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, β-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

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.Peer reviewe

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

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.Peer reviewe

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.Peer reviewe

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.Peer reviewe

Defense-related transcription factors WRKY70 and WRKY54 modulate osmotic stress tolerance by regulating stomatal aperture in Arabidopsis

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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. Keywords Plant signaling Arabidopsis thaliana Oligogalacturonides OG Trimers Transcriptomics Defense induction Growth inhibition Disease resistance Pectobacterium carotovorum Botrytis cinereaPeer reviewe

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

BackgroundStored 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 degrees C and 15 degrees C. The growth of the plants was also observed in a greenhouse after the incubation of tubers in wet conditions.ResultsWet 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.ConclusionsIn 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.Peer reviewe