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N-Acylethanolamines (NAEs), although are ubiquitous in plants and animals the occurrence of endocannabinoid ligands and the corresponding cannabinoid receptors was limited to mammals. Interestingly, bryophytes, unlike seed plants possess arachidonic acid (AA, 20:4) and eicosapentaenoic acid (EPA, 20:5), which are fatty acid precursors for endocannabinoid ligands. Here, we show that the moss Physcomitrella patens contains ~24 and 7 % of AA and EPA, respectively. Using selective lipidomic profiling, we identified polyunsaturated NAEs, including N-arachidonoyl ethanolamide (anandamide/AEA/NAE 20:4) and N-eicosapentaenoyl ethanolamide (EPEA) and also their corresponding N-acyl-phosphatidylethanolamine (NAPE) precursors in various developmental stages of Physcomitrella. Quantification of various NAPE and NAE species indicated the abundance of unsaturated species over saturated. In all haploid developmental stages analyzed, NAE 20:4 levels contributed to ~ 30 % (~ 26 ng mg-1 lipid) of the total NAE while NAE 20:5 remained as a minor component (~ 5 %; ~ 4.5 ng mg-1 lipid). Exogenous application of AEA, EPEA and their corresponding fatty acid precursors (AA and EPA, respectively) inhibited the growth of gametophytes and protonemata in a dose-dependent manner. AEA has shown the exclusive effect on the F-actin dynamics at the apex of protonemal cells, which was similar to the effect of abscisic acid (ABA) on protonemal growth inhibition. Additionally, we identified moss ortholog for NAPE-hydrolyzing phospholipase D (NAPE-PLD) enzyme that was responsive to exogenous ABA. Putative PpNAPE-PLD was expressed in E. coli for further characterization. Our data demonstrate the occurrence of evolutionarily conserved NAE metabolic pathway in the moss, with the occurrence of AEA and EPEA.

Im Rahmen der vorliegenden Dissertation wurden Enzyme des Oxylipinstoffwechsels in P. patens funktionell und strukturell charakterisiert. Dafür wurden die bifunktionelle PpLOX1 und zwei AOCs (PpAOC1 und PpAOC2) ausgewählt. Mittels verschiedener biochemischer, bioinformatischer und biophysikalischer Methoden wurden diese Enzyme bezüglich Funktion, Aktivität und Struktur charakterisiert. Desweiteren wurden nach erfolgreicher Kristallisation von PpAOC1 und PpAOC2 die hochaufgelösten Röntgenkristallstrukturen beider Enzyme im Grundzustand sowie im Komplex mit Substratanalogen gelöst. Für PpAOC2 wurden dabei zwei verschiedene Bindemodi des Liganden beobachtet. Der Einfluß der Aminosäurereste Arg-345, Arg-638 und Tyr-851 auf den Reaktionsmechanismus von PpLOX1 wurde durch zielgerichtete Mutagenese und nachfolgende Analyse der Produktbildung durch die erhaltenen Varianten untersucht. Es wurden keine signifikanten Unterschiede bei der Umsetzung verschiedener Fettsäuren durch das Ausgangsenzym oder die Varianten R345L bzw. R638L gefunden. Dagegen zeigte die Doppelvariante R345L/R638L eine stark verringerte Menge an gebildeten Produkten. Demnach scheint zumindest das Vorliegen einer dieser beiden positiv geladenen Reste wichtig für die Umsetzung der Substrate zu sein. Möglicherweise wird die negativ geladene Carboxylatgruppe der jeweiligen Fettsäure durch elektrostatische Wechselwirkungen über Arg-345 oder Arg-638 gebunden. Die Variante Y851I bildete geringere Mengen von 12-ODTE, Keto-Fettsäuren und auch weniger Produkt als das Ausgangsenzym. Demnach scheint auch dieser Rest an der Katalyse beteiligt zu sein. Da aber für die Variante Y851F sogar ein erhöhter Anteil an 12-ODTE gefunden wurde, scheint der voluminöse und hydrophobe aromatische Ring, und nicht die Hydroxyl-Gruppe des Tyrosin, wichtig zu sein. Die gereinigten Enzyme PpAOC1 und PpAOC2 wurden für Aktivitätstest mit verschiedenen C20-Fettsäure-Hydroperoxiden verwendet. Beide Enzyme zeigten Aktivität gegenüber den 15-Hydroperoxiden von EPA und ETA, jedoch nicht von AA. Darüber hinaus besitzt PpAOC2, aber nicht PpAOC1, Aktivität für die 12-Hydroperoxide welche sich von AA, EPA und DGLA ableiten. Es wurden zusätzlich zu 11-OPTA bislang nicht beschriebene zyklische Verbindungen gebildet, deren chemische Struktur durch Fragmentierung mittels ESI-MS/MS aufgeklärt wurde. In den vorliegenden Studien zu PpAOC1 und PpAOC2 wurde das Glutamat an Position 18 jeweils durch Glutamin oder Aspartat ausgetauscht. Es wurde gezeigt, dass der konservierte Glutamatrest und seine negative Carboxylatgruppe in beiden Enzymen essenziell für die Katalyse ist. Dagegen wurde für die Variante R22L lediglich ein Einfluß auf die Aktivität in PpAOC2 gefunden. Im aktiven Zentrum von PpAOC1 werden zwei Wassermoleküle von vier Aminosäureresten koordiniert, während in PpAOC2 ein Wassermolekül von zwei Aminosäureresten gebunden ist. Inwiefern diese Wassermoleküle an der Katalyse beteiligt sind, konnte bisher nicht eindeutig geklärt werden.

N-acylethanolamines (NAEs) including anandamide are lipid derivative molecules, which play vital roles in physiological and developmental processes in plants and animals and mediate stress responses. In mammals, NAEs are synthesized from hydrolysis of their precursor molecule N-acylphosphatidylethanolamine (NAPE) by NAPE-specific phospholipaseD (NAPE-PLD). All NAEs including anandamide (NAE20:4) are hydrolyzed by fatty acid amide hydrolase (FAAH) into free fatty acid and ethanolamine. To date, different NAEs including anandamide have been identified in Physcomitrella patens but its metabolic pathway remains undiscovered. It is hypothesized that NAE metabolic pathway in P. patens is conserved and is similar to that of other eukaryotic systems. To this extent, putative PpNAPE-PLD and PpFAAH were identified and cloned for heterologous expression and characterization. Expression of PpFAAH was further verified by Western blot analysis. Future studies will involve biochemical characterization of putative PpNAPE-PLD and PpFAAH, to establish the evolutionarily conserved nature of NAE functions in early land plants.

N-acylethanolamines (NAEs) with C12-C18 acyl chain are ubiquitous in seed plants and play a role in mediating abscisic acid (ABA)-dependent or -independent responses to stress. In moss Physcomitrella patens, using selective lipidomics approach, we recently identified the occurrence of anandamide or N-arachidonylethanolamide (NAE 20:4) and its precursors that were previously not reported in plants. Occurrence of anandamide in moss provides us with a unique opportunity to address if early land plants retained NAE-mediated signaling mechanism that is akin to animals but not to vascular plants. It is hypothesized that a distinctive NAE profile and metabolic pathway occurs in P. patens. To this extent, putative genes that might be responsible for anandamide metabolic pathway were identified and their expression levels were determined for three developmental stages of moss. The NAE metabolite levels and transcript levels for putative genes were higher in protonema stage and anandamide showed higher growth inhibitory effects, chlorophyll reduction, and putative gene induction than NAE 12:0, compared to ABA, when applied exogenously.

Since a proper balance between anabolic and catabolic reactions is essential for all eukaryotes, the basic mechanisms for regulation of the energy and carbon metabolism have been conserved throughout evolution. The moss Physcomitrella patens, which belongs to one of the basal clades among land plants, has many unique properties that make it an excellent plant model system. We have used a yeast two-hybrid system to identify novel possible regulators or targets of the moss Snf1-related kinases, previously shown to regulate energy homeostasis. The function of the identified interactors PpSki1 and PpSki2 was analyzed in order to better understand the biological role of plant Snf1-related kinases. The recently completed genome sequence of Physcomitrella was used in a comparative approach to study to what extent key enzyme and gene families involved in transport and metabolism of sugars and in regulation of the energy and carbon metabolism are conserved between mosses and vascular plants. It has long been known that transformed DNA can replicate episomally in Physcomitrella. We have now shown that such DNA can be rescued back into E. coli. Surprisingly, we found that the original plasmid can be recovered from moss transformants obtained with circular DNA. Plasmids rescued from transformants obtained with linearized DNA had been repaired either by homologous recombination or by cohesive end re-ligation. These findings suggest that methods using shuttle plasmids are feasible in Physcomitrella. Hexokinase, a key enzyme in the carbon metabolism, catalyzes the first step in hexose metabolism, but is also involved in sugar sensing and signaling. We have now made an initial characterization of the complete hexokinase family in Physcomitrella which is encoded by 11 genes. Two new types of plant hexokinases, types C and D, were found in addition to the previously described types A and B.

Plants produce a rich variety of natural products to face environmental constraints. Enzymes of the cytochrome P450 CYP98 family are key actors in the production of phenolic bioactive compounds. They hydroxylate phenolic esters for lignin biosynthesis in angiosperms, but also produce various other bioactive phenolics. We characterized CYP98s from a moss, a lycopod, a fern, a conifer, a basal angiosperm, a monocot and from two eudicots. We found that substrate preference of the enzymes has changed during evolution of land plants with typical lignin-related activities only appearing in angiosperms, suggesting that ferns, similar to lycopods, produce lignin through an alternative route. A moss CYP98 knock-out mutant revealed coumaroyl-threonate as CYP98 substrate in vivo and showed a severe phenotype. Multiple CYP98s per species exist only in the angiosperms, where we generally found one isoform presumably involved in the biosynthesis of monolignols, and additional isoforms, resulting from independent duplications, with a broad range of functions in vitro / Graduate / 2017-08-31

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O uso de plantas para a cura de males é um conhecimento milenar, sendo constituinte importante para a formação das bases da medicina atual. Atualmente 25% de todas as drogas mais prescritas no mudo advêm de plantas e o mercado chega a dois bilhões de dólares. As pesquisas na área de produtos naturais dependem da integração de diversas áreas, sendo, portanto multidisciplinar. As principais áreas envolvidas são a botânica, a química, farmacologia incluindo a toxicologia, bem como o emprego de avançadas técnicas analíticas. Em decorrência dessa integração surgiu a análise elementar em plantas como alvo de pesquisas pelo mundo, já que muitos desses elementos químicos participam dos constituintes ativos responsáveis pelas propriedades curativas. Estes constituintes ativos, em sua maior parte advêm do metabolismo secundário das plantas e são chamados de metabólitos secundários. Assim buscar relações entre a composição química elementar e os produtos de metabolismo secundário é etapa fundamental na compreensão das ações terapêuticas e curativas das plantas. Neste contexto a planta Talinum patens da família portulacaceae é utilizada pela cultura popular no tratamento de feridas e cortes, problemas gastrintestinais, edemas, coceiras, fadigas e cansaço mental. As folhas são utilizadas como hortaliças em alimentação humana. Com o objetivo de contribuir na elucidação das relações entre composição química elementar e metabólitos secundários da planta Talinum patens, realizou-se prospecção fitoquímica e análise por ativação neutrônica instrumental de amostras in-natura e amostras de extratos preparados com solventes de polaridade crescente. Os resultados mostraram tendência de concentração de certos elementos químicos em determinados extratos em detrimento de outros, evidenciando que a composição química elementar encontra-se interagida ou ligada a diferentes moléculas extraídas. Foi possível ainda avaliar as contribuições nutricionais das formas populares de uso e os resultados sugerem estudos epidemiológicos. / The use of plants for many diseases treatment is a millenary knowledge. These are the great importance for the formation of actual medicine bases. Actually, more than 25 % of the all drugs prescribed in the world are obtained from plants. The medicinal plants and other vegetable materials market reach to around two billions dollars. The researches of natural products depend on different scientific areas integration, therefore with multidisciplinary aspects. The mainly areas involved in these researches are botany, chemistry, pharmacology, and toxicology. In all cases are necessary the use of advanced analytical techniques. From the integration of these different areas appears the use of elemental analysis to determine the presence of some inorganic elements other than hydrogen, carbon and oxygen in plants. This analyze became a target of many researches because chemical elements are components of biologically active compounds, including those with curative properties. The bigger part of these active compounds, called secondary metabolites, is derived from the secondary plant metabolism. Than, search relations between chemical element composition and secondary metabolites represents fundamental step to understand the therapeutic actions of curative plants. Inserted in these context, Talinum patens of the Portulacaceae family is popularly used by the Patos de Minas peoples, for the cuts and wounds treatment, gastrointestinal problems, edemas, body and mental fatigue. The leaves of this plant are used in salads in human nutrition. To contribute for the relationships between elemental composition and secondary metabolites, the T. patens was submitted to phytochemical and elemental analyses. The phytochemical studies employed extraction methods using solvent of different polarity as well as chromatographic techniques. For the elemental analyses was used instrumental neutron activation analysis (INAA). The results showed a chemical elemental concentration tendency in some extract relatively to others. The data obtained demonstrated that elemental chemicals are included in the structure of molecules encountered in the extracts. It was possible evaluate the nutrition contributions for the popular use forms. The results suggests a realization of epidemiological study to verify the real nutrition contribution and/or undesirable effects of Talinum patens.

L'objectiu d'aquest treball ha estat l'estudi del gènere Kallymenia a la Península Ibèrica i a lesIlles Balears, el qual es troba representat per les espècies següents: K. reniformis (Turner) J.Agardh, K. feldmannii Codomier, K. lacerata Feldmann, K. requienii (J. Agardh) J. Agardh, K.patens (J. Agardh) P.G. Parkinson i K. spathulata (J. Agardh) P.G. Parkinson.En aquesta monografia es descriuen les principals característiques morfològiques i vegetatives itambé els tetrasporangis de totes les espècies de la Península Ibèrica i de les Illes Balears. Tambées descriuen les estructures reproductores i els estadis de postfertilització de totes, excepte de K. patens i K. spathulata, espècies més rares que les altres i per a les quals no hem pogut aportaraquesta informació. Les dades s'acompanyen amb una iconografia original a base de dibuixos ifotografies i amb una clau de determinació pels tàxons estudiats. També es detalla la corologiade les diferents espècies, el seu hàbitat i la seva fenologia.