SufD - Bestandteil eines essentiellen plastidär lokalisierten Systems in Cyanobakterien, Cryptomonaden und höheren Pflanzen

Durch die vorliegende Arbeit konnte ein Beitrag zur funktionellen Charakterisierung des Nucleomorph-kodierten und plastidär lokalisierten Proteins ORF467 aus Guillardia theta geleistet werden. Dieses Protein konnte durch Sequenzhomologien als Bestandteil SufD des plastidären SUF-Systems identifiziert werden. Mit immunologischen Methoden konnte SufD in der Plastide von Guillardia theta nachgewiesen werden. Ein Gen für eine weitere Komponente des SUF-Systems, SufS, wurde durch die Auswertung von EST-Daten im Kerngenom lokalisiert. Die Cryptomonade Guillardia theta enthält damit ein über drei Genome, Kern, Plastide und Nucleomorph, verteiltes plastidäres SUF-System. Die heterogenomische Knock-out Mutation von SufD in Synechocystis führte zu einem Defekt der Zellteilung, der durch die Bildung von aus vier Zellen bestehenden Teilungsstadien gekennzeichnet war. In Importexperimenten mit radioaktivem Eisen wurde eine erhöhte Eisenaufnahme der SufD-Mutante gemessen. Die Analyse einer T-DNA Mutante des SufD-Homologen ATNAP6 in Arabidospis thaliana zeigte Defekte in der Embryonalentwicklung, eine veränderte Ultrastruktur der Chloroplasten, sowie ein reduziertes Wurzelwachstum und verringerten Chlorophyllgehalt der Pflanzen. Durch die in vivo Expression von GFP-Fusionsproteinen von ATNAP6 in Protoplasten konnte eine plastidäre Lokalisation von ATNAP6 nachgewiesen werden. Das Lokalisationsmuster zeigte eine Verteilung der GFP-Fluoreszenz in abgegrenzten subplastidären Bereichen, die für die subplastidäre Lokalisation notwendige beta-helikale Proteindomäne von ATNAP6 wurde durch die Generierung und in vivo Expression von modifizierten ATNAP6-GFP Fusionsproteinen identifiziert. In einer Expressionsanalyse mit einem Oligonukleotid-Genchip Microarray konnten signifikante Änderungen der Expression von regulativen Genen und von Genen in verschiedenen Stoffwechselwegen der ATNAP6 Knock-out Mutante gemessen werden. Die Expressionsdaten der ATNAP6 Knock-out Mutante und der komplexe Phänotyp von atnap6 konnten als Folge eines grundlegenden Defekts des plastidären SUF-Systems zur Synthese von Eisen-Schwefel Clustern interpretiert werden und ermöglichten es, ein Modell für die Funktion von ATNAP6 in Arabidopsis thaliana zu formulieren

Nucleomorph and complex plastids

Endosimbiontska teorija o porijeklu plastida stara je više od sto godina i danas je prihvaćena kao jedina važeća teorija o postanku organela. Danas se pretpostavlja kako su mitohondriji podrijetlom α-protobakterije dok se pretkom plastida smatra cijanobakterija. Primarna endosimbioza spojila je endosimbiontsku bakteriju i njenog domaćinskog eukariota. Jednosmjernim transferom gena iz cijanobakterije u jezgru domaćina bakterija je s vremenom postala plastid. Sljedeći događaj koji je mogao je nastupiti sekundarna endosimbioza u kojoj je alga s cijanobakterijskim plastidom ušla u veću heterotrofnu stanicu. Tada je došlo do redukcije genetičkog materijala endosimbiota te nastanka nukleomorfa. Ukoliko je transfer gena koji je nastupio bio potpun, ostao je samo plastid okružen s tri ili četiri membrane koji se naziva kompleksnim plastidom. Može doći i do potpune degeneracije organela koji postaje neprepoznatljiv te ga tada nazivamo kriptičkim organelom. Osim transfera gena, drugi najvažniji mehanizam pri nastanku organela je organizacija transporta proteina preko novonastalih membrana. Ovaj rad se bavi karakteristikama organela nastalih sekundarnom endosimbiozom kao i nekim teorijama koje objašnjavaju njihov postanak.Endosymbiotic theory on the origin of plastids is over one hundred years old and today is accepted as the only valid theory on the origin of organelles. Today, it is assumed that mitochondria originated from α-proteobacteria and cyanobacteria are considered to be plastid ancestors. Primary endosymbiosis merged the endosymbiotic bacterium and its host eukaryote. One-way transfer of genes from cyanobacteria into the host nucleus eventually turned bacteria into a plastid. The next probable event was a secondary endosymbiosis in which the algae with cyanobacterial plastids entered the larger heterotrophic cell. The reduction of endosymbiont's genetic material occured and induced the creation of nucleomorph. If a total transfer of genes occured, only a plastid surrounded by three or four membranes – a complex plastid- remained, and then we call them complex plastids. A complete degeneration of organelles can also occure, organelle becomes unrecognizable and we name it cryptic organelle. In addition to gene transfer, the second most important mechanism in organelle formation is organization of protein transfer through the newly formed membranes. This seminar covers characteristics of organelles created as a product of secondary endosymbiotic event and some theories explaining their origin

Endosymbiosis and biodiversity

Endosimbioza ima važnu ulogu u postanku i evoluciji eukariotske stanice. Različiti plastidi nastali su nizom endosimbiotskih odnosa te uzrokovali raznolikost u svijetu algi. Endosimbioza hidre i Chlorelle predstavlja tipičan primjer dugotrajanog mutualističkog odnosa u kojem oba organizma imaju koristi. S druge strane, Elysia i Ambystoma pripadaju novootkrivenim nespecifičnim simbiozama. Fotosintetske simbioze osiguravaju organizmima svojevrsnu prednost jer mogu preživjeti u uvjetima bez hrane. Moguće je da ovakvi odnosi postoje u puno više organizama nego što je zabilježeno te su potrebna daljnja istraživanja koja će dati odgovore na neka, još otvorena pitanja te pružiti uvid u bioraznolikost koju osiguravaju ovi odnosi.Endosymbiotic relationships play an important role in the origin and evolution series of endosymbiotic events. Endosymbiosis between hydra and Chlorella represents a long-term mutualistic relationship in which both organisms benefit from each other. On the other hand, Elysia and Ambystoma are examples of the unusual symbioses, waiting yet to be fully understood. Associations with photosynthetic organisms provide an advatange to a host because it can survive in conditions without food. It is possible that these relationships exist in more organisms then that were observed so far. Further research is needed to provide the answers and give insight into the biodiversity that is shaped by symbiotic relationships

Phytoplankton of extreme habitats, example of the Rogoznica Lake

Rogozničko jezero je malo, slano, krško jezero 15 m dubine i veličine 5300 četvornih metara. Smješteno u kotlini, zaštićeno je od jakih vjetrova, te u njemu nema strujanja vode. Velike sezonske amplitude saliniteta, temperature i otopljene organske tvari čine ga ekstremnim staništem. Ono je meromiktičko jezero sa anoksičnim dubinskim slojevima. Životna zajednica u njemu sastoji se od malog broja fitoplanktonskih vrsta i beskralježnjaka. Krajem rujna i početkom listopada 1997. godine dolazi do naglog miješanja vodenog stupca i do anoksičnog stanja cijelog jezera. Nakon tog događaja dolazi do odumiranja gotovo cijele jezerske populacije fitoplanktona. Brojnim uzorkovanjima i istraživanjima praćen je oporavak fitoplanktona. Prvi primjerci mikroflagelata javljaju se već nakon dva tjedana na područjima povećane koncentracije amonijaka. Kasnija oksidacija vodenog stupca i stvaranje nitrata pogoduje pojavi većeg fitoplanktona kao dijatomeja i dinoflagelata. Uzorkovanje nakon godinu dana pokazuje kako je jezerska populacija fitoplanktona potpuno oporavljena te je jednaka onoj prije anoksije. U Rogozničkom jezeru nalazimo dvije rijetke vrste: Prorocentrum arcuatum Issel i Hermesium adriaticum Zaharis. Ove morske vrste fitoplanktona su rijetke u cijelom Sredozemlju. Možemo zaključiti kako u ovom malom slanom jezeru postoji specifična mikroklima koja odgovara upravo ovim rijetkim vrstama. Potpuna regeneracija životne zajednice ukazuje i na samoodrživost ove ekstremne mikroklime slanog, krškog Rogozničkog jezera.The Rogoznica Lake is a small, marine, karstic lake 15 m deep and the size of 5300 square meters. There is no fresh water inflow or drifts because it is situated in a valley, protected from strong winds. Large seasonal amplitudes of salinity, temperature and dissolved organic matter make it an extreme habitat. It is a meromictic lake with anoxic deeper layers. The biological community of the lake consists of a small number of phytoplankton species and invertebrates. In late September of 1997 there was a sudden mixing of the water column that caused anoxic conditions in the whole lake. After this event, almost the whole population of the lakes phytoplankton had died. With numerous sampling the recovery of phytoplankton was monitored. The first examples of microflagellates appear already two weeks after the mass mortality in areas of increased concentration of ammonia. Subsequent oxidation of the water column and larger nitrate concentrations favored the creation of larger phytoplankton, such as diatoms and dinoflagellates. Sampling a year after shows that the lake´s phytoplankton populations fully recovered and is the same as before the anoxia. In the Rogoznica Lake there are two rare species: Prorocentrum arcuatum Issel and Hermesinum adriaticum Zaharis. These marine species of phytoplankton are rare throughout the Mediterranean. We can conclude that specific microclimate in the lake favoures the growth of those rare species. The complete regeneration of 14 phytoplankton communities indicates that the Rogoznica Lake, is a selfsustainable environment

Some ultrastructural features of the vesicular-arbuscular mycorrhiza in the grapevine

A morphological analysis using transmission and scanning electron microscopy was carried out about the mycorrhizal roots of grapevine, grown in the field. Only the most common features of the endophyte, i.e. intercellular hyphae and arbuscules, were studied. The intercellular hyphae spreading the infection showed a protoplasm endowed with nuclei, mitochondria, vacuoles with dense globules and bacteria-like microorganisms. After the penetration inside the host cell, many fungal branches were found. The so formed arbuscule filled up the whole cortical cell. The different stages of the arbuscular deterioration were observed and described. The infected host cell showed beyond the usual organelles plastids with starch in close contact with the endophyte. Quelques caractéristiques ultrastructurelles des mycorrhizes du type vésiculairearbusculaire de la vigne On a étudié au moyen de microscopes electroniques à transmission et à balayage la morphologie de racines mycorrhizées prélevées dans les vignobles. L'attention a été concentrée sur les formes les plus répandues de l'endophyte, c'est-à-dire les hyphes intercellulaires et les arbuscules. Les hyphes intercellulaires, qui transmettent l'infection, ont un protoplasme muni de noyaux, de mitochondries, de vacuoles avec des globules sombres aux électrons et des micro-organismes semblables à des bactéries. A la suite de la pénétration dans la cellule de la plante-hôte, l'hyphe forme plusieures branches. L'arbuscule ainsi formé remplit complètement la cellule corticale. On a pu observer et décrire les différentes phases de dégradation de l'arbuscule. Les cellules de l'hôte infectées montrent outre les organelles normales des plastides avec de l'amidon en contact étmit avec l'endophyte