How dry is dry? Molecular mobility in relation to thallus water content in a lichen.

Lichens can withstand extreme desiccation to water contents of ≤ 0.1 g H2O g-1 DW, and in the desiccated state are among the most extremotolerant organisms known. Desiccation-tolerant life-forms such as seeds, mosses and lichens survive 'vitrification', that is the transition of their cytoplasm to a 'glassy' state, which causes metabolism to cease. However, our understanding of the mechanisms of desiccation tolerance is hindered by poor knowledge of what reactions occur in the desiccated state. Using Flavoparmelia caperata as a model lichen, we determined at what water contents vitrification occurred upon desiccation. Molecular mobility was assessed by dynamic mechanical thermal analysis, and the de- and re-epoxidation of the xanthophyll cycle pigments (measured by HPLC) was used as a proxy to assess enzyme activity. At 20 °C vitrification occurred between 0.12-0.08 g H2O g-1 DW and enzymes were active in a 'rubbery' state (0.17 g H2O g-1 DW) but not in a glassy state (0.03 g H2O g-1 DW). Therefore, desiccated tissues may appear to be 'dry' in the conventional sense, but subtle differences in water content will have substantial consequences on the types of (bio)chemical reactions that can occur, with downstream effects on longevity in the desiccated state.

Gene co-expression analyses: an overview from microarray collections in Arabidopsis thaliana.

Bioinformatics web-based resources and databases are precious references for most biological laboratories worldwide. However, the quality and reliability of the information they provide depends on them being used in an appropriate way that takes into account their specific features. Huge collections of gene expression data are currently publicly available, ready to support the understanding of gene and genome functionalities. In this context, tools and resources for gene co-expression analyses have flourished to exploit the 'guilty by association' principle, which assumes that genes with correlated expression profiles are functionally related. In the case of Arabidopsis thaliana, the reference species in plant biology, the resources available mainly consist of microarray results. After a general overview of such resources, we tested and compared the results they offer for gene co-expression analysis. We also discuss the effect on the results when using different data sets, as well as different data normalization approaches and parameter settings, which often consider different metrics for establishing co-expression. A dedicated example analysis of different gene pools, implemented by including/excluding mutant samples in a reference data set, showed significant variation of gene co-expression occurrence, magnitude and direction. We conclude that, as the heterogeneity of the resources and methods may produce different results for the same query genes, the exploration of more than one of the available resources is strongly recommended. The aim of this article is to show how best to integrate data sources and/or merge outputs to achieve robust analyses and reliable interpretations, thereby making use of diverse data resources an opportunity for added value.

Metabolomics approach based on NMR spectroscopy and multivariate data analysis to explore the interaction between the leafminer Tuta absoluta and tomato (Solanum lycopersicum).

INTRODUCTION: Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) is one of the most devastating and harmful pests of tomato (Solanum lycopersicum) crops causing up to 80-100% yield losses. A large arsenal of plant metabolites is induced by the leafminer feeding including defence compounds that could differ among varieties. OBJECTIVE: To compare the metabolomic changes of different genotypes of tomato (tolerant "T", susceptible "S" and "F1" hybrid obtained between T and S) after exposition to T. absoluta. METHODOLOGY: Nuclear magnetic resonance (NMR) spectroscopy followed by multivariate data analysis were performed to analyse the metabolic profiles of control and infested samples on three different tomato genotypes. RESULTS: Signals related to GABA (γ-aminobutyric acid) were relatively much higher in all infested samples compared to the non-infested plants used as control. Infested T genotype samples were the most abundant in organic acids, including fatty acids and acyl sugars, chlorogenic acid, neo-chlorogenic acid and feruloyl quinic acid, indicating a clear link between the exposure to leafminer. Results also showed an increase of trigonelline in all tomato varieties after exposition to T. absoluta. CONCLUSION: Metabolomics approach based on NMR spectroscopy followed by multivariate data analysis allowed for a detailed metabolite profile of plant defences, providing fundamental information for breeding programmes in plant crops.

Background element content in the lichen Pseudevernia furfuracea: a comparative analysis of digestion methods.

In bioaccumulation studies, the interpretation of pollutant contents in the target biomonitor has to be performed by assessing a deviation from an unaltered reference condition. A common strategy consists in the comparison with background element content (BEC) values, often built up by uncritically merging methodologically heterogeneous data. In this respect, the acid digestion of samples was identified as a major step affecting BEC data. Here, the analytical outcomes of two acid mixtures were compared on a set of matched paired samples of the lichen Pseudevernia furfuracea, a widely used biomonitor for which BEC values based on partial digestion were previously provided. The standard reference material BCR 482 (P. furfuracea) was used to validate analytical procedures consisting of either a HF total mineralization or an aqua regia partial one, both associated to ICP-MS multi-element analysis. In particular, the performance of the procedures was evaluated by comparing analytical results of field samples with the accuracy obtained on BCR aliquots (measured-to-expected percentage ratio). The total digestion showed a better performance for Al, As, Ba, Ca, Cd, Cu, Fe, Mn, Ni, Se, Sn, and Zn, whereas the opposite was found for Cr, Co, P, and S. Moreover, new BEC values were provided for P. furfuracea using a consolidated statistical approach, after a total sample digestion with hydrofluoric acid. The multivariate investigation of the background variability of 43 elements in 57 remote Italian sites led to the identification of geographically homogeneous areas for which BEC values are provided for use as reference in biomonitoring applications.

Comparing chemistry and bioactivity of burned vs. decomposed plant litter: different pathways but same result?

Litter burning and biological decomposition are oxidative processes co-occurring in many terrestrial ecosystems, producing organic matter with different chemical properties and differently affecting plant growth and soil microbial activity. We tested the chemical convergence hypothesis, i.e., materials with different initial chemistry converge toward a common profile, with similar biological effects, as the oxidative process advances, for burning and decomposition. We compared the molecular composition, assessed by 13 C NMR, of seven plant litter types either fresh, decomposed for 30, 90, 180 d in a microcosms incubation experiment, or heated at 100°C, 200°C, 300°C, 400°C, 500°C for 30 minutes. We used litter water extracts (5% dry weight) as treatments in bioassays on plant (Lepidium sativum) and fungal (Aspergillus niger) growth, and a washed quartz sand amended with litter (0.5% dw) to assess heterotrophic respiration by flux chamber (i.e., [µg of CO2 released]·[g added litter]-1 ·d-1 ). We observed different molecular variations for materials either burning (i.e., a sharp increase of aromatic C and a decrease of other fractions above 200°C) or decomposing (i.e., early increase of alkyl, methoxyl, and N-alkyl C and decrease of O-alkyl and di-O-alkyl C fractions). Soil respiration and fungal growth decreased with litter age and heating severity, down to 20% relative to fresh litter. Plants were inhibited on fresh litter (on average 13% of the control), but recovered on aged (180 d) and heated (30 min at 500°C) materials, up to 126% and 63% of the control, respectively. Correlation between the intensity of 13 C NMR signals in litter spectra and bioassay results showed that O-alkyl, methoxyl, and aromatic C fractions are crucial to understand organic matter effects, with plant response negatively affected by labile C but positively associated to lignification and pyrogenic C. The pattern of association of soil respiration and fungal growth to these C fractions was essentially opposite to that observed for plant root growth. Our findings suggest a functional convergence of decomposed and burned organic substrates, emerging from the balance between the bioavailability of labile C sources and the presence of recalcitrant and pyrogenic compounds, oppositely affecting different trophic levels.

Metabolomic Fingerprinting of Romaneschi Globe Artichokes by NMR Spectroscopy and Multivariate Data Analysis.

INTRODUCTION: Globe artichoke (Cynara cardunculus L. var. scolymus L. Fiori) and cardoon (Cynara cardunculus L. var. altilis DC) are sources of nutraceuticals and bioactive compounds. OBJECTIVES: To apply a NMR metabolomic fingerprinting approach to Cynara cardunculus heads to obtain simultaneous identification and quantitation of the major classes of organic compounds. METHODOLOGY: The edible part of 14 Globe artichoke populations, belonging to the Romaneschi varietal group, were extracted to obtain apolar and polar organic extracts. The analysis was also extended to one species of cultivated cardoon for comparison. The (1) H-NMR of the extracts allowed simultaneous identification of the bioactive metabolites whose quantitation have been obtained by spectral integration followed by principal component analysis (PCA). RESULTS: Apolar organic extracts were mainly based on highly unsaturated long chain lipids. Polar organic extracts contained organic acids, amino acids, sugars (mainly inulin), caffeoyl derivatives (mainly cynarin), flavonoids, and terpenes. The level of nutraceuticals was found to be highest in the Italian landraces Bianco di Pertosa zia E and Natalina while cardoon showed the lowest content of all metabolites thus confirming the genetic distance between artichokes and cardoon. CONCLUSION: Metabolomic approach coupling NMR spectroscopy with multivariate data analysis allowed for a detailed metabolite profile of artichoke and cardoon varieties to be obtained. Relevant differences in the relative content of the metabolites were observed for the species analysed. This work is the first application of (1) H-NMR with multivariate statistics to provide a metabolomic fingerprinting of Cynara scolymus. Copyright © 2016 John Wiley & Sons, Ltd.

Inhibitory and toxic effects of extracellular self-DNA in litter: a mechanism for negative plant-soil feedbacks?

Plant-soil negative feedback (NF) is recognized as an important factor affecting plant communities. The objectives of this work were to assess the effects of litter phytotoxicity and autotoxicity on root proliferation, and to test the hypothesis that DNA is a driver of litter autotoxicity and plant-soil NF. The inhibitory effect of decomposed litter was studied in different bioassays. Litter biochemical changes were evaluated with nuclear magnetic resonance (NMR) spectroscopy. DNA accumulation in litter and soil was measured and DNA toxicity was assessed in laboratory experiments. Undecomposed litter caused nonspecific inhibition of root growth, while autotoxicity was produced by aged litter. The addition of activated carbon (AC) removed phytotoxicity, but was ineffective against autotoxicity. Phytotoxicity was related to known labile allelopathic compounds. Restricted (13) C NMR signals related to nucleic acids were the only ones negatively correlated with root growth on conspecific substrates. DNA accumulation was observed in both litter decomposition and soil history experiments. Extracted total DNA showed evident species-specific toxicity. Results indicate a general occurrence of litter autotoxicity related to the exposure to fragmented self-DNA. The evidence also suggests the involvement of accumulated extracellular DNA in plant-soil NF. Further studies are needed to further investigate this unexpected function of extracellular DNA at the ecosystem level and related cellular and molecular mechanisms.

Inhibitory effects of extracellular self-DNA: a general biological process?

Self-inhibition of growth has been observed in different organisms, but an underlying common mechanism has not been proposed so far. Recently, extracellular DNA (exDNA) has been reported as species-specific growth inhibitor in plants and proposed as an explanation of negative plant-soil feedback. In this work the effect of exDNA was tested on different species to assess the occurrence of such inhibition in organisms other than plants. Bioassays were performed on six species of different taxonomic groups, including bacteria, fungi, algae, plants, protozoa and insects. Treatments consisted in the addition to the growth substrate of conspecific and heterologous DNA at different concentration levels. Results showed that treatments with conspecific DNA always produced a concentration dependent growth inhibition, which instead was not observed in the case of heterologous DNA. Reported evidence suggests the generality of the observed phenomenon which opens new perspectives in the context of self-inhibition processes. Moreover, the existence of a general species-specific biological effect of exDNA raises interesting questions on its possible involvement in self-recognition mechanisms. Further investigation at molecular level will be required to unravel the specific functioning of the observed inhibitory effects.

[Health risks associated with urban environmental pollution in Europe: results of the HEREPLUS multicentre study in Rome (Italy)]. / Rischi per la salute da inquinamento urbano nei sistemi urbani europei: approfondimento sui risultati dello studio multicentrico HEREPLUS nella città di Roma.

Urban air pollution is known to cause numerous health problems. The HEREPLUS project aims to evaluate the association between environmental air pollution and health and to provide guidelines for the management and organization of the urban environment. Risk maps were developed by georeferentiation, using hospitalization data and concentration levels of ozone and pollution matter. Data for four European cities involved in the project (Rome, Madrid, Dresden, and Athens) were analysed for the period 2003-2004. In this article, we report results for Rome only. Study findings demonstrated that the green area in a city reduces pollution in a city reduces pollution levels in the atmosphere, thus reducing health risks.

Negative plant-soil feedback in Arabidopsis thaliana: Disentangling the effects of soil chemistry, microbiome, and extracellular self-DNA.

Nutrient deficiency, natural enemies and litter autotoxicity have been proposed as possible mechanisms to explain species-specific negative plant-soil feedback (PSF). Another potential contributor to negative PSF is the plant released extracellular self-DNA during litter decay. In this study, we sought to comprehensively investigate these hypotheses by using Arabidopsis thaliana (L.) Heynh as a model plant in a feedback experiment. The experiment comprised a conditioning phase and a response phase in which the conditioned soils underwent four treatments: (i) addition of activated carbon, (ii) washing with tap water, (iii) sterilization by autoclaving, and (iv) control without any treatment. We evaluated soil chemical properties, microbiota by shotgun sequencing and the amount of A. thaliana extracellular DNA in the differently treated soils. Our results showed that washing and sterilization treatments mitigated the negative PSF effect. While shifts in soil chemical properties were not pronounced, significant changes in soil microbiota were observed, especially after sterilization. Notably, plant biomass was inversely associated with the content of plant self-DNA in the soil. Our results suggest that the negative PSF observed in the conditioned soil was associated to increased amounts of soilborne pathogens and plant self-DNA. However, fungal pathogens were not limited to negative conditions, butalso found in soils enhancing A.thaliana growth. In-depth multivariate analysis highlights that the hypothesis of negative PSF driven solely by pathogens lacks consistency. Instead, we propose a multifactorial explanation for the negative PSF buildup, in which the accumulation of self-DNA weakens the plant's root system, making it more susceptible to pathogens.

Extracellular Self-DNA Effects on Yeast Cell Cycle and Transcriptome during Batch Growth.

The cell cycle and the transcriptome dynamics of yeast exposed to extracellular self-DNA during an aerobic batch culture on glucose have been investigated using cytofluorimetric and RNA-seq analyses. In parallel, the same study was conducted on yeast cells growing in the presence of (heterologous) nonself-DNA. The self-DNA treatment determined a reduction in the growth rate and a major elongation of the diauxic lag phase, as well as a significant delay in the achievement of the stationary phase. This was associated with significant changes in the cell cycle dynamics, with slower exit from the G0 phase, followed by an increased level of cell percentage in the S phase, during the cultivation. Comparatively, the exposure to heterologous DNA did not affect the growth curve and the cell cycle dynamics. The transcriptomic analysis showed that self-DNA exposure produced a generalized downregulation of transmembrane transport and an upregulation of genes associated with sulfur compounds and the pentose phosphate pathway. Instead, in the case of the nonself treatment, a clear response to nutrient deprivation was detected. Overall, the presented findings represent further insights into the complex functional mechanisms of self-DNA inhibition.

Spatial scales of variation in lichens: implications for sampling design in biomonitoring surveys.

The variability of biological data is a main constraint affecting the quality and reliability of lichen biomonitoring surveys for estimation of the effects of atmospheric pollution. Although most epiphytic lichen bioindication surveys focus on between-site differences at the landscape level, associated with the large scale effects of atmospheric pollution, current protocols are based on multilevel sampling, thus adding further sources of variation and affecting the error budget. We test the hypothesis that assemblages of lichen communities vary at each spatial scale examined, in order to determine what scales should be included in future monitoring studies. We compared four sites in Italy, along gradients of atmospheric pollution and climate, to test the partitioning of the variance components of lichen diversity across spatial scales (from trunks to landscapes). Despite environmental heterogeneity, we observed comparable spatial variance. However, residuals often overcame between-plot variability, leading to biased estimation of atmospheric pollution effects.

Self-DNA Early Exposure in Cultivated and Weedy Setaria Triggers ROS Degradation Signaling Pathways and Root Growth Inhibition.

The accumulation of fragmented extracellular DNA reduces conspecific seed germination and plantlet growth in a concentration-dependent manner. This self-DNA inhibition was repeatedly reported, but the underlying mechanisms are not fully clarified. We investigated the species-specificity of self-DNA inhibition in cultivated vs. weed congeneric species (respectively, Setaria italica and S. pumila) and carried out a targeted real-time qPCR analysis under the hypothesis that self-DNA elicits molecular pathways that are responsive to abiotic stressors. The results of a cross-factorial experiment on root elongation of seedlings exposed to self-DNA, congeneric DNA, and heterospecific DNA from Brassica napus and Salmon salar confirmed a significantly higher inhibition by self-DNA as compared to non-self-treatments, with the latter showing a magnitude of the effect consistent with the phylogenetic distance between the DNA source and the target species. Targeted gene expression analysis highlighted an early activation of genes involved in ROS degradation and management (FSD2, ALDH22A1, CSD3, MPK17), as well as deactivation of scaffolding molecules acting as negative regulators of stress signaling pathways (WD40-155). While being the first exploration of early response to self-DNA inhibition at molecular level on C4 model plants, our study highlights the need for further investigation of the relationships between DNA exposure and stress signaling pathways by discussing potential applications for species-specific weed control in agriculture.

Self-DNA Inhibition in Drosophila melanogaster Development: Metabolomic Evidence of the Molecular Determinants.

We investigated the effects of dietary delivered self-DNA in the model insect Drosophila melanogaster. Self-DNA administration resulted in low but significant lethality in Drosophila larvae and considerably extended the fly developmental time. This was characterized by the abnormal persistence of the larvae in the L2 and L3 stages, which largely accounted for the average 72 h delay observed in pupariation, as compared to controls. In addition, self-DNA exposure affected adult reproduction by markedly reducing both female fecundity and fertility, further demonstrating its impact on Drosophila developmental processes. The effects on the metabolites of D. melanogaster larvae after exposure to self-DNA were studied by NMR, LC-MS, and molecular networking. The results showed that self-DNA feeding reduces the amounts of all metabolites, particularly amino acids and N-acyl amino acids, which are known to act as lipid signal mediators. An increasing amount of phloroglucinol was found after self-DNA exposure and correlated to developmental delay and egg-laying suppression. Pidolate, a known intermediate in the γ-glutamyl cycle, also increased after exposure to self-DNA and correlated to the block of insect oogenesis.

Extracellular DNA secreted in yeast cultures is metabolism-specific and inhibits cell proliferation.

Extracellular DNA (exDNA) can be actively released by living cells and different putative functions have been attributed to it. Further, homologous exDNA has been reported to exert species-specific inhibitory effects on several organisms. Here, we demonstrate by different experimental evidence, including 1H-NMR metabolomic fingerprint, that the growth rate decline in Saccharomyces cerevisiae fed-batch cultures is determined by the accumulation of exDNA in the medium. Sequencing of such secreted exDNA represents a portion of the entire genome, showing a great similarity with extrachromosomal circular DNA (eccDNA) already reported inside yeast cells. The recovered DNA molecules were mostly single strands and specifically associated to the yeast metabolism displayed during cell growth. Flow cytometric analysis showed that the observed growth inhibition by exDNA corresponded to an arrest in the S phase of the cell cycle. These unprecedented findings open a new scenario on the functional role of exDNA produced by living cells.

Urban ecosystem services: tree diversity and stability of tropospheric ozone removal.

Urban forests provide important ecosystem services, such as urban air quality improvement by removing pollutants. While robust evidence exists that plant physiology, abundance, and distribution within cities are basic parameters affecting the magnitude and efficiency of air pollution removal, little is known about effects of plant diversity on the stability of this ecosystem service. Here, by means of a spatial analysis integrating system dynamic modeling and geostatistics, we assessed the effects of tree diversity on the removal of tropospheric ozone (O3) in Rome, Italy, in two years (2003 and 2004) that were very different for climatic conditions and ozone levels. Different tree functional groups showed complementary uptake patterns, related to tree physiology and phenology, maintaining a stable community function across different climatic conditions. Our results, although depending on the city-specific conditions of the studied area, suggest a higher function stability at increasing diversity levels in urban ecosystems. In Rome, such ecosystem services, based on published unitary costs of externalities and of mortality associated with O3, can be prudently valued to roughly US$2 and $3 million/year, respectively.

Flooding and Soil Properties Control Plant Intra- and Interspecific Interactions in Salt Marshes.

The stress gradient hypothesis (SGH) states that plant-plant interactions shift from competition to facilitation in increasing stress conditions. In salt marshes, edaphic properties can weaken the application of the SGH by amplifying the intensity of flooding and controlling plant zonation. We identified facilitative and competitive interactions along flooding gradients and tested the role of edaphic properties in exacerbating stress and shaping plant-plant interactions. Morphological traits of two target halophytes (Limonium narbonense and Sarcocornia fruticosa), flooding intensity, soil texture and soil organic C were recorded. The relative plant fitness index was assessed for the two species based on the relative growth in plurispecific rather than monospecific plant communities. Plant fitness increased with increasing stress supporting the SGH. L. narbonense showed larger fitness in plurispecific stands whereas S. fruticosa performed better in conspecific stands. Significant intra- or interspecific interactions were observed along the stress gradient defined by the combination of flooding and clay content in soil. When considering the limited soil organic C as stressor, soil properties were more important than flooding in defining plant-plant interactions. We highlight the need for future improvements of the SGH approach by including edaphic stressors in the model and their possible interactions with the main abiotic drivers of zonation.

Little time left. Microrefuges may fail in mitigating the effects of climate change on epiphytic lichens.

Climate change is already causing considerable reductions in biodiversity in all terrestrial ecosystems. These consequences are expected to be exacerbated in biomes that are particularly exposed to change, such as those in the Mediterranean, and in certain groups of more sensitive organisms, such as epiphytic lichens. These poikylohydric organisms find suitable light and water conditions on trunks under the tree canopy. Despite their small size, epiphytic communities contribute significantly to the functionality of forest ecosystems. In this work, we surveyed epiphytic lichen communities in a Mediterranean area (Sardinia, Italy) and hypothesized that 1) the effect of microclimate on lichens at tree scale is mediated by the functional traits of these organisms and that 2) micro-refuge trees with certain morphological characteristics can mitigate the negative effects of future climate change. Results confirm the first hypothesis, while the second is only partially supported, suggesting that the capability of specific trees to host specific conditions may not be sufficient to maintain the diversity and ecosystem functionality of lichen communities in the Mediterranean.

Self-DNA Exposure Induces Developmental Defects and Germline DNA Damage Response in Caenorhabditis elegans.

All organisms, from bacteria to mammals, sense and respond to foreign nucleic acids to fight infections in order to survive and preserve genome integrity across generations. The innate immune system is an evolutionarily conserved defence strategy. Complex organisms have developed various cellular processes to respond to and recognise not only infections, i.e., pathogen-associated molecular patterns (PAMPs), but also to sense injury and tissue dysfunctions, i.e., damage-associated molecular patterns (DAMPs). Mis-localized self-DNA can be sensed as DAMP by specific DNA-sensing pathways, and self-DNA chronic exposure can be detrimental to the organisms. Here, we investigate the effects of dietary delivered self-DNA in the nematode Caenorhabditis elegans. The hermaphrodite worms were fed on Escherichia coli genomic libraries: a C. elegans library (self) and a legume (Medicago truncatula) library (non-self). We show that the self-library diet affects embryogenesis, larval development and gametogenesis. DNA damage and activation of p53/CEP-1-dependent apoptosis occur in gonadal germ cells. Studies of self-DNA exposure in this model organism were not pursued up to now. The genetic tractability of C. elegans will help to identify the basic molecular pathways involved in such mechanisms. The specificity of the adverse effects associated with a self-DNA enriched diet suggests applications in biological pest control approaches.

Metabolomics and molecular networking analyses in Arabidopsis thaliana show that extracellular self-DNA affects nucleoside/nucleotide cycles with accumulation of cAMP, cGMP and N6-methyl-AMP.

Extracellular DNA (exDNA) widely occurs in the environment due to release by either cell lysis or active secretion. The role of exDNA in plant-soil interactions has been investigated and inhibitory effects on the growth of conspecific individuals by their self-DNA have been reported. Transcriptome analysis in the model plant Arabidopsis thaliana showed a clear recognition by the plant roots of self- and nonself-exDNA, with inhibition occurring only after exposure to the former. In this study, an untargeted metabolomics approach was used to assess at molecular level the plant reactions to exDNA exposure. Thus, the effects on the metabolites profile of A. thaliana after exposure to self- and nonself-exDNA from plants and fish, were studied by NMR, LC-MS, chemometrics and molecular networking analyses. Results show that self-DNA significantly induces the accumulation of RNA constituents (nucleobases, ribonucleosides, dinucleotide and trinucleotide oligomers). Interestingly, AMP and GMP are found along with their cyclic analogues cAMP and cGMP, and in form of cyclic dimers (c-di-AMP and c-di-GMP). Also methylated adenosine monophosphate (m6AMP) and the dimeric dinucleotide N-methyladenylyl-(3'→5') cytidine (m6ApC) increased only in the self-DNA treatment. Such striking evidence of self-DNA effects highlights a major role of exDNA in plant sensing of its environment.