5-Aminothiazoles Reveal a New Ligand-Binding Site on Prolyl Oligopeptidase Which is Important for Modulation of Its Protein-Protein Interaction-Derived Functions.

A series of novel 5-aminothiazole-based ligands for prolyl oligopeptidase (PREP) comprise selective, potent modulators of the protein-protein interaction (PPI)-mediated functions of PREP, although they are only weak inhibitors of the proteolytic activity of PREP. The disconnected structure-activity relationships are significantly more pronounced for the 5-aminothiazole-based ligands than for the earlier published 5-aminooxazole-based ligands. Furthermore, the stability of the 5-aminothiazole scaffold allowed exploration of wider substitution patterns than that was possible with the 5-aminooxazole scaffold. The intriguing structure-activity relationships for the modulation of the proteolytic activity and PPI-derived functions of PREP were elaborated by presenting a new binding site for PPI modulating PREP ligands, which was initially discovered using molecular modeling and later confirmed through point mutation studies. Our results suggest that this new binding site on PREP is clearly more important than the active site of PREP for the modulation of its PPI-mediated functions.

Synthesis and import of GDP-l-fucose into the Golgi affect plant-water relations.

Land plants evolved multiple adaptations to restrict transpiration. However, the underlying molecular mechanisms are not sufficiently understood. We used an ozone-sensitivity forward genetics approach to identify Arabidopsis thaliana mutants impaired in gas exchange regulation. High water loss from detached leaves and impaired decrease of leaf conductance in response to multiple stomata-closing stimuli were identified in a mutant of MURUS1 (MUR1), an enzyme required for GDP-l-fucose biosynthesis. High water loss observed in mur1 was independent from stomatal movements and instead could be linked to metabolic defects. Plants defective in import of GDP-l-Fuc into the Golgi apparatus phenocopied the high water loss of mur1 mutants, linking this phenotype to Golgi-localized fucosylation events. However, impaired fucosylation of xyloglucan, N-linked glycans, and arabinogalactan proteins did not explain the aberrant water loss of mur1 mutants. Partial reversion of mur1 water loss phenotype by borate supplementation and high water loss observed in boron uptake mutants link mur1 gas exchange phenotypes to pleiotropic consequences of l-fucose and boron deficiency, which in turn affect mechanical and morphological properties of stomatal complexes and whole-plant physiology. Our work emphasizes the impact of fucose metabolism and boron uptake on plant-water relations.

Petunia dihydroflavonol 4-reductase is only a few amino acids away from producing orange pelargonidin-based anthocyanins.

Anthocyanins are responsible for the color spectrum of both ornamental and natural flowers. However, not all plant species produce all colors. For example, roses are not blue because they do not naturally possess a hydroxylase that opens the pathway for delphinidin and its derivatives. It is more intriguing why some plants do not carry orange or scarlet red flowers with anthocyanins based on pelargonidin, because the precursor for these anthocyanins should be available if anthocyanins are made at all. The key to this is the substrate specificity of dihydroflavonol 4-reductase (DFR), an enzyme located at the branch point between flavonols and anthocyanins. The most common example is petunia, which does not bear orange flowers unless the enzyme is complemented by biotechnology. We changed a few amino acids in the active site of the enzyme and showed that the mutated petunia DFR started to favor dihydrokaempferol, the precursor to orange pelargonidin, in vitro. When transferred to petunia, it produced an orange hue and dramatically more pelargonidin-based anthocyanins in the flowers.

Enzymatic assay for UDP-GlcNAc and its application in the parallel assessment of substrate availability and protein O-GlcNAcylation.

O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a ubiquitous and dynamic non-canonical glycosylation of intracellular proteins. Several branches of metabolism converge at the hexosamine biosynthetic pathway (HBP) to produce the substrate for protein O-GlcNAcylation, the uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Availability of UDP-GlcNAc is considered a key regulator of O-GlcNAcylation. Yet UDP-GlcNAc concentrations are rarely reported in studies exploring the HBP and O-GlcNAcylation, most likely because the methods to measure it are restricted to specialized chromatographic procedures. Here, we introduce an enzymatic method to quantify cellular and tissue UDP-GlcNAc. The method is based on O-GlcNAcylation of a substrate peptide by O-linked N-acetylglucosamine transferase (OGT) and subsequent immunodetection of the modification. The assay can be performed in dot-blot or microplate format. We apply it to quantify UDP-GlcNAc concentrations in several mouse tissues and cell lines. Furthermore, we show how changes in UDP-GlcNAc levels correlate with O-GlcNAcylation and the expression of OGT and O-GlcNAcase (OGA).

Nonpeptidic Oxazole-Based Prolyl Oligopeptidase Ligands with Disease-Modifying Effects on α-Synuclein Mouse Models of Parkinson's Disease.

Prolyl oligopeptidase (PREP) is a widely distributed serine protease in the human body cleaving proline-containing peptides; however, recent studies suggest that its effects on pathogenic processes underlying neurodegeneration are derived from direct protein-protein interactions (PPIs) and not from its regulation of certain neuropeptide levels. We discovered novel nonpeptidic oxazole-based PREP inhibitors, which deviate from the known structure-activity relationship for PREP inhibitors. These new compounds are effective modulators of the PPIs of PREP, reducing α-synuclein (αSyn) dimerization and enhancing protein phosphatase 2A activity in a concentration-response manner, as well as reducing reactive oxygen species production. From the best performing oxazoles, HUP-55 was selected for in vivo studies. Its brain penetration was evaluated, and it was tested in αSyn virus vector-based and αSyn transgenic mouse models of Parkinson's disease, where it restored motor impairment and reduced levels of oligomerized αSyn in the striatum and substantia nigra.

Mitochondrial complex III deficiency drives c-MYC overexpression and illicit cell cycle entry leading to senescence and segmental progeria.

Accumulating evidence suggests mitochondria as key modulators of normal and premature aging, yet whether primary oxidative phosphorylation (OXPHOS) deficiency can cause progeroid disease remains unclear. Here, we show that mice with severe isolated respiratory complex III (CIII) deficiency display nuclear DNA damage, cell cycle arrest, aberrant mitoses, and cellular senescence in the affected organs such as liver and kidney, and a systemic phenotype resembling juvenile-onset progeroid syndromes. Mechanistically, CIII deficiency triggers presymptomatic cancer-like c-MYC upregulation followed by excessive anabolic metabolism and illicit cell proliferation against lack of energy and biosynthetic precursors. Transgenic alternative oxidase dampens mitochondrial integrated stress response and the c-MYC induction, suppresses the illicit proliferation, and prevents juvenile lethality despite that canonical OXPHOS-linked functions remain uncorrected. Inhibition of c-MYC with the dominant-negative Omomyc protein relieves the DNA damage in CIII-deficient hepatocytes in vivo. Our results connect primary OXPHOS deficiency to genomic instability and progeroid pathogenesis and suggest that targeting c-MYC and aberrant cell proliferation may be therapeutic in mitochondrial diseases.

Metabolite Profiling of Paraquat Tolerant Arabidopsis thaliana Radical-induced Cell Death1 (rcd1)-A Mediator of Antioxidant Defence Mechanisms.

RADICAL-INDUCED CELL DEATH1 (RCD1) is an Arabidopsis thaliana nuclear protein that is disrupted during oxidative stress. RCD1 is considered an important integrative node in development and stress responses, and the rcd1 plants have several phenotypes and altered resistance to a variety of abiotic and biotic stresses. One of the phenotypes of rcd1 is resistance to the herbicide paraquat, but the mechanisms behind it are unknown. Paraquat causes a rapid burst of reactive oxygen species (ROS) initially in the chloroplast. We performed multi-platform metabolomic analyses in wild type Col-0 and paraquat resistant rcd1 plants to identify pathways conveying resistance and the function of RCD1 in this respect. Wild type and rcd1 plants were clearly distinguished by their abundance of antioxidants and specialized metabolites and their responses to paraquat. The lack of response in rcd1 suggested constitutively active defense against ROS via elevated flavonoid, glutathione, ß-carotene, and tocopherol levels, whereas its ascorbic acid levels were compromised under non-stressed control conditions when compared to Col-0. We propose that RCD1 acts as a hub that maintains basal antioxidant system, and its inactivation induces defense responses by enhancing the biosynthesis and redox cycling of low molecular weight antioxidants and specialized metabolites with profound antioxidant activities alleviating oxidative stress.

Gene expression and phytohormone levels in the asymptomatic and symptomatic phases of infection in potato tubers inoculated with Dickeya solani.

Dickeya solani is a soft rot bacterium with high virulence. In potato, D. solani, like the other potato-infecting soft rot bacteria, causes rotting and wilting of the stems and rotting of tubers in the field and in storage. Latent, asymptomatic infections of potato tubers are common in harvested tubers, and if the storage conditions are not optimal, the latent infection turns into active rotting. We characterized potato gene expression in artificially inoculated tubers in nonsymptomatic, early infections 1 and 24 hours post-inoculation (hpi) and compared the results to the response in symptomatic tuber tissue 1 week (168 hpi) later with RNA-Seq. In the beginning of the infection, potato tubers expressed genes involved in the detection of the bacterium through pathogen-associated molecular patterns (PAMPs), which induced genes involved in PAMPs-triggered immunity, resistance, production of pathogenesis-related proteins, ROS, secondary metabolites and salicylic acid (SA) and jasmonic acid (JA) biosynthesis and signaling genes. In the symptomatic tuber tissue one week later, the PAMPs-triggered gene expression was downregulated, whereas primary metabolism was affected, most likely leading to free sugars fueling plant defense but possibly also aiding the growth of the pathogen. In the symptomatic tubers, pectic enzymes and cell wall-based defenses were activated. Measurement of hormone production revealed increased SA concentration and almost no JA in the asymptomatic tubers at the beginning of the infection and high level of JA and reduced SA in the symptomatic tubers one week later. These findings suggest that potato tubers rely on different defense strategies in the different phases of D. solani infection even when the infection takes place in fully susceptible plants incubated in conditions leading to rotting. These results support the idea that D. solani is a biotroph rather than a true necrotroph.

Effects of Poty-Potexvirus Synergism on Growth, Photosynthesis and Metabolite Status of Nicotiana benthamiana.

Mixed virus infections threaten crop production because interactions between the host and the pathogen mix may lead to viral synergism. While individual infections by potato virus A (PVA), a potyvirus, and potato virus X (PVX), a potexvirus, can be mild, co-infection leads to synergistic enhancement of PVX and severe symptoms. We combined image-based phenotyping with metabolite analysis of single and mixed PVA and PVX infections and compared their effects on growth, photosynthesis, and metabolites in Nicotiana benthamiana. Viral synergism was evident in symptom severity and impaired growth in the plants. Indicative of stress, the co-infection increased leaf temperature and decreased photosynthetic parameters. In contrast, singly infected plants sustained photosynthetic activity. The host's metabolic response differed significantly between single and mixed infections. Over 200 metabolites were differentially regulated in the mixed infection: especially defense-related metabolites and aromatic and branched-chain amino acids increased compared to the control. Changes in the levels of methionine cycle intermediates and a low S-adenosylmethionine/S-adenosylhomocysteine ratio suggested a decline in the methylation potential in co-infected plants. The decreased ratio between reduced glutathione, an important scavenger of reactive oxygen species, and its oxidized form, indicated that severe oxidative stress developed during co-infection. Based on the results, infection-associated oxidative stress is successfully controlled in the single infections but not in the synergistic infection, where activated defense pathways are not sufficient to counter the impact of the infections on plant growth.

Mass Spectrometry Imaging of Arabidopsis thaliana Leaves at the Single-Cell Level by Infrared Laser Ablation Atmospheric Pressure Photoionization (LAAPPI).

In this study, we show that infrared laser ablation atmospheric pressure photoionization mass spectrometry (LAAPPI-MS) imaging with 70 µm lateral resolution allows for the analysis of Arabidopsis thaliana (A. thaliana) leaf substructures ranging from single-cell trichomes and the interveinal leaf lamina to primary, secondary, and tertiary veins. The method also showed its potential for depth profiling analysis for the first time by mapping analytes at the different depths of the leaf and spatially resolving the topmost trichomes and cuticular wax layer from the underlying tissues. Negative ion LAAPPI-MS detected many different flavonol glycosides, fatty acids, fatty acid esters, galactolipids, and glycosphingolipids, whose distributions varied significantly between the different substructures of A. thaliana leaves. The results show that LAAPPI-MS provides a highly promising new tool to study the role of metabolites in plants.

Red and blue light treatments of ripening bilberry fruits reveal differences in signalling through abscisic acid-regulated anthocyanin biosynthesis.

The biosynthesis of anthocyanins has been shown to be influenced by light quality. However, the molecular mechanisms underlying the light-mediated regulation of fruit anthocyanin biosynthesis are not well understood. In this study, we analysed the effects of supplemental red and blue light on the anthocyanin biosynthesis in non-climacteric bilberry (Vaccinium myrtillus L.). After 6 days of continuous irradiation during ripening, both red and blue light elevated concentration of anthocyanins, up to 12- and 4-folds, respectively, compared to the control. Transcriptomic analysis of ripening berries showed that both light treatments up-regulated all the major anthocyanin structural genes, the key regulatory MYB transcription factors and abscisic acid (ABA) biosynthetic genes. However, higher induction of specific genes of anthocyanin and delphinidin biosynthesis alongside ABA signal perception and metabolism were found in red light. The difference in red and blue light signalling was found in 9-cis-epoxycarotenoid dioxygenase (NCED), ABA receptor pyrabactin resistance-like (PYL) and catabolic ABA-8'hydroxylase gene expression. Red light also up-regulated expression of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) domain transporters, which may indicate involvement of these proteins in vesicular trafficking of anthocyanins during fruit ripening. Our results suggest differential signal transduction and transport mechanisms between red and blue light in ABA-regulated anthocyanin and delphinidin biosynthesis during bilberry fruit ripening.

Multi-modal meta-analysis of cancer cell line omics profiles identifies ECHDC1 as a novel breast tumor suppressor.

Molecular and functional profiling of cancer cell lines is subject to laboratory-specific experimental practices and data analysis protocols. The current challenge therefore is how to make an integrated use of the omics profiles of cancer cell lines for reliable biological discoveries. Here, we carried out a systematic analysis of nine types of data modalities using meta-analysis of 53 omics studies across 12 research laboratories for 2,018 cell lines. To account for a relatively low consistency observed for certain data modalities, we developed a robust data integration approach that identifies reproducible signals shared among multiple data modalities and studies. We demonstrated the power of the integrative analyses by identifying a novel driver gene, ECHDC1, with tumor suppressive role validated both in breast cancer cells and patient tumors. The multi-modal meta-analysis approach also identified synthetic lethal partners of cancer drivers, including a co-dependency of PTEN deficient endometrial cancer cells on RNA helicases.

Broad-range RNA modification analysis of complex biological samples using rapid C18-UPLC-MS.

Post-transcriptional RNA modifications play an important role in cellular metabolism with homoeostatic disturbances manifesting as a wide repertoire of phenotypes, reduced stress tolerance and translational perturbation, developmental defects, and diseases, such as type II diabetes, leukaemia, and carcinomas. Hence, there has been an intense effort to develop various methods for investigating RNA modifications and their roles in various organisms, including sequencing-based approaches and, more frequently, liquid chromatography-mass spectrometry (LC-MS)-based methods. Although LC-MS offers numerous advantages, such as being highly sensitive and quantitative over a broad detection range, some stationary phase chemistries struggle to resolve positional isomers. Furthermore, the demand for detailed analyses of complex biological samples often necessitates long separation times, hampering sample-to-sample turnover and making multisample analyses time consuming. To overcome this limitation, we have developed an ultra-performance LC-MS (UPLC-MS) method that uses an octadecyl carbon chain (C18)-bonded silica matrix for the efficient separation of 50 modified ribonucleosides, including positional isomers, in a single 9-min sample-to-sample run. To validate the performance and versatility of our method, we analysed tRNA modification patterns of representative microorganisms from each domain of life, namely Archaea (Methanosarcina acetivorans), Bacteria (Pseudomonas syringae), and Eukarya (Saccharomyces cerevisiae). Additionally, our method is flexible and readily applicable for detection and relative quantification using stable isotope labelling and targeted approaches like multiple reaction monitoring (MRM). In conclusion, this method represents a fast and robust tool for broad-range exploration and quantification of ribonucleosides, facilitating future homoeostasis studies of RNA modification in complex biological samples.

Enzymatically synthesized 2'-fluoro-modified Dicer-substrate siRNA swarms against herpes simplex virus demonstrate enhanced antiviral efficacy and low cytotoxicity.

Chemical modifications of small interfering (si)RNAs are used to enhance their stability and potency, and to reduce possible off-target effects, including immunogenicity. We have earlier introduced highly effective antiviral siRNA swarms against herpes simplex virus (HSV), targeting 653 bp of the essential UL29 viral gene. Here, we report a method for enzymatic production and antiviral use of 2'-fluoro-modified siRNA swarms. Utilizing the RNA-dependent RNA polymerase from bacteriophage phi6, we produced 2'-F-siRNA swarms containing either all or a fraction of modified adenosine, cytidine or uridine residues in the antisense strand of the UL29 target. The siRNA containing modified pyrimidines demonstrated high resistance to RNase A and the antiviral potency of all the UL29-specific 2'-F-siRNA swarms was 100-fold in comparison with the unmodified counterpart, without additional cytotoxicity. Modest stimulation of innate immunity signaling, including induced expression of both type I and type III interferons, as well as interferon-stimulated gene 54, by 2'-F-cytidine and 2'-F-uridine modified siRNA swarms occurred at early time points after transfection while the 2'-F-adenosine-containing siRNA was similar to the unmodified antiviral siRNA swarm in this respect. The antiviral efficacy of the 2'-F-siRNA swarms and the elicited cellular innate responses did not correlate suggesting that innate immunity pathways do not significantly contribute to the observed enhanced antiviral activity of the modified siRNAs. The results support further applications of enzymatically produced siRNA molecules with incorporated adenosine nucleotides, carrying fluoro-modification on ribose C2' position, for further antiviral studies in vitro and in vivo.

Primary Metabolite Responses to Oxidative Stress in Early-Senescing and Paraquat Resistant Arabidopsis thaliana rcd1 (Radical-Induced Cell Death1).

Rcd1 (radical-induced cell death1) is an Arabidopsis thaliana mutant, which exhibits high tolerance to paraquat [methyl viologen (MV)], herbicide that interrupts photosynthetic electron transport chain causing the formation of superoxide and inhibiting NADPH production in the chloroplast. To understand the biochemical mechanisms of MV-resistance and the role of RCD1 in oxidative stress responses, we performed metabolite profiling of wild type (Col-0) and rcd1 plants in light, after MV exposure and after prolonged darkness. The function of RCD1 has been extensively studied at transcriptomic and biochemical level, but comprehensive metabolite profiling of rcd1 mutant has not been conducted until now. The mutant plants exhibited very different metabolic features from the wild type under light conditions implying enhanced glycolytic activity, altered nitrogen and nucleotide metabolism. In light conditions, superoxide production was elevated in rcd1, but no metabolic markers of oxidative stress were detected. Elevated senescence-associated metabolite marker levels in rcd1 at early developmental stage were in line with its early-senescing phenotype and possible mitochondrial dysfunction. After MV exposure, a marked decline in the levels of glycolytic and TCA cycle intermediates in Col-0 suggested severe plastidic oxidative stress and inhibition of photosynthesis and respiration, whereas in rcd1 the results indicated sustained photosynthesis and respiration and induction of energy salvaging pathways. The accumulation of oxidative stress markers in both plant lines indicated that MV-resistance in rcd1 derived from the altered regulation of cellular metabolism and not from the restricted delivery of MV into the cells or chloroplasts. Considering the evidence from metabolomic, transcriptomic and biochemical studies, we propose that RCD1 has a negative effect on reductive metabolism and rerouting of the energy production pathways. Thus, the altered, highly active reductive metabolism, energy salvaging pathways and redox transfer between cellular compartments in rcd1 could be sufficient to avoid the negative effects of MV-induced toxicity.

Effect of wet storage conditions on potato tuber transcriptome, phytohormones and growth.

BACKGROUND: Stored potato (Solanum tuberosum L.) tubers are sensitive to wet conditions that can cause rotting in long-term storage. To study the effect of water on the tuber surface during storage, microarray analysis, RNA-Seq profiling, qRT-PCR and phytohormone measurements were performed to study gene expression and hormone content in wet tubers incubated at two temperatures: 4 °C and 15 °C. The growth of the plants was also observed in a greenhouse after the incubation of tubers in wet conditions. RESULTS: Wet conditions induced a low-oxygen response, suggesting reduced oxygen availability in wet tubers at both temperatures when compared to that in the corresponding dry samples. Wet conditions induced genes coding for heat shock proteins, as well as proteins involved in fermentative energy production and defense against reactive oxygen species (ROS), which are transcripts that have been previously associated with low-oxygen stress in hypoxic or anoxic conditions. Wet treatment also induced senescence-related gene expression and genes involved in cell wall loosening, but downregulated genes encoding protease inhibitors and proteins involved in chloroplast functions and in the biosynthesis of secondary metabolites. Many genes involved in the production of phytohormones and signaling were also affected by wet conditions, suggesting altered regulation of growth by wet conditions. Hormone measurements after incubation showed increased salicylic acid (SA), abscisic acid (ABA) and auxin (IAA) concentrations as well as reduced production of jasmonate 12-oxo-phytodienoic acid (OPDA) in wet tubers. After incubation in wet conditions, the tubers produced fewer stems and more roots compared to controls incubated in dry conditions. CONCLUSIONS: In wet conditions, tubers invest in ROS protection and defense against the abiotic stress caused by reduced oxygen due to excessive water. Changes in ABA, SA and IAA that are antagonistic to jasmonates affect growth and defenses, causing induction of root growth and rendering tubers susceptible to necrotrophic pathogens. Water on the tuber surface may function as a signal for growth, similar to germination of seeds.

Arabidopsis RCD1 coordinates chloroplast and mitochondrial functions through interaction with ANAC transcription factors.

Reactive oxygen species (ROS)-dependent signaling pathways from chloroplasts and mitochondria merge at the nuclear protein RADICAL-INDUCED CELL DEATH1 (RCD1). RCD1 interacts in vivo and suppresses the activity of the transcription factors ANAC013 and ANAC017, which mediate a ROS-related retrograde signal originating from mitochondrial complex III. Inactivation of RCD1 leads to increased expression of mitochondrial dysfunction stimulon (MDS) genes regulated by ANAC013 and ANAC017. Accumulating MDS gene products, including alternative oxidases (AOXs), affect redox status of the chloroplasts, leading to changes in chloroplast ROS processing and increased protection of photosynthetic apparatus. ROS alter the abundance, thiol redox state and oligomerization of the RCD1 protein in vivo, providing feedback control on its function. RCD1-dependent regulation is linked to chloroplast signaling by 3'-phosphoadenosine 5'-phosphate (PAP). Thus, RCD1 integrates organellar signaling from chloroplasts and mitochondria to establish transcriptional control over the metabolic processes in both organelles.

Western Diet Deregulates Bile Acid Homeostasis, Cell Proliferation, and Tumorigenesis in Colon.

Western-style diets (WD) high in fat and scarce in fiber and vitamin D increase risks of colorectal cancer. Here, we performed a long-term diet study in mice to follow tumorigenesis and characterize structural and metabolic changes in colon mucosa associated with WD and predisposition to colorectal cancer. WD increased colon tumor numbers, and mucosa proteomic analysis indicated severe deregulation of intracellular bile acid (BA) homeostasis and activation of cell proliferation. WD also increased crypt depth and colon cell proliferation. Despite increased luminal BA, colonocytes from WD-fed mice exhibited decreased expression of the BA transporters FABP6, OSTß, and ASBT and decreased concentrations of secondary BA deoxycholic acid and lithocholic acid, indicating reduced activity of the nuclear BA receptor FXR. Overall, our results suggest that WD increases cancer risk by FXR inactivation, leading to BA deregulation and increased colon cell proliferation. Cancer Res; 77(12); 3352-63. ©2017 AACR.

Short oligogalacturonides induce pathogen resistance-associated gene expression in Arabidopsis thaliana.

BACKGROUND: Oligogalacturonides (OGs) are important components of damage-associated molecular pattern (DAMP) signaling and influence growth regulation in plants. Recent studies have focused on the impact of long OGs (degree of polymerization (DP) from 10-15), demonstrating the induction of plant defense signaling resulting in enhanced defenses to necrotrophic pathogens. To clarify the role of trimers (trimeric OGs, DP3) in DAMP signaling and their impact on plant growth regulation, we performed a transcriptomic analysis through the RNA sequencing of Arabidopsis thaliana exposed to trimers. RESULTS: The transcriptomic data from trimer-treated Arabidopsis seedlings indicate a clear activation of genes involved in defense signaling, phytohormone signaling and a down-regulation of genes involved in processes related to growth regulation and development. This is further accompanied with improved defenses against necrotrophic pathogens triggered by the trimer treatment, indicating that short OGs have a clear impact on plant responses, similar to those described for long OGs. CONCLUSIONS: Our results demonstrate that trimers are indeed active elicitors of plant defenses. This is clearly indicated by the up-regulation of genes associated with plant defense signaling, accompanied with improved defenses against necrotrophic pathogens. Moreover, trimers simultaneously trigger a clear down-regulation of genes and gene sets associated with growth and development, leading to stunted seedling growth in Arabidopsis.

Peroxidase-Generated Apoplastic ROS Impair Cuticle Integrity and Contribute to DAMP-Elicited Defenses.

Cuticular defects trigger a battery of reactions including enhanced reactive oxygen species (ROS) production and resistance to necrotrophic pathogens. However, the source of ROS generated by such impaired cuticles has remained elusive. Here, we report the characterization of Arabidopsis thaliana ohy1 mutant, a Peroxidase 57 (PER57) - overexpressing line that demonstrates enhanced defense responses that result both from increased accumulation of ROS and permeability of the leaf cuticle. The ohy1 mutant was identified in a screen of A. thaliana seedlings for oligogalacturonides (OGs) insensitive/hypersensitive mutants that exhibit altered growth retardation in response to exogenous OGs. Mutants impaired in OG sensitivity were analyzed for disease resistance/susceptibility to the necrotrophic phytopathogens Botrytis cinerea and Pectobacterium carotovorum. In the ohy1 line, the hypersensitivity to OGs was associated with resistance to the tested pathogens. This PER57 overexpressing line exhibited a significantly more permeable leaf cuticle than wild-type plants and this phenotype could be recapitulated by overexpressing other class III peroxidases. Such peroxidase overexpression was accompanied by the suppressed expression of cutin biosynthesis genes and the enhanced expression of genes associated with OG-signaling. Application of ABA completely removed ROS, restored the expression of genes associated with cuticle biosynthesis and led to decreased permeability of the leaf cuticle, and finally, abolished immunity to B. cinerea. Our work demonstrates that increased peroxidase activity increases permeability of the leaf cuticle. The loss of cuticle integrity primes plant defenses to necrotrophic pathogens via the activation of DAMP-responses.