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Oxylipins are important biological active compounds that play essential roles in defense, growth, development, and reproduction of plants and animals. Oxylipins are formed either by enzymatic pathways or radical catalyzed reaction from polyunsaturated fatty acids. Products of oxidation of arachidonic acid (C20:4) in animals by enzymatic and non-enzymatic pathways are prostaglandins and isoprostanes, respectively. In plants, radical catalyzed reaction of -linolenic acid (C18:3) forms phytoprostanes and enzymatic oxidation of this fatty acid produces OPDA and jasmonic acid. Like plants, cyanobacterial membranes contain a high ratio of polyunsaturated fatty acid, about 25% of total fatty acids. Oxylipin biosynthesis and function was studied in two model cyanobacteria, Anabaena PCC 7120 and Synechocystis PCC 6803, for the first time: 1. The filamentous cyanobaterium Anabaena PCC 7120 can naturally produce phytoprostanes type I and II as well as hydroxy fatty acids like in plants but lacks the enzymatic capacity to form jasmonates (12-oxo-phytodienoic acid and jasmonic acid) and prostaglandins. Data obtained provide the first evidence for the occurence of phytoprostanes in cyanobacteria as well as in the baterial kingdom. 2. By GC-MS analysis, the E1- and F1-phytoprostanes in Anabaena PCC 7120 were detected both in free and esterified form. Their levels are comparable with those in plants, in the range of ng/g DW. In one week old cultures, there was no evidence of PPF1 in the medium but its level accumulated up to 142 ng/l in six weeks old cultures. In contrast, PPE1 was stable over time, about 20 ng/g DW. Free cellular PPE1 was found about 4 times higher than that of PPF1, 80.5  23.6 and 24.1  10.9 ng/g DW, respectively. However, there was no significant difference in the total cellular levels of PPF1 and PPE1, ranging from 150 to about 200 ng/g DW. 3. Phytoprostanes are inducible in Anabaena. In the combination of oxidative stress (200 µM H2O2 or 10 µM CuSO4) with high light intensity (330 µE.m-2.s-1) for 8 h, levels of total cellular PPE1 and PPF1 were increased about 2 to 4 times. Interestingly, unlike in higher plants, application of oxidative stress or high light intensity alone showed no phytoprostaneous induction in this cyanobacterium. 4. When Anabaena cells were treated with phytoprostanes, Anabaena cells became remarkably resistant against subsequently applied – otherwise lethal – oxidative stress. All phytoprostanes displayed a high protective effect except for PPE1. The highest protection level was contributed by a mixture of PPA1 type I and II. After preincubation of Anabena cells with 100 µM PPA1–type I/II for 16 h followed by application of 1 mM H2O2 or 50 µM CuSO4 for 5 h, A1-phytoprostane pre-treatment protected 84.2% and 77.5% of the cells from cell death, respectively. Without oxylipins pre-treatment, about 98% of the cells were dead. Surprisingly, preincubation of Anabaena with other oxylipins derived from enzymatic pathway in plants and animals showed also an effect, however, the protection effect was low and ranged from 10 to 30%. In contrast, phytoprostanes did not protect Pseudomonas syringae and Escherichia coli from the toxicity of hydrogen peroxide. However, these bacteria do not synthesize polyunsaturated fatty acids and are therefore devoid of and not exposed to endogenously formed oxidized lipids. 5. Exogenous application of 100 µM PPF1 or 1.5 mM H2O2 for 90 min did not activate the expression of isiA in Anabaena. Oxylipins also displayed no effect on shinorine and tocopherol levels in Anabaena. However, application of 100 µM PPF1 for 6 h altered the protein expression in Anabaena. Most PPF1-modulated proteins are down-regulated and related to photosynthesis. Since oxidative stress only in combination with high light intensity increased lipid peroxidation, down-regulation of photosynthesis after recognition of oxidised lipids (phytoprostanes) may be a survival strategy of Anabaena to avoid damage by peroxidized lipids. 6. Dead plants may be the main source of (exogenous) phytoprostanes in the natural environment of Anabaena. Dry hay releases PPE1 and PPF1 (11 µg/g DW) into an aqueous environment. Anabaena is the typical cyanobacterium in paddy rice fields. After harvesting, most of uneconomical parts of rice plants are abundant on the field, which may release phytoprostanes that in turn might have an impact on cyanobacteria in the rice ecosystems. However, field research is needed to clarify this suspection. 7. A new class of oxylipins, phytoprostanes type III and IV, was identified and quantified in vitro. The two main phytoprostanes, PPE1 and PPF1 (type III and IV), can be obtained by autoxidation of -linolenic acid or Borage oil (containing 25% esterified -linolenic acid). After 12 days of autoxidation and subsequent hydrolysis, 1 g of Borage oil yielded 112.71 ± 1.93 µg of PPF1 and 3.80 ± 0.14 mg of PPE1. PPB1 and PPA1 (type III and IV) were prepared by isomerization and dehydration of PPE1 (type III and IV). The overall yield of PPB1 was 1.71 ± 0.04 mg/g oil (type III) and 2.09 ± 0.12 mg/g oil (type IV). Those of PPA1 were 8.38 ± 0.35 µg/g and 10.18 ± 0.30 µg/oil, respectively. 8. A rapid HPLC-MS/MS method for phytoprostane and phytohormone analysis has been developed. This method was applied to quantify free and esterified E1- and F1-phytoprostanes type III and IV in Synechocystis PCC 6803. The in vivo phytoprostanes type III and IV are present both in free and esterified form. The total cellular level of PPE1 type III and IV in Synechocystis is at least 2 times higher than that of PPF1. Unlike Anabaena, PPE1 and PPF1 were detectable in the medium of one week old Synechocystis cultures. Free levels of PPF1 in the medium (231.8 ± 36.2 ng/l) and in the cells (164.9 ± 15.2 ng/g DW) are lower than those of PPE1 (1003.3 ± 365.2 ng/l and 2331.0 ± 87.7 ng/g DW).
Lipidperoxidation kann entweder durch Lipoxygenasen oder reaktive Sauerstoffspezies ausgelöst werden. Enzymatische Oxidation von alpha-Linolensäure kann zur Biosynthese von zyklischen Oxylipinen vom Typ der Jasmonate führen, wohingegen durch freie Radikal-katalysierte Oxidation von alpha-Linolensäure mehrerere Klassen zyklischer Oxylipine, den Phytoprostanen entstehen können. Eine dieser Phytoprostanklassen, Phytoprostane E1 (PPE1), kommen ubiquitär in Pflanzen vor. In der vorliegenden Arbeit wird gezeigt, dass PPE1 in planta in neuartige Cyclopentenon-Phytoprostane, die PPA1 und PPB1 umgewandelt werden. Eine gesteigerte Bildung von PPE1, PPA1 und PPB1 wurde sowohl nach Peroxid-Behandlung von Tabak-Zellkulturen als auch nach Behandlung von Tomatenpflanzen mit dem nekrotrophen Pilz Botrytis cinerea beobachtet. Darüberhinaus besitzen PPA1 und PPB1 biologische Wirkung. Sie stimulierten beispielsweise die Bildung von Phytoalexinen in mehreren Zellkulturen. Diese Daten implizieren, dass die Bildung von Phytoprostanen eine Folge von oxidativem Stress in Pflanzen ist und dass Phytoprostane pflanzliche Abwehrmechanismen induzieren können.
Phytoprostane F1
(2001)
Isoprostane F2 sind Autoxidationsprodukte der Arachidonsäure, die über radikal-katalysierte Oxidation entstehen. Bei einigen Erkrankungen im Tier konnte gezeigt werden, daß die Konzentration von Isoprostanen F2 mit dem verstärkten Vorkommen von freien Radikalen korreliert, weshalb Isoprostane heute als Marker des oxidativen Streß genutzt werden. Darüber hinaus weisen einige Isoprostane eine biologische Aktivität auf, weshalb sie heute als Signalstoffe des oxidativen Streß im Tier diskutiert werden. Pflanzen hingegen können keine Isoprostane F2 synthetisieren, da ihnen der Precursor Arachidonsäure fehlt. In der vorliegenden Arbeit wird gezeigt, daß analog zu den Isoprostanen F2 Phytoprostane F1 in Pflanzen aus Linolensäure gebildet werden. Hierfür wurden HPLC- und Gaschromatographie-Massenspektroskopie-Methoden entwickelt, die eine Quantifizierung von Phytoprostanen F1 in Pflanzen ermöglichten. In frischen Pflanzenorganen wurden Phytoprostane F1 sowohl in freier als auch in veresterter Form detektiert. Darüber hinaus stieg die Konzentration sowohl freier als auch veresterter Phytoprostane F1 in Pfefferminzblättern nach Verwundung und in pflanzlichen Zellkulturen nach Zusatz von Agentien, von denen bekannt ist, daß sie pflanzliche Zellen oxidativ schädigen, an. In getrockneten Pflanzenmaterialien wurden extrem hohe Konzentrationen an Phytoprostanen F1 quantifiziert. Daher steht die Vermutung nahe, daß Phytoprostane F1 ähnlich wie die Isoprostane F2 im Tier als sensitiver Marker der oxidativen Zellverletzung in der Pflanze eingesetzt werden können. Darüber hinaus konnte gezeigt werden, daß der Zusatz von Phytoprostanen F1 zu Eschscholzia californica-, Crotalaria cobalticola- and Thalictrum tuberosum-Zellsuspensionskulturen zu einer Phytoalexinakkumulation führte.