Ozone and nitrogen dioxide regulate similar gene expression responses in Arabidopsis but natural variation in the extent of cell death is likely controlled by different genetic loci

High doses of ozone (O-3) and nitrogen dioxide (NO2) cause damage and cell death in plants. These two gases are among the most harmful air pollutants for ecosystems and therefore it is important to understand how plant resistance or sensitivity to these gases work at the molecular level and its genetic control. We compared transcriptome data from O-3 and NO2 fumigations to other cell death related treatments, as well as individual marker gene transcript level in different Arabidopsis thaliana accessions. Our analysis revealed that O-3 and NO2 trigger very similar gene expression responses that include genes involved in pathogen resistance, cell death and ethylene signaling. However, we also identified exceptions, for example RBOHF encoding a reactive oxygen species producing RESPIRATORY BURST OXIDASE PROTEIN F. This gene had increased transcript levels by O-3 but decreased transcript levels by NO2, showing that plants can identify each of the gases separately and activate distinct signaling pathways. To understand the genetics, we conducted a genome wide association study (GWAS) on O-3 and NO2 tolerance of natural Arabidopsis accessions. Sensitivity to both gases seem to be controlled by several independent small effect loci and we did not find an overlap in the significantly associated regions. Further characterization of the GWAS candidate loci identified new regulators of O-3 and NO2 induced cell death including ABH1, a protein that functions in abscisic acid signaling, mRNA splicing and miRNA processing. The GWAS results will facilitate further characterization of the control of programmed cell death and differences between oxidative and nitrosative stress in plants.Peer reviewe

Adaptation of aphid stylectomy for analyses of proteins and mRNAs in barley phloem sap

Sieve tubes are transport conduits not only for photoassimilates but also for macromolecules and other compounds that are involved in sieve tube maintenance and systemic signalling. In order to gain sufficient amounts of pure phloem exudates from barley plants for analyses of the protein and mRNA composition, a previously described stylectomy set-up was optimized. Aphids were placed in sealed cages, which, immediately after microcauterization of the stylets, were flooded with water-saturated silicon oil. The exuding phloem sap was collected with a capillary connected to a pump. Using up to 30 plants and 600 aphids (Rhopalosiphum padi) in parallel, an average of 10 μl of phloem sap could be obtained within 6 h of sampling. In first analyses of the macromolecular content, eight so far unknown phloem mRNAs were identified by cDNA-amplified fragment length polymorphism. Transcripts in barley phloem exudates are related to metabolism, signalling, and pathogen defence, for example coding for a protein kinase and a pathogen- and insect-responsive WIR1A (wheat-induced resistance 1A)-like protein. Further, one-dimensional gel electrophoresis and subsequent partial sequencing by mass spectrometry led to the identification of seven major proteins with putative functions in stress responses and transport of mRNAs, proteins, and sugars. Two of the discovered proteins probably represent isoforms of a new phloem-mobile sucrose transporter. Notably, two-dimensional electrophoresis confirmed that there are >250 phloem proteins awaiting identification in future studies

Synthesis and transport of signals in the phloem after wounding, induction of pathogen resistance and abiotic stress

In dieser Arbeit wurden physiologische und molekulare Muster von Reaktionen des Phloems auf diverse Reize untersucht. Im ersten Teil der Arbeit wurden mit Hilfe des cDNA-AFLP (cDNA-amplified fragment length polymorphism) Teilsequenzen von 11 Transkripten im Phloemsaft aus Gerstenpflanzen ermittelt. Drei Transkripte, zu denen auch die mRNA des Abwehrproteins Carboanhydrase gehört, wurden ausschließlich nach chemischer Induktion von Pathogenresistenz gefunden. Die Transkripte sind damit Kandidaten für systemisch transportierte Resistenzsignale. Ein weiterer Teil der Arbeit befasste sich mit Funktionen des Signalmoleküls NO im Phloem. In mikroskopischen Studien mit dem NO-spezifischen Fluoreszenzfarbstoff Diaminofluoresceindiacetat konnte in den Geleitzellen des Phloems von Vicia faba eine starke NO-Synthese beobachtet werden. Diese NO-Synthese wurde durch exogene Applikation von Salicylsäure und H2O2 ausgelöst. Ein pilzlicher Elicitor und Jasmonsäure bewirkten dagegen eine vergleichsweise schwache NO-Synthese. Pharmakologische Analysen zeigten, dass im Phloem eine Ca2+- und L-Arginin-abhängige NO-Synthase (NOS) aktiv ist. Während Vicia faba gut geeignet ist für die in vivo-Mikroskopie des Phloems, können aus dieser Pflanze jedoch nur sehr geringe Mengen Phloemsaft gewonnen werden. Daher wurden biochemische Untersuchungen mit Phloemproben aus Kürbispflanzen vorgenommen. Nach Verwundung, Hitzestress und Behandlung mit dem Resistenzinduktor BION konnte in Western Blots mit Nitrotyrosin-Antikörpern eine zunehmende Nitrierung von Phloem-proteinen festgestellt werden. Außerdem wurden mit der Biotin switch-Methode potenzielle nitrosylierbare Proteine im Phloemsaft nachgewiesen. Insgesamt wurden 12 Proteine identifiziert, darunter die Phloemproteine 16 und 36 sowie zwei Peroxidasen, die Glyoxalase I und die 25-26 kD große Isoform des Phloemprotein 2 (PP2). Das letztere Protein wurde wahrscheinlich sowohl an Cysteinresten nitrosyliert als auch an Tyrosinresten nitriert. Durch Behandlung von Kürbis-Phloemproben mit NO-Donoren wurden die antioxi-dativen Enzyme Ascorbatperoxidase (APX) und die Superoxiddismutase (SOD) in vitro inhibiert. Einen ähnlichen Effekt hatten Verwundung, Resistenzinduktion und Hitzestress in vivo. Wenn vor dem Hitzestress die Versuchspflanzen mit einem NOS-Inhibitor behandelt wurden, konnte die Hemmung der APX und SOD fast vollständig verhindert werden. Das deutet an, dass diese Enzyme nach Hitzestress direkt oder indirekt durch NO gehemmt wurden. Mit dem gleichen Inhibitor wurde außerdem die partielle Fällung des Phloem-proteins 1 und einer 24,5 kD großen PP2-Isoform nach Reizung der Pflanzen unterdrückt. PP1 und PP2 sind redoxsensitive Proteine, deren Fällung wahrscheinlich ein Absinken des Redoxpotentials im Siebelement anzeigt. Die Ergebnisse werden in Bezug auf eine mögliche Rolle von NO in Redox-abhängigen systemischen Signalkaskaden diskutiert.In this work, physiological and molecular patterns of responses of the phloem to various stimuli were investigated. Utilising cDNA-AFLP (cDNA-amplified fragment length poly-morphism) partial sequences of 11 gene transcripts in barley phloem exudates were received. Three transcripts, of which one coded for the defence-related carboanhydrase, were detected exclusively after chemical induction of pathogen resistance. These mRNAs are candidates for systemically translocated resistance signals. The second part of the PhD project dealt with functions of the signal molecule nitric oxide in the phloem. Microscopic studies with the NO-specific fluorescent dye diaminofluoresceine diacetate revealed a strong NO-synthesis in companion cells of the phloem of Vicia faba. The NO burst was induced by exogenously applied salicylic acid and H2O2. A fungal elicitor and jasmonic acid were less effective inducers. Pharmacological approaches demonstrated that a Ca2+- and L-Arginin-dependent NO synthase (NOS) is active in the phloem. After wounding of leaves, heat stress, and treatment with the resistance inducing chemical BION an increase in nitration of proteins in pumpkin phloem sap was recognised using western blot analyses with nitrotyrosine antibodies. Furthermore, potentially nitrosylated phloem proteins were detected by the biotin switch method. 12 proteins were identified, among them the phloem proteins 16 and 36, two peroxidases, glyoxalase I, and a 25-26 kD isoform of the phloem protein 2 (PP2). The latter protein was probably nitrosylated at cysteine residues as well as nitrated on tyrosine residues. Treatment of pumpkin phloem sap with NO donors caused a decrease in activity of the antioxidant enzymes ascorbate peroxidase (APX) and superoxide dismutase (SOD), wounding, resistance induction, and heat stress had a similar effect. Pretreatment of plants with a NOS inhibitor suppressed the inhibition of APX and SOD after heat stress, indicative for a regulation of the enzymes by NO. The NOS inhibitor also prevented the partial precipitation of the phloem protein 1 and of the 24,5 kD PP2 isoform after diverse treatments of pumpkin plants. PP1 and PP2 are redoxsensitive proteins whose precipitation could indicate a drop of the redox potential in the sieve elements. These results are discussed in terms of a potential role of NO in redox-dependent systemic signal cascades

Interplay between NO Signalling, ROS, and the Antioxidant System in Plants

Over the last decades, nitric oxide (NO) has emerged as an essential player in redox signalling. Reactive oxygen species (ROS) also act as signals throughout all stages of plant life. Because they are potentially harmful for cellular integrity, ROS and NO levels must be tightly controlled, especially by the classical antioxidant system and additional redox-active metabolites and proteins. Recent work provided evidence that NO and ROS influence each other’s biosynthesis and removal. Moreover, novel signalling molecules resulting from the chemical reaction between NO, ROS and plant metabolites have been highlighted, including N2O3, ONOO-, NO2, S-nitrosoglutathione and 8-NO2 cGMP. They are involved in diverse plant physiological processes, the best characterized being stomata regulation and stress defense. Taken together, these new data demonstrate the complex interactions between NO, ROS signalling and the antioxidant system. This Frontiers in Plant Science Research Topic aims to provide an updated and complete overview of this important and rapidly expanding area through original article and detailed reviews

A T-DNA mutant screen that combines high-throughput phenotyping with the efficient identification of mutated genes by targeted genome sequencing.

Background: Nitrogen dioxide (NO2) triggers hypersensitive response (HR)-like cell death in Arabidopsis thaliana. A high-throughput mutant screen was established to identify genes involved in this type of programmed cell death.Results: Altogether 14,282 lines of SALK T-DNA insertion mutants were screened. Growing 1000 pooled mutant lines per tray and simultaneous NO2 fumigation of 4 trays in parallel facilitated high-throughput screening. Candidate mutants were selected based on visible symptoms. Sensitive mutants showed lesions already after fumigation for 1 h with 10 ppm (ppm) NO2 whereas tolerant mutants were hardly damaged even after treatment with 30 ppm NO2. Identification of T-DNA insertion sites by adapter ligation-mediated PCR turned out to be successful but rather time consuming. Therefore, next generation sequencing after T-DNA-specific target enrichment was tested as an alternative screening method. The targeted genome sequencing was highly efficient due to (1.) combination of the pooled DNA from 124 candidate mutants in only two libraries, (2.) successful target enrichment using T-DNA border-specific 70mer probes, and (3.) stringent filtering of the sequencing reads. Seventy mutated genes were identified by at least 3 sequencing reads. Ten corresponding mutants were re-screened of which 8 mutants exhibited NO2-sensitivity or -tolerance confirming that the screen yielded reliable results. Identified candidate genes had published functions in HR, pathogen resistance, and stomata regulation.Conclusions: The presented NO2 dead-or-alive screen combined with next-generation sequencing after T-DNAspecific target enrichment was highly efficient. Two researchers finished the screen within 3 months. Moreover, the target enrichment approach was cost-saving because of the limited number of DNA libraries and sequencing runs required. The experimental design can be easily adapted to other screening approaches e.g. involving high-throughput treatments with abiotic stressors or phytohormones