The underestimated fraction: diversity, challenges and novel insights into unicellular cyanobionts of lichens.

Lichens are remarkable and classic examples of symbiotic organisms that have fascinated scientists for centuries. Yet, it has only been for a couple of decades that significant advances have focused on the diversity of their green algal and/or cyanobacterial photobionts. Cyanolichens, which contain cyanobacteria as their photosynthetic partner, include up to 10% of all known lichens and, as such, studies on their cyanobionts are much rarer compared to their green algal counterparts. For the unicellular cyanobionts, i.e. cyanobacteria that do not form filaments, these studies are even scarcer. Nonetheless, these currently include at least 10 different genera in the cosmopolitan lichen order Lichinales. An international consortium (International Network of CyanoBionts; INCb) will tackle this lack of knowledge. In this article, we discuss the status of current unicellular cyanobiont research, compare the taxonomic resolution of photobionts from cyanolichens with those of green algal lichens (chlorolichens), and give a roadmap of research on how to recondition the underestimated fraction of symbiotic unicellular cyanobacteria in lichens.

Population genomics resolves cryptic species of the ecologically flexible genus Laspinema (cyanobacteria).

Cyanobacterial taxonomy is entering the genomic era, but only a few taxonomic studies have employed population genomics, which provides a framework and a multitude of tools to understand species boundaries. Phylogenomic and population genomic analyses previously suggested that several cryptic lineages emerged within the genus Laspinema. Here, we apply population genomics to define boundaries between these lineages and propose two new cryptic species, Laspinema olomoucense and L. palackyanum. Moreover, we sampled soil and puddles across Central Europe and sequenced the 16S rRNA gene and 16S-23S ITS region of the isolated Laspinema strains. Together with database mining of 16S rRNA gene sequences, we determined that the genus Laspinema has a cosmopolitan distribution and inhabits a wide variety of habitats, including freshwater, saline water, mangroves, soil crusts, soils, puddles, and the human body.

Fish stocks as phosphorus sources or sinks: Influenced by nutritional and metabolic variations, not solely by dietary content and stoichiometry.

The study provides a descriptive understanding of when fish (Cyprinus carpio model) are the source or sink of phosphorus. Dissolved reactive phosphorus (DRP; PO4-P) losses (51.1 ± 5.9 % of intake-P) increase at excess of bioavailable P (>0.83 g 100 g-1 dry matter, DM fed) or when food (digestible) N:P mass ratio (≤4.4:1) approaches organismal storage threshold (~4:1). This is known, however, even at a sub-threshold food P content (0.57 g 100 g-1 DM) and food N:P mass ratio (7.3:1), DRP losses (57.8 ± 4.5 % of intake-P) may be extraordinary if two indispensable amino acids are biologically insufficient (lysine ≤1.43 g, methionine ≤0.39 g 100 g-1 DM fed). Given that methionine and lysine are sufficient, DRP losses cease (≈0 %) and even some P from water is absorbed, given there is support from non-protein energy (NPE). Insufficient NPE (<180 kcal 100 g-1 DM fed) may drive DRP losses (81.6 ± 4.3 % of intake-P) beyond expected levels (46-59 % of intake-P) at a given food P content (0.91 g 100 g-1 DM). Natural food seldom fulfills low P, high lysine + methionine, and high NPE contents simultaneously, thus keeping fish in a perpetual P recycling for algae (scaleless carp > scaly carp). Such P recycling ceases only during basal metabolism. During feeding state, the richness of lysine + methionine bound N and lipid + carbohydrate bound C in the food base may enhance the fishes' threshold of P storage. P storage can be diminished when they are insufficient. We show that for fish, the decision of P recycling or not recycling (for algae) may change based on the supply of specific fractions of N or C from the food web or metabolic variations (basal metabolism, presence of scales). NOVELTY STATEMENT: The ecological stoichiometry theory is better connected to fish nutritional bioenergetics for better understanding and biomanipulation of eutrophication processes.

Population genomics and morphological data bridge the centuries of cyanobacterial taxonomy along the continuum of Microcoleus species.

The filamentous cyanobacterium Microcoleus is among the most important global primary producers, especially in hot and cold desert ecosystems. This taxon represents a continuum consisting of a minimum of 12 distinct species with varying levels of gene flow and divergence. The notion of a species continuum is poorly understood in most lineages but is especially challenging in cyanobacteria. Here we show that genomic diversification of the Microcoleus continuum is reflected by morphological adaptation. We compiled a dataset of morphological data from 180 cultured strains and 300 whole genome sequences, including eight herbarium specimens and the type specimen of Microcoleus. We employed a combination of phylogenomic, population genomic, and population-level morphological data analyses to delimit species boundaries. Finally, we suggest that the shape of the filament apices may have an adaptive function to environmental conditions in the soil.

The global speciation continuum of the cyanobacterium Microcoleus.

Speciation is a continuous process driven by genetic, geographic, and ecological barriers to gene flow. It is widely investigated in multicellular eukaryotes, yet we are only beginning to comprehend the relative importance of mechanisms driving the emergence of barriers to gene flow in microbial populations. Here, we explored the diversification of the nearly ubiquitous soil cyanobacterium Microcoleus. Our dataset consisted of 291 genomes, of which 202 strains and eight herbarium specimens were sequenced for this study. We found that Microcoleus represents a global speciation continuum of at least 12 lineages, which radiated during Eocene/Oligocene aridification and exhibit varying degrees of divergence and gene flow. The lineage divergence has been driven by selection, geographical distance, and the environment. Evidence of genetic divergence and selection was widespread across the genome, but we identified regions of exceptional differentiation containing candidate genes associated with stress response and biosynthesis of secondary metabolites.

Synergistic digestibility effect by planktonic natural food and habitat renders high digestion efficiency in agastric aquatic consumers.

A digestibility enhancing effect of natural food on stomachless fish model (Cyprinus carpio) was verified by fluorogenic substrate assays of enzymatic activities in experimental pond carp gut flush and planktonic food over a full vegetative season. Then compared with size-matched conspecific grown artificially (tank carp) and an advanced omnivore species possessing true stomach (tilapia, Oreochromis niloticus). Results suggested activities of digestive enzymes (except amylolytic) were significantly higher in pond carp (p ≤ 0.05) than in the size-matched tank carp. Even compared to tilapia, pond carp appeared superior (p < 0.05; proteolytic or chitinolytic activities) or comparable (p > 0.05; phosphatase or cellulolytic activities). Amylolytic, chitinolytic, and phosphatases activities in pond carp gut significantly increased (p ≤ 0.01) over season. Several orders-of-magnitude higher enzymatic activities were detected in planktonic natural food than expressed in carp gut. Amino acid markers in planktonic food revealed a higher share of zooplankton (microcrustaceans), but not phytoplankton, synchronized with higher activities of complex polysaccharide-splitting enzymes (cellulolytic and chitinolytic) in fish gut. Periods of clear water phase low in chlorophyll-a and nutrients, but high in certain zooplankton (preferably cladocerans), may create a synergistic digestibility effect in pond carp. We conclude aquatic ecosystem components (natural food, water, microbiota) enhance fishes' hydrolyzing capabilities of C/N/P macromolecules and even their complex polymers such as cellulose, chitin, and maybe phytate (to be validated), to the extent that being stomachless is not an issue. Aquatic nutritional ecologists may consider that laboratory-based understandings of digestibility may underestimate digestion efficiency of free-ranging fish in ponds or lakes.

Runoff of foliar-applied natural fungicides in aquaponics: Implications for fish and nitrification.

Aquaponics is a method of producing food in a sustainable manner through the integration of aquaculture and hydroponics, which allows simultaneous cultivation of fish and economic crops. The use of natural fungicides are crucial to the sustainable control of diseases in aquaponics. We assessed the potential impacts of natural fungicides, such as clove oil and lecithin, as well as a synthetic fungicide, tebuconazole, following foliar application in aquaponics. This study examined the runoff rates of the fungicides in decoupled aquaponics, and the subsequent effects of the runoffs on nitrification processes and Nile tilapia (Oreochromis niloticus). The runoffs of the foliar-applied fungicides, clove oil, lecithin, and tebuconazole, were detected in aquaponics water at a percentage runoff rate of 0.3 %, 2.3 %, and 0.3-0.8 % respectively. In the biofilter, lecithin altered the ammonium levels by increasing ammonium-nitrogen levels by 7 mg L-1, 6 h post application. Clove oil, on the other hand, showed no significant effect on ammonium, nitrite, and nitrate-nitrogen. Similarly, the toxicity test showed that eugenol had no significant effects on the hematological, biochemical and antioxidative activities of O. niloticus. Conversely, tebuconazole exhibited significant and persistent effects on various biochemical parameters, including lactate, albumin, and total protein, as well as hematological parameters like hemoglobin and MCH. The use of lecithin and tebuconazole should only be limited to decoupled aquaponics.

Methyl viologen-induced changes in the Arabidopsis proteome implicate PATELLIN 4 in oxidative stress responses.

The photosynthesis-induced accumulation of reactive oxygen species in chloroplasts can lead to oxidative stress, triggering changes in protein synthesis, degradation, and the assembly/disassembly of protein complexes. Using shot-gun proteomics, we identified methyl viologen-induced changes in protein abundance in wild-type Arabidopsis and oxidative stress-hypersensitive fsd1-1 and fsd1-2 knockout mutants, which are deficient in IRON SUPEROXIDE DISMUTASE 1 (FSD1). The levels of proteins that are localized in chloroplasts and the cytoplasm were modified in all lines treated with methyl viologen. Compared with the wild-type, fsd1 mutants showed significant changes in metabolic protein and chloroplast chaperone levels, together with increased ratio of cytoplasmic, peroxisomal, and mitochondrial proteins. Different responses in proteins involved in the disassembly of photosystem II-light harvesting chlorophyll a/b binding proteins were observed. Moreover, the abundance of PATELLIN 4, a phospholipid-binding protein enriched in stomatal lineage, was decreased in response to methyl viologen. Reverse genetic studies using patl4 knockout mutants and a PATELLIN 4 complemented line indicate that PATELLIN 4 affects plant responses to oxidative stress by effects on stomatal closure.

Rapid diversification of a free-living protist is driven by adaptation to climate and habitat.

Microbial eukaryotes (protists) have major functional roles in aquatic ecosystems, including the biogeochemical cycling of elements as well as occupying various roles in the food web. Despite their importance for ecosystem function, the factors that drive diversification in protists are not known. Here, we aimed to identify the factors that drive differentiation and, subsequently, speciation in a free-living protist, Synura petersenii (Chrysophyceae). We sampled five different geographic areas and utilized population genomics and quantitative trait analyses. Habitat and climate were the major drivers of diversification on the local geographical scale, while geography played a role over longer distances. In addition to conductivity and temperature, precipitation was one of the most important environmental drivers of differentiation. Our results imply that flushing episodes (floods) drive microalgal adaptation to different niches, highlighting the potential for rapid diversification in protists.

Spatiotemporal distribution of reactive oxygen species production, delivery, and use in Arabidopsis root hairs.

Fluorescent selective probes for reactive oxygen species (ROS) detection in living cells are versatile tools for the documentation of ROS production in plant developmental or stress reactions. We employed high-resolution live-cell imaging and semiquantitative analysis of Arabidopsis (Arabidopsis thaliana) stained with CM-H2DCFDA, CellROX Deep Red, and Amplex Red for functional characterization of the spatiotemporal mode of ROS production, delivery, and utilization during root hair formation. Cell viability marker fluorescein diacetate served as a positive control for dye loading and undisturbed root hair tip growth after staining. Using a colocalization analysis with subcellular molecular markers and two root hair mutants with similar phenotypes of nonelongating root hairs, but with contrasting reasons for this impairment, we found that: (i) CM-H2DCFDA is a sensitive probe for ROS generation in the cytoplasm, (ii) CellROX Deep Red labels ROS in mitochondria, (iii) Amplex Red labels apoplastic ROS and mitochondria and shows high selectivity to root hairs, (iv) the root hair defective 2-1 (rhd2-1) mutant with nonfunctional NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN C/ROOT HAIR-DEFECTIVE 2 (AtRBOHC/RHD2) has a low level of CM-H2DCFDA-reactive ROS in cytoplasm and lacks Amplex Red-reactive ROS in apoplast, and (v) the ACTIN2-deficient deformed root hairs1-3 (der1-3) mutant is not altered in these aspects. The sensitivity of CellROX Deep Red was documented by discrimination between larger ROS-containing mitochondria and small, yet ROS-free premature mitochondria in the growing tip of root hairs. We characterized spatial changes in ROS production and compartmentalization induced by external ROS modulators, ethylene precursor 1-aminocyclopropane-1-carboxylic acid, and ionophore valinomycin. This dynamic and high-resolution study of ROS production and utilization opens opportunities for precise speciation of particular ROS involved in root hair formation.

Nutrient footprint versus EPA + DHA security in land-locked regions-more of local pond farmed, imported marine fish or fish oil capsules?

EPA + DHA intake in land-locked central Europe (CE) is barely fulfilled. Imported marine fish/farmed salmonids are likely the backbone of an ailing EPA + DHA security. Supplementing with captured marine fish oil capsules (~0.5 g up to 1.6 g CO2-eq. mg EPA + DHA-1) could be comparable in GHG emissions with fish consumption itself (~1 g to as low as 0.6 g CO2-eq. mg EPA + DHA-1). But synergistic benefits of EPA + DHA intake by consuming fish protein need consideration too. Taking semi-intensive pond carp and intensively farmed salmon as models, we analyzed footprint, eco-services, and resource use efficiency perspectives of achieving EPA + DHA security in a CE region. Despite a lower production footprint, pond-farmed fish greatly lag in EPA + DHA supply (carp 101-181 mg 100 g-1 < salmon 750-1300 mg 100 g-1). It doubles-to-quadruples footprint 'per mg' of EPA + DHA: nitrogen (carp 18.3 > salmon 8.7 mg N), phosphorus (carp 6.8 > salmon 1.6 mg P), and climate change (carp 1.84 > salmon 0.8 g CO2-eq.). With enhancements in pond carp (>300 mg EPA + DHA 100 g-1), these differences may cease to exist. Harnessing EPA + DHA bioaccumulation pathways active in ponds, finishing feeding strategies, and polyculture, the EPA + DHA content in pond fish may be increased. Ecosystem services with EPA + DHA mining from pond food web or high EPA + DHA output-to-input ratio (pond carp 1-200 > RAS salmon 0.75) make ponds an eco-efficient system. As fish consumption in CE must improve, pond-farmed fish would be needed to complement (but not substitute) salmonid/marine fish/oil capsules consumption. Achieving EPA + DHA security with minimum pressure on the environment or global resources.

Correlative Imaging of Individual CsPbBr3 Nanocrystals: Role of Isolated Grains in Photoluminescence of Perovskite Polycrystalline Thin Films.

We report on the optical properties of a CsPbBr3 polycrystalline thin film on a single grain level. A sample composed of isolated nanocrystals (NCs) mimicking the properties of the polycrystalline thin film grains that can be individually probed by photoluminescence spectroscopy was prepared. These NCs were analyzed using correlative microscopy allowing the examination of structural, chemical, and optical properties from identical sites. Our results show that the stoichiometry of the CsPbBr3 NCs is uniform and independent of the NCs' morphology. The photoluminescence (PL) peak emission wavelength is slightly dependent on the dimensions of NCs, with a blue shift up to 9 nm for the smallest analyzed NCs. The magnitude of the blueshift is smaller than the emission line width, thus detectable only by high-resolution PL mapping. By comparing the emission energies obtained from the experiment and a rigorous effective mass model, we can fully attribute the observed variations to the size-dependent quantum confinement effect.

Percutaneous CT-guided Biopsy of Focal Liver Lesions - Long- term Experience with more than 300 Procedures.

BACKGROUND AND AIMS: Despite the undeniable ongoing development of cross-sectional imaging methods, not all focal liver lesions (FLLs) have a typical pattern. An image-guided biopsy using a percutaneous approach might safely provide a final histological diagnosis of the FLLs. We aimed to evaluate the accuracy, efficiency, complication rate, technical features, and relationships between the followed parameters of computed tomography (CT)-guided percutaneous biopsies of FLLs using a retrospective approach. METHODS: 303 percutaneous biopsy procedures in 295 patients were carried out in patients with suspected or indeterminate FLLs over a 10-year period. The median size of the tumors was 44 mm (15 - 144 mm). Median age of patients was 67 years (25 to 87 years). Skin-to-lesion distance was variable, from 30 mm to 138 mm (median length 59 mm). In 200 procedures (66%) malignant disease was known from the patients´ clinical history. RESULTS: In 288 biopsies (95%) the results were true positive or true negative; 15 procedures (4.95%) resulted in a histologically false negative and had to be confirmed using other approaches. Metastatic disease to hepatic parenchyma of various origins was the most frequent histological diagnosis (55.4%). Cholangiocarcinoma was the most common individual result (13.5%). In total 14 complications (4.6%) were confirmed, 4 of which were severe haemorrhages that needed angiographic treatment and in one case surgical revision. The mortality rate in our group was 0.3%. A statistically significant relationship between lesion size and diagnostic accuracy (p < 0.01) was revealed. The use of a 16 G needle calibre and at least two samples were suitable for hypo- and hypervascular lesions without a significant increase in the complication rate. CONCLUSIONS: Core needle biopsy using a percutaneous approach and a CT-guidance performed on patients with indetermined FLLs had a high overall accuracy in determining the final histological diagnosis including subtyping. Concurrently, the complication incidence was low.

Possibilities of Interpreting the Night-to-Day Ratio Specified by 24-Hour Blood Pressure Monitoring.

Aim: Specify the risk rate of incorrect patient classification based on the night-to-day ratio specification from singular 24-h ABPM in comparison to the results of 7-day ABPM monitoring. Materials and Methods: 1197 24 h cycles were enrolled in 171 subjects in the study and divided into 4 groups: group 1 (40 healthy men and women without exercise), group 2 (40 healthy exercise-training men and women), group 3 (40 patients with ischemic coronary artery disease without exercise), and group 4 (51 patients with ischemic coronary artery disease following cardiovascular rehabilitation). The subject of the evaluation was the percentage rate of incorrect subject classification (dipper, nondipper, extreme dipper, and riser) based on the mean blood pressure values for 7 days and from seven independent 24-hour cycles (the mean value mode). Results: In the case of the individuals included in the monitored groups, the mean night-to-day ratio-based (mode for the 7 days versus the individual days of 24-hour monitoring) classification accordance ranged between 59% and 62%. Only in singular cases did the accordance reach 0% or 100%. The accordance size was not dependent on the health or cardiovascular disease (p < 0.594; 56% vs. 54%) or physical activity (p < 0.833; 55% vs. 54%) of the monitored individuals. Conclusion: The specification of the night-to-day ratio of each individual for each day of the 7-day ABPM monitoring would be the most convenient option. In many patients, diagnosing could thus be based on the most frequently occurring values (mode specification).

High genomic differentiation and limited gene flow indicate recent cryptic speciation within the genus Laspinema (cyanobacteria).

The sympatric occurrence of closely related lineages displaying conserved morphological and ecological traits is often characteristic of free-living microbes. Gene flow, recombination, selection, and mutations govern the genetic variability between these cryptic lineages and drive their differentiation. However, sequencing conservative molecular markers (e.g., 16S rRNA) coupled with insufficient population-level sampling hindered the study of intra-species genetic diversity and speciation in cyanobacteria. We used phylogenomics and a population genomic approach to investigate the extent of local genomic diversity and the mechanisms underlying sympatric speciation of Laspinema thermale. We found two cryptic lineages of Laspinema. The lineages were highly genetically diverse, with recombination occurring more frequently within than between them. That suggests the existence of a barrier to gene flow, which further maintains divergence. Genomic regions of high population differentiation harbored genes associated with possible adaptations to high/low light conditions and stress stimuli, although with a weak diversifying selection. Overall, the diversification of Laspinema species might have been affected by both genomic and ecological processes.

New cyanobacterial genus Argonema is hidding in soil crusts around the world.

Cyanobacteria are crucial primary producers in soil and soil crusts. However, their biodiversity in these habitats remains poorly understood, especially in the tropical and polar regions. We employed whole genome sequencing, morphology, and ecology to describe a novel cyanobacterial genus Argonema isolated from Antarctica. Extreme environments are renowned for their relatively high number of endemic species, but whether cyanobacteria are endemic or not is open to much current debate. To determine if a cyanobacterial lineage is endemic is a time consuming, elaborate, and expensive global sampling effort. Thus, we propose an approach that will help to overcome the limits of the sampling effort and better understand the global distribution of cyanobacterial clades. We employed a Sequencing Read Archive, which provides a rich source of data from thousands of environmental samples. We developed a framework for a characterization of the global distribution of any microbial species using Sequencing Read Archive. Using this approach, we found that Argonema is actually cosmopolitan in arid regions. It provides further evidence that endemic microbial taxa are likely to be much rarer than expected.

Arabidopsis Iron Superoxide Dismutase FSD1 Protects Against Methyl Viologen-Induced Oxidative Stress in a Copper-Dependent Manner.

Iron superoxide dismutase 1 (FSD1) was recently characterized as a plastidial, cytoplasmic, and nuclear enzyme with osmoprotective and antioxidant functions. However, the current knowledge on its role in oxidative stress tolerance is ambiguous. Here, we characterized the role of FSD1 in response to methyl viologen (MV)-induced oxidative stress in Arabidopsis thaliana. In accordance with the known regulation of FSD1 expression, abundance, and activity, the findings demonstrated that the antioxidant function of FSD1 depends on the availability of Cu2+ in growth media. Arabidopsis fsd1 mutants showed lower capacity to decompose superoxide at low Cu2+ concentrations in the medium. Prolonged exposure to MV led to reduced ascorbate levels and higher protein carbonylation in fsd1 mutants and transgenic plants lacking a plastid FSD1 pool as compared to the wild type. MV induced a rapid increase in FSD1 activity, followed by a decrease after 4 h long exposure. Genetic disruption of FSD1 negatively affected the hydrogen peroxide-decomposing ascorbate peroxidase in fsd1 mutants. Chloroplastic localization of FSD1 is crucial to maintain redox homeostasis. Proteomic analysis showed that the sensitivity of fsd1 mutants to MV coincided with decreased abundances of ferredoxin and photosystem II light-harvesting complex proteins. These mutants have higher levels of chloroplastic proteases indicating an altered protein turnover in chloroplasts. Moreover, FSD1 disruption affects the abundance of proteins involved in the defense response. Collectively, the study provides evidence for the conditional antioxidative function of FSD1 and its possible role in signaling.

On the relativity of species, or the probabilistic solution to the species problem.

For centuries, both scientists and philosophers have discussed the nature of species resulting in c. 35 species concepts proposed to date. However, in our opinion, none of them incorporated neither recent advances in evolutionary genomics nor dimensionality of species in befitting depth. Our attempt to do so resulted in the following conclusions. Due to the continuous nature of evolution (regardless of its rate and constancy), species are inevitably undefinable as natural discontinuous units (except those originating in saltatory speciation) whenever the time dimension is taken into consideration. Therefore, the very existence of species as a natural discontinuous entity is relative to its dimensionality. A direct consequence of the relativity of species is the duality of speciators (e.g., incipient species) meaning that, in a given time, they may be perceived as both being and not being a species. Finally, the most accurate way to reflect both the relativity of species and the duality of speciators in species delimitation is probabilistic. While the novelty of these ideas may be questionable, they still deserve more extensive attention from the biological community. Here, we hope to draw such attention by outlining one of the possible pathways towards a new kind of probabilistic species delimitation methods based on the probability of irreversible divergence of evolutionary lineages. We anticipate that our probabilistic view of speciation has the potential to facilitate some of the most serious and universal issues of current taxonomy and to ensure unity of the species-level taxonomy across the tree of life.

Zebularine induces enzymatic DNA-protein crosslinks in 45S rDNA heterochromatin of Arabidopsis nuclei.

Loss of genome stability leads to reduced fitness, fertility and a high mutation rate. Therefore, the genome is guarded by the pathways monitoring its integrity and neutralizing DNA lesions. To analyze the mechanism of DNA damage induction by cytidine analog zebularine, we performed a forward-directed suppressor genetic screen in the background of Arabidopsis thaliana zebularine-hypersensitive structural maintenance of chromosomes 6b (smc6b) mutant. We show that smc6b hypersensitivity was suppressed by the mutations in EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 3 (ENT3), DNA METHYLTRANSFERASE 1 (MET1) and DECREASE IN DNA METHYLATION 1 (DDM1). Superior resistance of ent3 plants to zebularine indicated that ENT3 is likely necessary for the import of the drug to the cells. Identification of MET1 and DDM1 suggested that zebularine induces DNA damage by interference with the maintenance of CG DNA methylation. The same holds for structurally similar compounds 5-azacytidine and 2-deoxy-5-azacytidine. Based on our genetic and biochemical data, we propose that zebularine induces enzymatic DNA-protein crosslinks (DPCs) of MET1 and zebularine-containing DNA in Arabidopsis, which was confirmed by native chromatin immunoprecipitation experiments. Moreover, zebularine-induced DPCs accumulate preferentially in 45S rDNA chromocenters in a DDM1-dependent manner. These findings open a new avenue for studying genome stability and DPC repair in plants.