The circadian clock is an endogenous timer that oscillates with a period of approximately 24 hours and is reset upon environmental time cues such as the daily light/ dark or temperature cycles. Chlamydomonas reinhardtii is an ideal model organism for research on the circadian clock, because it shows several well-characterized behaviors that exhibit a circadian rhythm. Its circadian rhythm of phototaxis (swimming toward light) has been automated. Former action spectrum studies using the circadian phototaxis rhythm as an indicator surprisingly found that pulses of blue light were not effective in resetting the circadian clock of dark-adapted cells. This may have been because of the particular strain used in the study (the cell wall-deficient strain CW15). It may also have been due to the additional phase shift caused by the act of placing the cultures into the monitoring machine at particular times during their circadian cycle. This additional phase shift was most likely the result of the white background light present when monitoring the rhythm of phototaxis. The phototaxis monitoring process was improved by using narrow-wavelength LEDs specific for phototaxis as test lights and by omitting the background light between test light cycles. This study demonstrates that the modifications prevent any phase shifts due to the cultures being placed into the monitoring machine. Using a further improved experimental set-up and the wild-type strain CC124, this study unambiguously shows that blue light of 440nm is effective in resetting the circadian clock in Chlamydomonas reinhardtii. Because of this difference in blue light response to the earlier study, the action spectrum of the entire visible light range was also evaluated. Effective wavelengths for resetting the circadian clock in wild-type C. reinhardtii were found to occur at 400nm, 440nm, 540nm, and 640-660nm, corresponding to near UV-A, blue, green, and red light, respectively. With the exception of 440nm, these findings are congruent with previous action spectrum studies for the cell wall-deficient strain CW15.

In h?heren Pflanzen vermittelt der Blaulicht-sensitive Photorezeptor Phototropin verschiedene Reaktionen wie Phototropismus, Chloroplastendrehung und die ?ffnung von Schlie?zellen. Alle Reaktionen haben mit der Optimierung der pflanzlichen Lichtaufnahme und damit einer angepa?ten Photosynthese sowie der Vermeidung von Lichtsch?digung zu tun. In C.reinhardtii haben alle Phototropinreaktionen mit dem Sexualleben dieser Alge zu tun, d.h. Gametogenese, sexueller Kompetenz sowie der Keimung von Zygoten. Diese Doktorarbeit wurde Ende 2001 begonnen und verlief parallel zu den Arbeiten von Huang. Im Unterschied zu den Ergebnissen von Huang war das Chlamydomonas Phototropin in meinen H?nden unl?slich. Das Protein war nicht komplett membranassoziiert, sondern ein Teil des Proteins blieb immer l?slich. Die Phototropinmenge sowie die Verteilung waren in vegetativen Zellen, die unter Stark- oder Schwachlicht gewachsen waren, unterschiedlich. Deshalb wurde fš¹r diesen Unterschied Licht als wesentlicher Faktor verantwortlich gemacht. Jedoch zeigen verschiedene St?mme bei vegetativem Wachstum unter identischen Lichtbedingungen unterschiedliche Phototropinmengen. Das deutet auf weitere Faktoren hin, die Konzentration und Verteilung von Phototropin beeinflussen. In Chlamydomonas wurde neben dem Volll?ngenprodukt noch eine c-terminal verkš¹rzte Phototropinvariante gefunden. Licht wurde als Verursacher der Verkš¹rzung identifiziert. Aber nur lange Belichtungen von ca. 48 h fš¹hrten je nach Intensit?t zu klaren Abbaumustern. Fš¹r das Studium der Beteiligung des Phototropins am Sexualleben von Chlamydomonas, sollte ein Phot- Stamm generiert werden. Dazu wurde ein RNAi-Konstrukt hergestellt, mit dem es m?glich war, im Stamm cw15 arg- A das Phototropin bis auf 10% des Originalniveaus zu reduzieren. Leider funktionierte das Konstrukt in anderen St?mmen nicht zuverl?ssig. Im Stamm CC32pab1mt(+) konnte nur ein Klon mit einer Reduktion auf 15% des Originalniveaus erreicht werden. Au?erdem war die Silencing-Effizienz stark von den Wachstumsbedingungen abh?ngig. Die beste Reduktion wurde bei niedrigen Lichtintensit?ten gefunden. Es wurde ein weiterer Kreuzungstest etabliert, der fš¹r die Analyse der Zygotenkeimung verschiedene Vorteile gegenš¹ber bekannten Tests liefert. Aus den durchgefš¹hrten Tests konnte geschlossen werden, dass Licht auch ein wesentlicher Faktor fš¹r die Zygotenkeimung ist. Bei mittleren Lichtintensit?ten keimen Zygoten mit wenig Phototropin sp?ter. Starklicht kann diesen Mangel weitgehend kompensieren. Zur biochemischen Analyse des Phototropins in vitro war die Expression eines markierten Phototropins notwendig. Zur Analyse des Phototropinabbaus und fš¹r die sp?tere Reinigung wurde auch versucht, Phototropin in verschiedenen Gastorganismen zu exprimieren. In dieser Arbeit wurde Phototropin in Xenopus Oocyten und Diatom?en exprimiert. Diese Versuche haben best?tigt, dass das Phototropinabbauprodukt vom selben Gen wie das Volll?ngenprotein resultiert. Durch Expression der Phot-Mutante S57S/C250S konnte auch gezeigt werden, dass die Aktivierung des Phototropins keine Voraussetzung fš¹r den Abbau ist. Erstmalig konnte auch Phototropin als Fusionsprotein in Chlamydomonas exprimiert werden. Das Fusionsprotein wurde gereinigt und die Identit?t massenspektrometrisch verifiziert. Es wurde ein Stamm gefunden, der nur eine verkš¹rzte Variante des Phototropins exprimiert. Das Produkt war besser l?slich als die Volll?ngenversion. Eine Gro?produktion sollte fš¹r die Reinigung und nachfo! lgende Kristallisation angesetzt werden. Tandem Affinit?tsreinigungen sollten fš¹r die Identifizierung von Reaktionspartnern durchgefš¹hrt werden. / Abstract In higher plants, phototropin is in charge of phototropism (Liscum, 2002), chloroplast relocation (Wada et al., 2003) and stomatal opening (Schroeder et al., 2001). All its functions are connected with plants' modulation to the surrounding light conditions so that plants can make the best use of light for photosynthesis and dodge harmful strong light. In C.reinhardtii, all its reported functions are connected to the sexual life of this green alga, i.e. gametogenesis, maintenance of gamete competence and zygote germination. This PhD work started at the end of 2001 and went on in parallel with Huang's work. Different from Huang's observation that phototropin was insoluble (Huang et al., 2002), it was found that phototropin existed not only as a membrane associated protein, a portion of phototropin always remained soluble. Phototropin levels and distributions were different between vegetative cells grown in strong light or in darkness. Light was thought to be the essential factor that caused this difference. But, different strains grown vegetatively under same light conditions showed that the levels of phototropin and its distributions varied. This suggested the existence of other factors in the determination of its level and distribution. A C-terminus degradation product of phototropin was found as a stable component in C. reinhardtii. Light was found as a reason that caused the degradation. However, short time of illumination with strong light (up to 2 h) did not evoke the degradation machinery. In light gradient experiment, long time illumination (~48h) with different light intensity showed a clear degradation pattern. To study phototropin involvement in Chlamydomonas sexual life, a Phot1- strain was needed. An RNAi phototropin construct was made. It managed to reduce the level of phototropin in strain cw15 arg- A down to 10% of its original level. However, the construct did not work properly in other strains. Only one transformant of strain CC32pab1mt(+) with a reduction of the phototropin level to around 15% of the original level was found. Under different growth conditions, the silencing efficiency varied. The best silencing result appeared when cells were grown under low light conditions. A new mating assay was established in this work, which has many benefits over the traditional way of studying zygote germination. The conclusion was drawn from this assay that light was the primary factor that determines zygote germination; under moderate light conditions, zygotes with higher phototropin level would germinate earlier; strong illumination could compensate the difference caused by the low phototropin level in zygote germination. For phototropin function analysis in vitro, a Chlamydomonas strain that over-expressed phototropin was in need. To prove that the suspected degradation product originated from the phototropin gene and to purify phototropin for crystallization, trials to express C.r. phototropin cDNA in different organisms were also made. Phototropin was expressed in Xenopus oocytes and diatom in this work. The expression pattern confirmed that both the full-length and the suspected degradation product did originate from the same phototropin gene. It was also found that the degradation was independent on phototropin activation state by expressing C.r. Phot1 (C57S, C250S) in oocytes. For the first time, recombinant phototropin got expressed in Chlamydomonas by fusion expression strategy. The fusion product was purified and the identity was confirmed by Mass Spectrometry analysis. One strain which expressed only a C-terminus truncation version of the fusion product was found. Compared with the full length product, this mutant had better solubility and was easier to be purified. Large scale of purification should be performed to obtain enough material for crystallographic studies. Tandem affinity purification (TAP) tag should also be performed in Chlamydomonas proteomic studies about phototropin.

Die einzellige Grünalge Chlamydomonas reinhardtii ist ein vielseitiger Modellorganismus sowohl in der Grundlagenforschung als auch für biotechnologische Anwendung. Für die genetische Veränderung wurden verschiedene Methoden entwickelt, jedoch ist die gezielte Modifikation kerncodierter Gene immernoch sehr schwierig. In dieser Arbeit wird eine Strategie vorgestellt, die es ermöglicht, kerncodierte Gene in Chlamydomonas gezielt mit sequenzspezifischen Zinkfinger-Nukleasen zu verändern. Das COP3-Gen, welches den lichtaktivierbaren Ionenkanal Kanalrhodopsin-1 codiert, diente hierbei als Zielsequenz der für die Deletion hergestellten Zinkfinger-Nukleasen. Um eine Charakterisierung der ZFNs zu ermöglichen, wurde ein Modelstamm generiert, der ein inaktiviertes Markergen enthält. Die Inaktiverung erfolgte hierbei durch Insertion der COP3-ZFN Zielsequenz. Die Transformation dieses Modellstamms mit ZFN codierender Plasmid-DNA und einem Reparatur-Template ermöglichte die Wiederherstellung der Markeraktivität und eine Selektion Antibiotika-resistenter Kolonien. Wenn in diesen Experimenten zusätzlich ein COP3 veränderndes Template benutzt wurde, enthielt 1% der analysierten Klone ein mutiertes COP3-Gen. Der Chlamydomonas Augenfleck ist ein lichtsensitives Organell mit entscheidender Funktion für die phototaktische Orientierung der Alge. Eine Deletionsmutante des Blaulicht-Photorezeptors Phototropin zeigte in Experimenten eine veränderte Regulation der lichtabhängigen Augenfleckgröße. Durch Komplementierung der Phototropin-Dysfunktion konnte der lichtabhängige Regulationsprozess wiederhergestellt werden. Die Expression der Phototropin-Kinasedomäne führte zu einer lichtunabhängigen Reduktion der Augenfleckfläche. Interessanterweise führte auch die Expression der N-terminalen LOV-Domänen zu einer geänderten Regulation des Augenflecks und der Phototaxis. Dies deutet, zusätzlich zur Lichtregulation der Kinasedomäne, auf eine zelluläre Signalfunktion der LOV-Domänen hin. / The unicellular green alga Chlamydomonas reinhardtii is a versatile model for fundamental and biotechnological research. A wide toolset for genetic manipulation has been developed for this alga, but specific modification of nuclear genes is still not routinely possible. Here we present a nuclear gene targeting strategy for Chlamydomonas that is based on the application of zinc-finger nucleases (ZFNs). Initially, we designed a set of ZFNs for targeting the COP3 gene that encodes the light-activated ion channel channelrhodopsin-1. To evaluate the designed ZFNs, we constructed a model strain by inserting a non-functional selection marker interspaced with a short COP3 target sequence into the nuclear genome. Upon co-transformation of this recipient strain with the engineered ZFNs and a DNA repair template, we were able to restore marker activity and select antibiotic resistant clones with active nucleases. In cases where cells were co-transformed with a modified COP3 template, 1% of these clones contained a modified COP3 locus as well. The eyespot of Chlamydomonas is a light-sensitive organelle important for phototactic orientation of the alga. Here we found that eyespot size is downregulated in light. In a strain in which the blue light photoreceptor phototropin was deleted, the light regulation of the eyespot size was affected. We restored this dysfunction in different phototropin complementation experiments. Complementation with the phototropin kinase fragment reduced the eyespot size, independent of light. Interestingly, overexpression of the N-terminal LOV-domains alone also affected eyespot size and phototaxis, suggesting that aside from activation of the kinase domain, they fulfill an independent signaling function in the cell. We propose that phototropin is a light regulator of phototaxis that desensitizes the eyespot when blue light intensities increase.

<p>Plants use an array of photoreceptors to measure the quality, quantity, and direction of light in order to respond to ever-changing light environments. Photoreceptors not only determine how and when individual plants complete their life cycles, but they also have a profound and long-term macroevolutionary influence on species diversification. Despite their significances, very little is known about photoreceptors across plants as whole, and we lack a comprehensive view of photoreceptor evolution. </p><p> In my dissertation, I investigate the origin and evolution of three of the most prominent photoreceptor gene families in plants: phytochromes, phototropins and neochromes. Using newly available transcriptomic and genomic data, I completed the first in-depth survey of these photoreceptor families across land plants, green algae, red algae, glaucophytes, cryptophytes, haptophytes, and stramenopiles. </p><p> Phytochromes are red/far-red photoreceptors that play essential roles in seed germination, seedling photomorphogenesis, shade-avoidance, dormancy, circadian rhythm, phototropism, and flowering. Here, I show that the canonical plant phytochromes originated in a common ancestor of streptophytes (charophyte green algae plus land plants), and I identify the most likely sequence whereby the plant phytochrome structure evolved from its ancestral phytochrome. Phytochromes in charophyte algae are structurally diverse, including canonical and non-canonical forms, whereas in land plants, phytochrome structure is highly conserved. Liverworts, hornworts, and Selaginella apparently possess a single phytochrome gene copy, whereas independent gene duplications occurred within mosses, lycopods, ferns, and seed plants, leading to diverse phytochrome families in these clades. My detailed phylogeny encompasses all of green plants and enables me to not only uncover new phytochrome lineages, but also to make links to our current understanding of phytochrome function in Arabidopsis and Physcomitrella (the major model organism outside of flowering plants). Based on this robust evolutionary framework, I propose new hypotheses and discuss future directions to study phytochrome mechanisms.</p><p> Phototropins are blue-light photoreceptors that regulate key adaptive physiological responses, including shoot-positive phototropism, root-negative phototropism, chloroplast accumulation/avoidance, stomatal opening, circadian rhythm, leaf expansion, and seedling elongation I show that phototropins originated in the common ancestor of Viridiplantae (all green algae [charophytes, chlorophytes, prasinophytes] plus land plants). Phototropins repeatedly underwent independent duplications in all major plant lineages (mosses, lycopods, ferns and seed plants), except for liverworts and hornworts, where phototropin is a single-copy gene. Following each major duplication event, phototropins subsequently differentiated in parallel, resulting in two specialized (yet partially overlapping) functional forms that primarily mediate either low- or high-light responses. My gene phylogeny further suggests that phototropins have co-evolved with phytochromes, as is evident from their molecular interactions and strikingly similar gene duplication patterns. I hypothesize that the co-evolution of phototropins with phytochromes, together with their subsequent convergent functional divergences in phototropic responses, contributed to the success of plants in adapting to diverse and heterogeneous habitats.</p><p> Neochromes are chimeric photoreceptors that, by fusing phytochrome and phototropin modules into a single protein, are able to use both red/far-red and blue light to modulate phototropic responses. Neochromes were first discovered in ferns, and the evolution of neochromes was implicated as a key innovation that facilitated fern diversification under the low-light angiosperm canopies. Despite its significance from an evolutionary standpoint, the origin of neochromes has remained a mystery. Here I present the first evidence for neochrome in hornworts (a bryophyte lineage) and demonstrate that ferns acquired neochrome from hornworts via horizontal gene transfer (HGT). Fern neochromes are nested within hornwort neochromes in my large-scale phylogenetic reconstructions of phototropin and phytochrome gene families. Divergence date estimates further support the HGT hypothesis, with fern and hornwort neochromes diverging 179 MYA, long after the split between the two plant lineages (at least 400 MYA). By analyzing the draft genome of the Anthoceros punctatus hornwort, I also discovered a novel phototropin gene that likely represents the ancestral lineage of the neochrome phototropin module. Thus, a neochrome originating in hornworts was horizontally transferred to ferns, where it may have played a significant role in the diversification of modern ferns. </p><p> In summary, my studies identified the molecular origins of phytochromes, phototropins and neochromes, and reconstructed their respective evolutionary histories. This new framework for photoreceptor evolution will stimulate new research linking ecology, evolution, and photochemistry to understand how plants adapt to variable light environments.</p> / Dissertation

The main aim of this diploma thesis was creation of functional experiments and supporting teaching materials for high school students which would clearly demonstrate the processes of photomorphogenesis in plants. Proposed experiments had to be verified in practice on the basis of their reliability, repeatability and reproducibility in schools. It was necessary to drew up the educational materials for each experiment focused on observation of photomorphogenesis plant's reactions. These materials give students the basics of scientific work and specifically they increase the interest in experimental plant biology. The first part of the thesis is literary introduction that provides the theoretical background to the topic of photomorphogenesis for teachers. It summarizes the current of the art mechanisms and manifestations of photomorphogenesis processes of plants. The second didactic part of the introduction deals with the framing up teaching of plant physiology into current curricula of education in the Czech Republic. For the formativ of educational materials experiments had to be designed to demonstrate reliably basic photomorphogenetical processes. Three experiments were designe dat: 1. plant phototropism, 2. negative plant phototropism and 3. plant scotomorphogenesis.To select these experiments I...

Die lichtaktivierte Kinase Phototropin aus Chlamydomonas reinhardtii, die photoaktivierte Adenylatcyclase (PAC) aus Euglena gracilis und das BLUF-Protein Slr1694 aus Synechocystis sp. PCC 6803 wurden in Hinblick auf die molekularen Details der primären photochemischen Prozesse sowie der Signalweiterleitung untersucht. Phototropin wurde mit Hilfe von Arginin aus Escherichia coli in Milligramm Mengen isoliert. Ohne Arginin wurde E. coli cAMP Rezeptorprotein assoziiert aufgefunden, welches eine hohe Homologie zu einer cAMP aktivierten Kinase aus C. reinhardtii besitzt. Volllängen Phototropin bildet wie einzelne LOV-Domänenkonstrukte ohne Kinasedomäne den Flavin-Triplettzustand und das kovalente Cysteinyl-Addukt. Der Zerfall des Signalzustandes ist in Anwesenheit von ATP beschleunigt und deutet auf Photorezeptor-Kinase Interaktion hin. Strukturelle Änderungen in der Kinasedomäne wurden durch FTIR-Differenzspektroskopie gezeigt. Über ELDOR-Spektroskopie wurde der Abstand der Photorezeptordomänen auf etwa 25 Angstrom bestimmt. Mutationen in Slr1694 an S28, N31 und W91 zeigten keine konservierten Einfluss auf die Dynamik des Signalzustands. Die Entfernung der Seitenkette von S28 führte zu einer 15 nm Rotverschiebung des Absorptionsspektrums aufgrund veränderter Wasserstoffbrückenkoordination des Kofaktors. Die Einführung von positiv geladenen Seitenketten an Stelle von N31 erhöhte die Kofaktorbindung von phosphorylierten Flavinen. Künstliche Kofaktoren wie Roseoflavin konnten in Slr1694 durch Koexpression eines prokaryotischen Flavintransporters erreicht werden. Die Rolle von M152 in PAC für die Signalweiterleitung wurde anhand der lichtaktivierten cAMP Synthese-Aktivität gezeigt. Durch ultraschnelle IR-Spektroskopie wurde die Beteiligung der Seitenketten von Y8 sowie Q50 bestätigt und eine genauere Beschreibung der Wasserstoffbrücken im langlebigen Signalzustand ermöglicht. / The light activated kinase Phototropin from Chlamydomonas reinhardtii, the photoactivated adenylylcyclase (PAC) from Euglena gracilis and the BLUF protein Slr1694 from Synechocystis sp. PCC 6803 were investigated concerning the molecular details of the primary photochemistry as well as signal transduction. Phototropin was isolated from Escherichia coli in mg amounts after solubilization with arginine. Without arginine E. coli cAMP receptor protein, which shows high homology to a cAMP activated kinase from C. reinhardtii, was copurified. Full length Phototropin shows similar photochemistry to LOV-domain containing proteins without the kinase including triplet and covalent cysteinyl adduct formation. Signaling state decay is accelerated in the presence of ATP and suggests photoreceptor-kinase interaction. FTIR spectroscopy showed light induced structural changes in the kinase domain. The distance of the photoreceptor domains of 25 Angstrom was determined by ELDOR spectroscopy. Mutation of the side chains of S28, N31 and W91 in Slr1694 showed no conserved influence on the dynamic of the signaling state. Removal of the hydroxyl group of S28 lead to a 15 nm red shift of the absorption spectrum as a result of altered hydrogen bond coordination of the cofactor. Introduction of positively charged side chains at the position of N31 strengthened the binding of phosphorylated flavins. An artificial flavin like roseoflavin was introduced in Slr1694 by coexpression of a bacterial flavin transporter. The essential role of M152 in PAC for signal transduction was shown by determination of light activated cAMP synthesis activity. Ultrafast IR spectroscopy confirmed the contribution of Y8 and Q50 in the photocycle and gave a more detailed description of the hydrogen bonding situation in the signaling state.