. (LeIBP)24, T. ishikariensis isoform 6 (TisAFP6)25, Antarctomyces psychrotrophicus (AnpIBP1a)26, and Fragilariopsis cylindrus (FcIBP)16 show moderate activity. X-ray crystallographic studies revealed that all these AFPs adopt a similar β-helical structure. The putative IBS of this protein family is located on the flat surface of one β-helix25. The level of IBS sequence identity shared by various AFPs is similar to that shared by other regions of these proteins; however, no conserved sequence motifs in IBS have been identified. Based on phylogenetic analysis, microbial AFP genes are presumably propagated via horizontal gene transfer27. Less is known, however, about the key determinants of hyperactivity and moderate activity of this AFP family.Most microbial AFPs, produced by such microorganisms as fungi29. On the array, Thr residues are aligned at constant intervals, matching the distance between the water molecules on the ice plane28. Further, the hydroxy groups in the side chain of IBS Thr residues anchor the bound water molecules at constant intervals, which match the basal and prism plane of the ice surface29. According to a molecular dynamics simulation study, regularly ordered water clathrate gives rise to the highest affinity for the ice basal plane30. Crystal structure analysis of MpAFP_RIV from the Antarctic bacterium Marinomonas primoryensis revealed an array of ordered water molecules aligned along the IBS residues. The bound water molecules are anchored to a repetitive motif of IBS residues (Thr–Gly–Asn/Asp) to form ice-like structure, which led to the proposal of the “anchored clathrate water” mechanism for AFP–ice interaction31.Three-dimensional structural analyses of hyperactive AFPs from arthropods and bacteria have revealed unique features of the IBS and bound water structure. For instance, insect AFPs form a β-helical structure with repetitive sequence motifs. Conserved residues in these motifs  are organized into two parallel arrays along the β-helical axis, constructing the IBS on one flat face of the β-helix25, which implies that a different architecture drives their hyperactivity. Based on studies of ColAFP and TisAFP8, Hanada et al.20 and Cheng et al.22, respectively, proposed that the b-face of IBS be subdivided into the β-sheet and adjacent loop regions, termed a compound IBS, which stick to the discrete ice plane. Based on the crystal structure analysis and ice-docking modeling combined with a mutation study, IBS loop residues were proposed as crucial for the affinity to the basal plane, conferring hyperactivity22. Nevertheless, it is still not clear how the compound IBS recognizes the specific ice plane.IBS of microbial AFPs with the DUF3494 fold lack the repetitive amino acid sequence and consensus motifT. ishikariensis secretes AFP (TisAFP) at 0 °C15. Culture filtrates contain seven AFP isoforms (TisAFP2–8), 223 amino acid residues each. We have previously shown that TisAFP8 is hyperactive22 whereas TisAFP6 is only moderately active25, despite high sequence identity (83%) shared with TisAFP8. Another isoform, TisAFP7, shares an even higher identity with TisAFP8 (91%) and TisAFP6 (87%). We reasoned that TisAFP7 might be a suitable target molecule for gaining insight into the relationship between AFP antifreeze activity and IBS structure. In the current study, we characterized TisAFP7 using TH measurements, ice crystal morphology observation, and visualization of AFP-bound ice planes by fluorescence-based ice plane affinity (FIPA) analysis. We also analyzed the crystal structures of TisAFP7 and its defective mutants. The analysis revealed a distinct bound-water network on IBS, which presumably defines the binding affinity of microbial AFPs for the basal plane. The findings broaden the understanding of cold-adaptation mechanism of psychrophilic and cold-tolerant microorganisms, and will inform the design of artificial compounds with antifreeze properties.The snow mold fungus TisAFP7 was first evaluated by TH measurements, at a series of protein concentrations  at 0.11 mM, and TisAFP6 exhibited the lowest TH (approximately 0.6 °C) at 0.35 mM. Comparing the TH values of these AFPs at the same protein concentration, e.g., 0.11 mM, TisAFP7 TH was approximately 35% that of TisAFP8, and 1.15-fold that of TisAFP6. Based on the TH values, the antifreeze activity of the three isoforms could be ordered as TisAFP8 > TisAFP7 > TisAFP6 (highest to lowest). TisAFP7 shares 91% and 87% sequence identity with TisAFP8 and TisAFP6, respectively, which supports its intermediate position among the isoforms. Based on the TH value, TisAFP7 is an intermediately active AFP. The denaturation profile monitored by CD spectra from 20 to 70 °C is shown in Supplementary Fig. Tm) was estimated as a midpoint of the profile as 47.5, 50.0, 53.5 °C for TisAFP7, TisAFP8, and TisAFP6, respectively. Besides, defective mutants of TisAFP7 exhibited no change in their Tm, as shown in Supplementary Fig. Tm shows that the order of the thermal stability is not correlated with that of TH values. Contrary to our results, higher Tm was reported for moderately active LeIBP21 (61.0 °C) and EfcIBP32 (66.4 ± 2.7 °C), and lower Tm was reported for hyperactive FfIBP21 (56.4 °C) and IBPv33 (53.5 °C). On the other hand, thermal denaturation experiments for chimeric proteins for LeIBP and FfIBP21 demonstrated that denaturation temperature of microbial AFP might be affected by various structural segments including capping head regions, which cover both ends of β-helix. Therefore, the authors of the referenced paper 21 suggested that Tm is not directly related to antifreeze activity. For TisAFP6, most of the replaced residues (7 residues out of 11) from TisAFP7 and TisAFP8 are situated in the capping head regions, which seems to originate the highest Tm among the isoforms.Antifreeze activity of TisAFP7 (TH 0.9 °C), the single ice crystal retained its original size below Tm  perpendicular to the c-axis, with a hexagonal-stellar pattern with dendritic branches . At a relatively low concentration (0.01 mM), the seed ice crystal changed shape to a hexagonal bipyramid with truncated tips in the TH gap, and then grew rapidly, maintaining the bipyramidal shape , as shown in Fig. Choristoneura fumiferana)34 and SfIBP_123. On the other hand, TisAFP6 supported ice bursting along the c-axis even at a high protein concentration 37, and TmAFP (Tenebrio molitor)37. Some moderately active AFPs, when assayed at a relatively high concentration (0.1 mg/ml), also exhibit whole-hemisphere binding, e.g., BpAFP14 and AFPII38 (Brachyopsis rostratus), with a concentration-dependent multiple-plane binding. In the current study, FIPA analysis of TisAFP7 was performed using a low protein concentration (0.007 mg/ml); the protein’s multiple-plane binding ability was higher than those of the typical moderately active AFPs. The ability to bind the basal plane has been also reported for moderately active AFPs, such as TisAFP625 and LpIBP39 (Lolium perenne), but with the basal and primary prism planes covered by separate hemisphere protein patches, which was different from the whole-plane binding observed for TisAFP7. Based on the above, we propose that TisAFP7 binds all ice planes, including the basal plane, with an intermediate binding strength, positioned between those of hyperactive and moderately active AFP. This is a unique ice-binding property, reflecting the sequence identity shared by the hyperactive and moderately active TisAFP isoforms.To visualize the AFP-bound planes of ice crystal Fig. , we prepTisAFP7 was collected using a crystal grown as a thin plate-like shape. The crystal belongs to the orthorhombic space group P212121, with unit cell parameters of a = 57.12 Å, b = 62.99 Å, and c = 101.21 Å, with two molecules in an asymmetrical unit. The crystal structure of TisAFP7 was determined at 1.54 Å resolution by a molecular replacement method. The statistics for data collection and refinement are summarized in Table R factor of 0.195 and Free R factor of 0.229, and was composed of 446 (223 × 2) residues and 746 solvent molecules. The refined TisAFP7 structure exhibited the root-mean–squared deviation (RMSD) of 0.37 Å and 0.34 Å from Cα atoms of TisAFP6 and TisAFP8, respectively, reflecting high sequence identities shared by the isoforms.Diffraction data for wild-type TisAFP7. The structure is dominated by a right-handed β-helical domain, a typical DUF3494 structure  connect the β-strands and loop segments. Figure TisAFP7 viewed along the β-helix axis. A triangular cross section is seen, with three flat surfaces  composed of parallel β-sheets. The a-face is covered with a long α-helix and the N-terminal segment, while the b- and c-faces are exposed to the solvent. In Fig. Figure TisAFP7, which is located on the flat b-face of the β-helix. IBS consists of 26 residues of a six-stranded parallel β-sheet and 21 residues of the adjacent loop region connected to the a-face of the β-helix. Only five residues differentiate IBS of TisAFP7 from the hyperactive TisAFP8 isoform, all of which are located in the loop region. To assess their role in ice binding, Thr20 in the loop region, reported to be one of the residues key for TisAFP8 hyperactivity22, was replaced with Tyr. The crystal structure of TisAFP7 T20Y was determined at 1.72 Å resolution. The crystal was isomorphous with the wild-type crystal, and the final structure was refined with R-factor of 0.208 and Free R of 0.246, containing 446 residues and 554 solvent molecules.Figure TisAFP7, accordingly  simulation. Table TisAFP isoforms. For TisAFP7 and TisAFP8, water residence time on IBS was longer than that on other areas, suggesting that IBS of TisAFP7 and TisAFP8 retain more water molecules. On the other hand, for TisAFP6, non-IBS residues exhibited longer residence time than IBS, which might be attributable to the substituted residues located in the capping head regions, similarly to higher thermal stability estimated by CD measurement. For the bound water molecules on IBS residues, hyperactive TisAFP8 exhibited the longest residence time and TisAFP6 exhibited the shortest, which is the same order for their antifreeze activities. Our current MD calculation offers a simple estimation of the relationship between the water coverage on IBS and its ice plane affinity. Therefore, discussion with the careful calculation should be necessary in the separated report.Further, on the top edge of the IBS loop of 30, terahertz spectroscopy46, and sum frequency generation spectroscopy47. The current study provides basic insights that can be utilized in these experimental approaches.Detailed analysis of water-molecule dynamics might be required for further elucidation of the relationship between the bound-water structure and ice-binding property of AFP. The unique hydration structure of surface water of AFP has been reported by utilizing molecular dynamics simulationTisAFP isoforms are associated with the bound water structure around IBS. The IBS residues act like a platform on a β-helical scaffold, organizing the hydration waters. This is an astounding example of evolution of the antifreeze properties of microbial AFPs, namely, tuning the hydration structure, as a means to adapt to the microbial habitat.To conclude, we here showed that the different activities of TisAFP7 (Genbank accession number BAD02892) gene was codon-optimized for Escherichia coli and synthesized by GENEWIZ . It was then inserted into the expression vector pET38b . The vector was used to transform E. coli BL21 (DE3) (Novagen), and the transformants were plated onto Luria–Bertani (LB) agar containing 30.0 μg/ml kanamycin, and incubated at 37 °C overnight. Site-directed mutagenesis of the TisAFP7 gene was performed using the KOD plus mutagenesis kit  with primers listed in Supplementary Table TisAFP7 and its mutants were prepared as described by Cheng et al.22, with a slight modification. Specifically, a bacterial colony was inoculated into 50 ml of LB medium containing 30.0 μg/ml kanamycin, and pre-cultured overnight at 37 °C, with shaking at 130–140 rpm. The culture was then transferred into 1.0 L of fresh LB medium supplemented with 30.0 μg/ml kanamycin, and cultured at 30 °C until the culture OD600 reached 0.4–0.8 units. Expression of TisAFP7 was induced by the addition of 0.5 mM isopropyl-β-d-thiogalactopyranoside. The cells were further cultured at 15 °C for 24 h, harvested by centrifuging, and disrupted using high-pressure homogenizer EmulsiFlex-C3 . Cell debris was removed by centrifuging, and the supernatant was dialyzed against 10 mM glycine–HCl (pH 3.0). The dialysate was filtered and loaded onto a cation exchange column Macro-Prep High S  equilibrated with the same buffer. The adsorbed sample was eluted using a linear gradient of 0–300 mM NaCl. TisAFP7-containing fractions were identified by checking antifreeze activity and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and were then dialyzed against 25 mM ammonium bicarbonate (ABC) buffer (pH 7.9). The sample was loaded onto an anion exchange column Macro-Prep High Q (Bio-Rad) equilibrated with same buffer. TisAFP7 was collected in the flow-through fraction. The purity of the sample was confirmed by SDS-PAGE and analytical size-exclusion chromatography (SEC) using Sephadex G-75 . Purified TisAFP preparation was concentrated by using an ultrafiltration device Amicon Ultra . Protein concentration was determined based on the optical absorbance at 280 nm. Wild-type TisAFP7 eluted as a major fraction after cation-exchange chromatography, and migrated as a single band on SDS-PAGE gel and a single peak after SEC  equipped with a temperature-controlled sample holder. TisAFP samples were prepared at 0.25 mg/ml in 10 mM sodium phosphate buffer (pH 7.4) and measured at 4 °C in a quartz cuvette of 1 mm optical path length. As shown in Supplementary Fig. TisAFP sample was observed by monitoring the ellipticity (θ) at 220 nm with heating the samples at 0.2 °C interval from 20 to 60 °C at a rate of 1 °C/min. During the temperature scanning, CD spectra were recorded at 4, 20, 30, 40, 50, 60 °C to record the spectral change upon the thermal denaturing process. Typical spectral change is shown in Supplementary Fig. TisAFP7, which was fully denatured at 60 °C. Fraction of unfolded protein was estimated as T) with 1.0 °C intervals in Supplementary Fig. Tm) was estimated as a midpoint temperature at which 50% of the sample is unfolded. For TisAFP6, the heating process was applied up to 70 °C, and TisAFP6 exhibited slightly higher thermostability than other isoforms. After the temperature scanning, the sample was cooled back to 4 °C. For all TisAFP samples, the denaturated protein was precipitated in the cuvette even after the cooling back process. Also, CD spectrum observed at 4 °C after heating process revealed the most of samples remain denaturated, as shown in Supplementary Fig. TisAFP is the irreversible process whereas the reversible process was reported for fish AFPs49.The The antifreeze activity of protein samples was evaluated by observing ice crystal growth in AFP solutions by using Leica DMLB100 photomicroscope  equipped with Linkam THMS600 sample temperature controller . A color video 3CCD camera  was used to capture the images of ice crystals and the temperature of the solution displayed on the screen..35. Briefly, approximately 0.75 μl of AFP sample was placed in a center of a glass capillary tube (10–15 mm long), which was then sealed at both ends with mineral oil. The capillary was placed in a copper holder on the cooling stage. To ensure the thermal conductivity, the clearance gap between the capillary and the stage was filled with 20 μl of ethylene glycol. The sample solution was frozen by cooling to approximately –30 °C, subsequently warmed up to obtain a single particle of ice crystal. The melting point was determined as the temperature at which the ice started to melt. The sample was then incubated for 3 min at 0.05 °C below the Tm. Finally, the ice crystal was cooled at a rate of 0.1 °C/min until rapid growth of the ice crystal was observed. The temperature at which the rapid growth started was recorded as the freezing point, Tf, of the solution. TH measurement was repeated at least three times for each sample at the specified protein concentrations.TH of protein solutions was determined as described by Takamichi et alTisAFP7-bound ice plane, FIPA analysis was performed, as described by Garnham et al.50 and Basu et al.37. Briefly, a single ice crystal, approximately 40 mm in diameter and 30–40-mm high, was prepared in a suitably sized polyvinyl chloride pipe by gradually growing from a single seed ice. The single crystallinity of the ice crystal was checked by using in-house assembled crossed polarizer. The orientation of the crystallographic a-axis was confirmed by the direction of a star shape poked on the ice surface after lyophilization for 10 min. The macroscopic ice crystal was then mounted on a brass cold finger cooled at –1 °C, with the basal or primary prism plane perpendicular to the cold finger. The mounted ice crystal was immersed in chilled milli-Q water and trimmed to a hemispherical shape with 10-mm diameter. Wild-type TisAFP7 was labeled with an orange fluorescent dye  . The three mutants, TisAFP7 T20Y, A214Y and T20Y/A214Y, were labeled with NHS-Fluorescein (Thermo Fisher Scientific). AFP samples were incubated with the fluorescent dyes in 0.1 M NaHCO3 at 25 °C for 3 h, and then dialyzed against water to remove free dye. The mounted ice crystal was grown in 35–40 ml of a solution of fluorescently-labeled AFP (0.007 mg/ml), which was slowly incorporated into the specific ice plane in the hemisphere. The AFP solution was stirred every 20 min. The cooling temperature of the cold finger was gradually lowered to between –7 °C and –8 °C over 3–4 h. The ice hemisphere was detached from the cold finger after achieving a 45-mm diameter, and kept refrigerated at –30 °C for 20–30 min prior to observation. The fluorescence of the ice hemisphere was observed under UV light and photographed.To determine the TisAFP7 and T20Y mutant were crystallized by hanging-drop vapor diffusion method51. The initial crystallization conditions were determined by using screening kits, Crystal Screen, Crystal Screen II, and Index , and Wizard . For the crystallization trial, the protein solution was concentrated to 30.0 mg/ml for wild-type TisAFP7 and 28.0 mg/ml for the T20Y mutant in milli-Q water. Then, 1 μl of the protein solution was mixed with an equal volume of a mother liquor of the crystallization solution and incubated at 20 °C. A thin plate-like crystal was grown under various conditions, as specified in the kits; all reactions contained polyethylene glycol and polyethylene glycol monomethyl ether 5000. The wild-type TisAFP7 crystal used for the data collection was grown in 0.2 M ammonium sulfate, 0.1 M 2-(N-morpholino)ethanesulfonic acid monohydrate (pH 6.5), and 30% (w/v) polyethylene glycol monomethyl ether 5000. For TisAFP7 T20Y, the diffraction data were collected using a crystal grown in 0.2 M MgCl2, 0.1 M Tris–HCl (pH 8.5), and 30% (w/v) polyethylene glycol 4000. For diffraction data collection, the crystals were picked up and flash-cooled in liquid nitrogen. The diffraction data for both proteins were collected at the beamline BL-1A in Photon Factory, KEK, Tsukuba, Japan52, using 1.1000 Å radiation. The diffraction data were processed using XDS53 and CCP454 software suites version 7.0.055 (http://ccp4.ac.uk). The crystal structure of TisAFP7 was determined by the molecular replacement method using PHENIX55 and the coordinates of TisAFP8 (Protein Data Bank 5B5H) as a search model. The initial model was examined and corrected by using Coot56, and further refined by using PHENIX55 and REFMAC557. The crystal structure of T20Y mutant was determined by molecular replacement by using the refined structure of TisAFP7 as a search model and refined by the same procedure as that used for wild-type TisAFP7. To survey the bound water molecules of TisAFPs, which were crystallized in different space groups, the water molecules were rearranged in equivalent positions around the IBS by applying crystallographic symmetry operation of each space group by using Coot56.TisAFP7 and a set of water molecules comprising the basal plane of ice were superposed. Eleven water molecules at the IBS loop were sampled for superposition with the basal plane of ice crystal. The optimum orientation of the ice plane was determined by using program LSQKAB version 7.0.05560 in CCP4 suite, which calculates the presumable minimum distances between bound water molecules and the corresponding ice-plane water molecules. The same calculation was performed for TisAFP8 and TisAFP6. In these analyses, the same numbers of bound water molecules and in positions equivalent to those on TisAFP7 were sampled.To assess the structural similarity of IBS waters and ice waters, the water molecules bound at the IBS loop of TisAFP7, TisAFP8, and TisAFP6, we calculated the residence time for water molecules by molecular dynamics (MD) simulations using the program GROMACS 2020 version 2020.1-Ubuntu-2020.1–1 (http://gromacs.org) 61. The crystal structure of each TisAFP isoform was solvated in a simulation box and neutralized by adding counter ions (Na+ or Cl−). After energy minimization and short position-restrained MD simulation, production MD simulation in 5 ns was performed. Berendsen temperature and pressure coupling were applied, along with the OPLS all-atom force field62 and TIP4P water model63. The trajectory of the MD simulation was applied to the hbond tool in GROMACS to calculate the residence time of water molecules that are forming hydrogen bonds with the protein surface. The surface of TisAFP was divided into a whole molecule, IBS, and non-IBS to compare the water residence time of each region. The water residence time was estimated as a “forward lifetime” of hydrogen bonds, based on the model by references65.To compare the hydration network on the molecular surface of Supplementary Information


The Younger Dryas (YD) is recognized as a cool period that began and ended abruptly during a time of general warming at the end of the last glacial. New multi-proxy data from a sediment gravity core from Storfjordrenna  reveals that the onset of the YD occurred as a single short-lived dramatic environment deterioration, whereas the subsequent warming was oscillatory. The water masses in the western Barents Sea were likely strongly stratified at the onset of the YD, possibly due to runoff of meltwater combined with perennial sea-ice cover, the latter may last up to several decades without any brake-up. Consequently, anoxic conditions prevailed at the bottom of Storfjordrenna, leading to a sharp reduction of benthic biota and the appearance of vivianite microconcretions which formation is favoured by reducing conditions. While the anoxic conditions in Storfjordrenna were transient, the unfavorable conditions for benthic foraminifera lasted for c. 1300 years. We suggest that the Pre-Boreal Oscillation, just after the onset of the Holocene, may have been a continuation of the oscillatory warming trend during the YD. Numerous climate predictions using numerical and theoretical models of ocean circulation suggest that the AMOC will weaken over the coming century due to glacial meltwater runoff and decreases in sea ice cover under global warming2. However, its consequences3 as well as its other hypothetic oceanic2, extraterrestrial4, volcanic5, and atmospheric6 causes are still debated.One of the most striking examples of AMOC weakening from the geological past is the Bølling-Allerød (B-A)–Younger Dryas  transition. The YD constituted a return to near-glacial conditions after the Earth’s climate began to shift from a cold glacial world to a warmer interglacial state at the end of the last glacial. It is generally accepted that the YD cold event resulted from a slowdown in the AMOC8. As earlier investigations focused mainly on overviews of Late Glacial and Holocene paleoceanography of the Nordic Seas, the YD interval in marine records was presented in low temporal resolution . Although environmental variability during the YD is documented in records from the northern Atlantic Ocean11, high-resolution records from the northern part of the Nordic Seas—where the effects of ongoing global climate change are most pronounced12—remain absent.Numerous marine sedimentary records attest that the northern hemisphere was subjected to rapid cooling over circa 1000 years during the YD13. In this paper, we present a new multi-proxy record of the YD from core JM09-020-GC with higher temporal resolution than that presented in Łącka et al.13 consisting of sedimentological (ice-rafted debris counts), mineralogical , micropaleontological (benthic foraminifera counts) and geochemical  analyses and we compare our results to other paleoclimatic records. Our new findings, as well as new data concerning the Arctic Ocean circulation at that time, prompted us to resume the discussion on the YD trigger and its evolution. The aim of this study is to provide a more detailed understanding of the oceanographic variability that occurred in the western Barents Sea during this stadial.An earlier study of sediment gravity core JM09-020-GC from Storfjordrenna  were found in three adjacent 1-cm thick sediment samples (293–295 cm) dated to approximately 12.80–12.85 ka BP  is a mineral formed in various aquatic environments25. The vivianite nodules found at the YD onset in Storfjordrenna are interpreted as authigenic in origin due to the absence of abrasion and dissolution features on mineral surfaces, which could suggest their formation in different environments  and/or long transport. Vivianite microconcretions from Arctic sediments have hitherto been reported exclusively from the Laptev Sea, where their appearance accompanied by rhodochrosite concretions (not present in our study site) has been linked to enhanced water stratification caused by surface water freshening and the development of a thick ice cover for large portions of the year18. Taldenkova et al.18 assumed that water freshening was largely due to glacial meltwater input and proposed that concretion formation reflects meltwater events. A similar interpretation may apply for Storfjordrenna. Increased δ18O just after the onset of the YD may reflect high-saline water near the bottom  studies performed hitherto lack sufficient temporal resolution; (2) whereas some studies reflected open-ocean conditions, Storfjordrenna was a semi-enclosed glacial bay at that time29, permitting bottom-water mass stagnation, strong stratification, and large supplies of Fe-oxides in meltwater—factors that are essential for vivianite nodule formation32. However, the likely anoxic conditions in near-bottom waters in the western Barents Sea at that time are also confirmed by Sternal et al.19 in a study from the continental shelf offshore southwestern Svalbard reporting high authigenic pyrite, organic carbon and sulphur content around the onset of the YD. Our observation of vivianite is the second ever observation of vivianite in the sediments of the Arctic shelf areas. Therefore still more studies are needed to better understand its formation in a generally well-oxygenated environment.The appearance of vivianite in three adjacent sediment samples deposited likely in the earliest YD confirms a dramatic transition from ameliorated conditions during B-A with constant AW inflow AW Fig. . The YD 17 or partly open33, so the only effective freshwater outflow from Siberian rivers and melting ice caps was through the Fram Strait and the Barents Sea. According to empirical and modeling data34, a warming in the northwestern part of the North Atlantic occurred at the beginning of the YD. Warmer sea-surface conditions in Baffin Bay throughout the YD 35. The YD conditions, in contrast to the environment in the modern North Atlantic Ocean, suggest that the lid of the freshwater from Siberian rivers and melting ice sheets possibly contributed to the reduction of the inflow of AW to the eastern part of the North Atlantic Ocean, forcing these water masses to spread along the eastern coast of North America , opportunistic benthic foraminifera, such as .7 ka BP,26 13. The bottom waters were likely much better ventilated than during the early and late YD (higher Mn/Fe). By contrast, the beginning of the Holocene in Baffin Bay was characterized by a marked decrease in SST16  is marked by a pronounced increase in ice rafting, greater foraminifera abundance, and concomitant higher foraminiferal biodiversity likely corresponding to the increasing SST Figs.  and 4. H5–11.3 kaT16 Fig. A. Benthi17 and the further decline in SST in Baffin Bay  from Storfjordrenna  in November 2009 from 253 m water depth13 Fig. .13 supplemented with three additional accelerator mass spectrometer (AMS) 14C measurements . All values are calibrated to the PeeDee Belemnite (PDB) scale and corrected for ice volume changes45.Oxygen stable isotope compositions of tests of the infaunal foraminifer species −2 ka−1) using the sediment accumulation rate.The ice-rafted debris  were counted under a stereo-microscope and expressed as flux values , UiT with an Avaatech X-ray fluorescence (XRF) core scanner using the following settings: 10 kV, 1000 µA, 10-s measuring time, and no filter. The manganese/iron ratio (Mn/Fe) was used as an indicator of reducing conditionsThe morphologies and semiquantitative chemical compositions of concretions were studied with a Hitachi S-3700N Scanning Electron Microscope with Energy Dispersive Spectrometer  at Faculty of Geographical and Geological Sciences in Poznań. The X-ray diffraction (XRD) analysis of powdered samples were run on a ARL X’tra  from Thermo Electron at Institute of Geology, Adam Mickiewicz University, Poznań.Supplementary Figures.


We studied the induction of TaeIF2α phosphorylation in germinated wheat (Triticum aestivum) embryos subjected to different adverse conditions. Data confirmed that formation of TaeIF2(αP) was not a general response, as no phosphorylation was observed under salt, oxidative, or heat stress. Nevertheless, treatment by salicylic acid, UV-light, cold shock and histidinol did induce phosphorylation of TaeIF2α of wheat as has been established previously for AteIF2α in Arabidopsis . The influence of TaeIF2α phosphorylation on translation of reporter mRNA with different 5′-untranslated regions (5′UTRs) was studied in wheat germ cell-free system (WG-CFS), in which TaeIF2α was first phosphorylated either by heterologous recombinant human protein kinase, HsPKR (activated by double-stranded (ds)RNA), or by endogenous protein kinase TaGCN2 (activated by histidinol). Pretreatment of WG-CFS with HsPKR in the presence of dsRNA or with histidinol resulted in intense phosphorylation of TaeIF2α; however, the translation levels of all tested mRNAs decreased by only 10–15% and remained relatively high. In addition, factor OceIF2 from rabbit (Oryctolagus cuniculus) bound GDP much more strongly than the homologous factor TaeIF2 from wheat germ. Furthermore, factor OceIF2B was able to stimulate guanine nucleotide exchange (GDP→GTP) on OceIF2 but had no effect on a similar exchange on TaeIF2. These results suggest that the mechanism of stress response via eIF2α phosphorylation is not identical in all eukaryotes, and further research is required to find and study in detail new plant-specific mechanisms that may inhibit overall protein synthesis in plants under stress.A mechanism based on reversible phosphorylation of the  Plants, being immobile organisms, are compelled to withstand various stresses, such as extreme temperatures, nutritional deficiencies, injuries, drought, and salinization, without being able to avoid or weaken them. For these stresses, plants respond by programmed changes in expression of genes at the levels of transcription, processing and translation of mRNA . Investiα-subunit at Ser51. This is brought about by the action of one or more eIF2α kinases  and results in a rapid and profound suppression of protein synthesis  with GTP and initiator methionyl-tRNA [GTP*eIF2*Met-tRNAiMet], which then binds to the 40S ribosomal subunit  is much higher than for the unphosphorylated meIF2α. In mammalian cells, the amount of meIF2 is 3–5 times higher than meIF2B; therefore, phosphorylation of only 20–30% of meIF2 leads to the capture of all the available meIF2B molecules, thus preventing new TC formation even with unphosphorylated meIF2. Thus, phosphorylation of eIF2α results in rapid inhibition of translation initiation and the shutdown of overall protein synthesis in mammalian and yeast cells  lacking AtGCN2 activity are quite viable, although they demonstrate higher sensitivity to herbicides that disrupt synthesis of amino acids and purines (AteIF2 subunits in Arabidopsis plants between night (low protein synthesis level) and day (high protein synthesis) (α was observed under osmotic or oxidative stresses (α using a specific kinase (PERK) in order to reduce overall translation as part of the unfolded protein response. In contrast, the unfolded protein response in Arabidopsis is not accompanied by eIF2α phosphorylation, and no changes in protein synthesis level were observed  is not linked to general protein synthesis arrest (αP) in plants relative to other eukaryotes, at least with respect to general protein synthesis.Consistent with this, neither the biochemical activity nor genes encoding a peIF2B-like factor have been found in plants . In compn plants , and gcn purines . Convincnthesis) . Likewisstresses , viral istresses , or heatstresses . Moreoveobserved . Furthers arrest . These rα is phosphorylated by pGCN2 in plants subjected to amino acid  embryos subjected to different adverse conditions. In addition, the effect of TaeIF2α phosphorylation on mRNA translation was studied in a wheat germ cell-free system.In this work, we studied the induction of T. aestivum, var. Kazakhstanskaya 4) by the method of Johnston and Stern (2O2 (oxidative stress imitation); 4) 4 h at 26°C with 0.6 mM salicylic acid ; 5) 110 min at 26°C + 10 min under UV light (in UV cross-linker at 2 mJ/cm2) + 2 h at 26°C; 6) 4 h at 4°C (cold shock); 7) 4 h at 37°C (heat shock); 8) 4 h at 26°C in the presence of 2 mM L-histidinol (simulation of amino acid starvation). L-histidinol (HisOH) is a potent inhibitor of protein synthesis in mammalian cells, acting not only as an inhibitor of histidine synthesis but also competitively inhibiting histidyl-tRNAHis synthetase  containing Cantharidin, (−)-p-Bromotetramisole oxalate and Calyculin A (Santa Cruz), 2.5 mM sodium fluoride, 1 mM sodium orthovanadate, 0.5 mM 3-phosphoglycerate, 1.15 mM sodium molybdate. The crude extract was filtered through nylon and centrifuged at 23,000 × g  for 20 min to remove the nuclei, plastids, and mitochondria , 100 mM KCI, 2 mM MgCIchondria . The res2 and 20 V using 0.22 μm pore NitroBind nitrocellulose membranes (GVS Life Sciences).Proteins were analyzed by SDS-PAGE (12.5%) with 0.1% SDS, according to HsPKR with a carboxy-terminal 6His-tag was constructed by polymerase chain reaction (PCR) amplification of HsPKR cDNA gene from pUNO-hPRKR plasmid (InvivoGen) using oligonucleotides hPKR-Nde-FW (5′GGCCCATATGGCTGGTGATCTTTCAGCAGG) and hPKR-Xho-Rev (5′ATTGGATCCTTACTCGAGACATGTGTGTCGTTCATTTTTCT). The PCR product was digested with NdeI and XhoI and ligated into pET23c vector digested with the same enzymes, resulting in ‘Pet23c-HsPKR-6His' plasmid.Plasmid expressing Escherichia coli strain BL21(DE3) transformed with Pet23c-HsPKR-6His plasmid were grown in 100 ml of LB medium containing ampicillin (100 μg/ml) at 30°C to 0.5 optical unit of A600. The expression of recombinant protein was induced by 0.5 mM IPTG at 30°C for 4 h. Cells were collected by centrifugation, suspended in His-buffer  containing 10 mM imidazole and then lysed by addition of lysozyme (1 mg/ml) and sonication. The cell debris was removed by centrifugation at 10,000 × g for 20 min at 4°C, and the supernatant was combined with PerfectPro Ni–NTA resin suspension , shaken at 4°C for 1 h followed by washing of unbound proteins from the column. The resin was washed twice by His-buffer containing 20 mM imidazole at 4°C. The HsPKR-6His bound to the resin was eluted with His-buffer containing 250 mM imidazole and dialyzed against buffer (20 mM Tris(Ac) pH 7.6, 90 mM K(Ac), 2 mM Mg(Ac)2) at 4°C for 12 h to remove the imidazole. After dialysis, the HsPKR-6His was concentrated using 10,000 MWCO HY membrane centrifuge columns (Sartorius) according to the manufacturer's manual. Protein concentration was determined by Bradford protein assay  in agarose gel revealed that the average length was in the 350–400 nucleotides range, permitting the calculation that 1 μg/ml approximately corresponded to 15 nM concentration.Poly(rI–rC)—polyinosinic–polycytidylic acid (Sigma) was used as an activator of HsPKR was measured in 20 μl reaction mixtures that contained 10 mM Tris(HCl) pH 7.6; 1 mM DTT; 50 mM K(Ac); 2 mM Mg(Ac)2; 10 μM ATP; 20 ng of purified HsPKR and poly(rI:rC) to a final concentration from 1 ng/ml to 10 μg/ml. WG-extract was added so that the total protein concentration was 2.5 μg/μl. The mixtures were incubated for 30 min at 26°C, and then the proteins were separated by SDS-PAGE and the gels subjected to blotting and development as described.Activity of TaeIF2α, commercially-available phospho-eIF2α(S51) polyclonal antibodies were used ; these had been raised in a rabbit against human phosphorylated eIF2α. This antibody has been used successfully in previous studies for the detection of the phosphorylated form of AteIF2α in Arabidopis  in TBST buffer  Tween 20) for 1 h at room temperature with gentle shaking. Phospho-eIF2α(S51) polyclonal antibodies were used at 1:1,000 dilution in TBST containing 5% BSA then horseradish peroxidase-coupled donkey-anti-rabbit secondary antibodies (ECL) were used as the secondary antibodies in 1:2,000 dilution in a blocking solution. Finally, blot-membrane was developed by a chemiluminescent substrate for peroxidase (Promega) according to the instruction of manufacturer.In order to detect phosphorylated abidopis . The amiHsPKR-6His, the blots were incubated in blocking buffer (5% skimmed milk powder (Sigma) in TBST) overnight at 4°C. The blocked membrane was then incubated with first antibodies  for 1 h at 25°C. After three successive washings in TBST for 20 min each, the membrane was incubated with anti-mouse HRP-conjugate (“5-Prime”) in a 1:2,000 dilution in blocking buffer for 1 h at 25°C. After washing the membrane twice for 20 min in TBST and washing twice for 10 min in TBS, the blot-membrane was developed by a chemiluminescent substrate for peroxidase (Promega) according to the manufacturer's manual.To detect in vitro translation were obtained by in vitro transcription as described earlier (in vitro of EcoRI-linearized plasmid DNAs was carried out using bacteriophage T7 RNA-polymerase (Thermo Fisher Scientific). After incubation for 2 h at 37°C, RNAs were precipitated in the presence of 3 M LiCl followed by double washing with 70% ethanol.All mRNAs used for  earlier . Transcrin vitro was carried out in WG-CFS as described previously  of WG-extract (S23), and 2 µg of in vitro-transcribed mRNA. The translation efficiency was determined by fluorometric measuring of the activity of reporter protein β-glucuronidase (GUS), which was the translation product, as described (β-D-glucuronide (MUG) as the substrate. The amount of the fluorescent product  in each assay was measured with a TKO 100 fluorimeter (Hoefer).Translation eviously  with sliescribed  with 1 mTaeIF2) was isolated as described previously (OceIF2) and RR-eIF2B (OceIF2B) were isolated from a rabbit reticulocyte lysate (RRL) according to the procedure described by The WG-eIF2   (NEN), and ~200 µg/ml of the respective eIF2 and were kept at 30°C for 10 min. GTP was added in final concentrations from 1 to 400 μM, and reactions were incubated for 10 more min, after which samples were diluted by 3 ml of ice-cold buffer C  and were filtered through wet nitrocellulose discs. Filters were washed well with the same buffer, dried, and counted in liquid scintillation counter LS-10012 (Beckman Coulter).The strength of GDP interaction with WG-eIF2 or RR-eIF2 was determined by measuring the loss of [ 400 μM) . Incubat3H]GDP]; 20 mM Tris/HCl, pH 7.6; 100 mM KCl; 1 mM MgC12; 1 mM DTT; 10 μM GTP and RR-eIF2B at amounts from 10 to 200 ng/20 μl assay. After 5 min at 30°C, samples were diluted with 3 ml of ice-cold buffer C and filtered through nitrocellulose. Filters were washed, dried, and counted as above.Assay of RR-eIF2B activity was carried out according to the procedure of AtGCN2 phosphorylates AteIF2α in Arabidopsis plants in response to herbicides that impair biosynthesis of amino acids and purines , oxidative stress (1 mM H2O2), or heat shock , while the same stresses do induce phosphorylation of SceIF2α in yeast cells (Saccharomyces cerevisiae), which, like plants, have only one protein kinase for this activity—ScGCN2 , which is a potent inhibitor of protein synthesis in mammalian cells. HisOH acts not only as an inhibitor of histidine synthesis but also as a competitive inhibitor of histidyl-tRNAHis synthetase (HARS), thereby increasing the level of deacylated tRNAHis  may be involved in the global protein synthesis inhibition observed under these conditions  and purines (8-azaadenine) (AtGCN2 (acifluorfen and diuron) and phosphorylation of AteIF2α (diuron)  . It is n(diuron) . In the  AteIF2α .AtGCN2 non-essential for viability of plants and indicate that phosphorylation of peIF2α cannot be considered as a universal mechanism for the inhibition of initiation of most mRNA translation in plants that survive multiple stresses. This notion is consistent with many observations that peIF2α phosphorylation does not correlate with changes in protein synthesis level in vivo. For instance, in a study of Arabidopsis plants grown either in the dark (when protein synthesis level is low) or under daylight (high protein synthesis), no phosphorylation of AteIF2α was observed at all  is much less important for plants compared with other eukaryotes   , at leasα. Thus, for example, overall protein synthesis has been shown to be strongly inhibited (to 20% level) after treatment of Arabidopsis plants with 8-azaadenine (inhibitor of purine synthesis), a treatment that also induced pronounced AteIF2α phosphorylation  while the amount of smaller polysomes stayed unaffected or even slightly increased  reduction of protein synthesis , to rabbit reticulocytes (RR)-lysate results in the phosphorylation of RReIF2 effects . It has ynthesis . Such anynthesis .α phosphorylation on initiation of mRNA translation, we used a wheat germ cell-free system (WG-CFS) in which TaeIF2α was first phosphorylated in two different ways: (i) by exogenous recombinant human PKR (HsPKR) that was activated by double stranded (ds)RNA–poly(rI–rC); (ii) by endogenous TaGCN2 activated by L-histidinol (HisOH).In order to study the influence of peIF2HsPKR that was partially purified from HeLa cells and activated by poly(rI–rC) has been utilized as a heterologous tool to demonstrate specific phosphorylation of TaeIF2α and concomitant inhibition of Brome mosaic virus (BMV) RNA translation in WG-CFS , which was tagged by C-terminal 6His, contained a substantial amount of a degradation product (~59 kDa) that was truncated from the N-terminus (hence still recognized by Penta-His-antibodies). To verify its functional activity, a constant amount of recombinant HsPKR-6His was supplemented with poly(rI–rC) in different concentrations and tested for its ability to phosphorylate TaeIF2 in wheat germ cell-free extract. After separation of the reaction mixtures in SDS-PAGE and subsequent blotting, the nitrocellulose membrane was developed with commercial antibodies that recognize the phosphorylated form of HseIF2α  or less) of poly(rI–rC) were sufficient (HsPKR-6His kinase was achieved at a concentration of 0.1 μg/ml (1.5 nM) poly(rI–rC). Interestingly, the HsPKR-6His could phosphorylate a small amount of TaeIF2α even in the absence of exogenously-added poly(rI–rC)   poly(rI–rC) without addition of HsPKR-6His (Cowpea mosaic virus (CMV) dsRNA to inhibit translation of different mRNAs in WG-CFS (Turnip yellow mosaic virus (TYMV) or Turnip crinkle virus (TCV) that infected Arabidopsis were unable to induce phosphorylation of AteIF2α, implying the absence of plant kinase equivalent to PKR  concentrations that would be minimal though sufficient to phosphorylate the maximum amount of TaeIF2α molecules in WG-CFS reaction mixtures (HsPKR-6His and 0.15 nM of poly(rI–rC)  and thereby increase the level of deacylated tRNAHis . Uncapped in vitro-transcribed reporter mRNAs coding for β-glucuronidase (GUS) were used, with the following 5′UTRs: 5′UTR of Tobacco mosaic virus (TMV) genomic (g) RNA (5′Ω); 5′UTR of Potato virus Y gRNA (5′PVY), and artificial translation enhancer 3хARC1 (r 3хARC1 . The 5′Ur 3хARC1 . The 5′Ur 3хARC1 .HsPKR-6His (plus poly(rI-rC)) or with histidinol and incubated for 30 min at 26°C, while the reference reactions stayed without such additions. After this incubation, equal aliquots of reactions were analyzed by SDS-PAGE and subsequent western blotting to check the phosphorylation state of TaeIF2α (TaeIF2α occurred in reaction mixtures containing active HsPKR or 2 mM histidinol (HisOH), whereas no phosphorylation occurred without these additions (Ctrl). To study the effect of TaeIF2α phosphorylation on protein synthesis level, equal amounts of reporter mRNAs were added to the pre-incubated WG-CFS reactions and incubation was continued further for 60 min. After completion of translation, equal aliquots of the reactions were tested for the TaeIF2 phosphorylation state, which was found to be the same as that demonstrated in TaeIF2α are presented in HsPKR or histidinol. In either case, the levels of reporter protein synthesis remained rather high (85–90%), confirming our previously made supposition that protein synthesis in plants may proceed efficiently irrespective of peIF2α phosphorylation  of poly(rI–rC), completely inhibited translation of BMV RNA in WG-CFS. However, our results agree with the outcomes of in vitro experiments done in WG-CFS supplemented by purified rabbit reticulocyte heme-regulated inhibitor (RR-HRI)  . While Wn WG-CFS .HsPKR-6His, but also with the endogenous TaGCN2 activated by histidinol. Also, our results agree with recent findings that, while Arabidopsis AtGCN2, together with AtGCN1 and AtGCN20, does mediate phosphorylation of AteIF2α, formation of AteIF2(αP) is not accompanied by inhibition of general protein synthesis  is present in Arabidopsis, but the gene of transcription factor GCN4 has not been found in plants  mechanism . The GCNno acids . The GCNn plants . This cin plants .Introduction), together with our results, suggest that phosphorylation of peIF2α in plants cannot be considered as a universal mechanism for inhibition of general protein synthesis. Then, why is pGCN2 conserved in plants and why does it phosphorylate peIF2α in response to a lack of amino acids, purines, and some other stresses? One possible explanation may be that pGCN2 serves as a sensor of deficiency of amino acids and nucleotides, though it can be also activated in response to some adverse factors. Phosphorylation of peIF2α does not prevent translation of most mRNAs, while it may serve as a signal for other mechanisms, which do inhibit general protein synthesis. If so, then the finding and investigation of such mechanisms become an important task.Thus, data available in the literature , and then the partially purified RR-eIF2B was added in increasing amounts. As expected, RR-eIF2B greatly increased the replacement of [3H]GDP by GTP at 10 μM on factor RR-eIF2; the exchange that was impossible without the addition of RR-eIF2B: indeed, 0.2 μg stimulated almost complete exchange  and RR-eIF2B. The authors of that study concluded that the ability of WG-eIF2 to exchange guanine nucleotides independently of any eIF2B-like protein and the inability of phosphorylated WG-eIF2 to interact with RR-eIF2B indicated that WG-eIF2 differs from RR-eIF2 by amino acid sequence and mechanisms regulating its activity  is not a universal stress response in wheat, in particular under salt, oxidative, and heat stress. Nevertheless, we confirm also that treatment by salicylic acid (stress and defense hormone), histidinol (simulation of amino acid starvation), cold shock, and UV light does induce phosphorylation of TaeIF2α in wheat in a similar way as AteIF2α of Arabidopsis  inhibits translation by phosphorylating AteIF4E and AteIFiso4E at two conserved SnRK1 consensus target sites, which are present in all tested plant factors but absent from homologous mammalian factors (AteIF4E nor AteIFiso4E and AteIFiso4G undergo any phosphorylation when plants are subjected to light (high level of protein synthesis) or dark (low level of protein synthesis) (Recently it was shown that the energy-sensing protein kinase of Arabidopsis ( factors . The autnthesis) .2+/calmodulin-dependent meEF2-kinase in response to starvation, hypoxia, and oxidative stress  of translation elongation factor 2 (meEF2), catalyzed by specific Cae stress . Phosphoin vitro or in vivo. Furthermore, peEF2-kinase activity was not found in different organs of wheat or other higher plants (both monocots and dicots)  were not phosphorylated during seed development, germination, or heat shock (It was shown earlier that WG-eEF2 can be readily phosphorylated by the RR-eEF2-kinase and this resulted in the inhibition of WG-eEF2 activity in poly(U)-dependent cell-free translation system . Thus, t dicots) . Other g2-kinase . Moreoveat shock .In conclusion, the results obtained in this work together with previous literature data show that plants do not conform to the mammalian and yeast paradigm concerning the regulation of global protein synthesis by reversible phosphorylation of translation initiation and elongation factors. These differences may originate from the autotrophic mode of plant nutrition. Therefore, it is important to look for alternative plant-specific mechanisms that do inhibit protein synthesis in plants under stress conditions . Such alThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.in vitro translation reactions and their analyses. AA performed the plant growth, treatments, sample preparation and analyses. AN performed in vitro transcription and preparation of mRNA samples. GS and NP prepared rabbit reticulocytes, RReIF2, RReIF2B. OK and AZ prepared TaeIF2 from wheat germ. RK and AZ performed guanine nucleotide exchange reactions and statistical analyses of the data. AZ and BI are the leaders of projects АР05130800 and AP05132532, respectively; BI and NH prepared the manuscript. All authors contributed to the article and approved the submitted version.AZ prepared recombinant DNAs, HsPKR, WG-CFS, and performed via the Designing Future Wheat Programme (BB/P016855/1). BBSRC is part of UK Research and Innovation.The work was carried out in the framework of the following scientific grants provided by SC MES RK: AP05132532 to BI; АР05130800 to AZ; AP05131133 to OK; AP05132066 to GS. NH is supported at Rothamsted Research by the Biotechnology and Biological Sciences Research Council (BBSRC) The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.