2017 The growth of plants directed by light or gravity is referred as phototropism or gravitropism. They are generally subdivided into stimulus perception, signal transduction and a final phototropic or gravitropic reaction. In higher plants such as Arabidopsis thaliana, light is detected by the blue-light receptor phototropin. The perception of gravity, on the other hand, is supported by the sedimenta-tion of starch-containing statoliths. But the genuine gravity receptor is presently unknown. Further details on the subsequent early signal transduction of photo- and gravitropism are also unknown. Only the ARF-controlled redistribution of PIN proteins via the trans-Golgi network is the next known intermediate step in the common signal transduction of photo- and gravitropism. The results are changes of the auxin transport, the development of an auxin gradient, and asymmetric growth, final-ly. The outcome of this work shows that AGD12 and EHB1 act as a positive and negative effector of pho-to- and gravitrope signal transduction. Thus, it was shown that the loss-of-function mutant agd12-9 compared with wildtype has a reduced photo- and gravitropism. A phenotype that is contrary to the hypergravitropic mutant ehb1-2. In addition, it was shown that both proteins interact in vivo with the NPH3 protein essential for phototropism. Similarly, yeast two-hybrid system interactions of AGD12 and EHB1 with various ARF proteins were demonstrated in vivo, which are essential for the common signal propagation of photo- and gravitropism. Within the scope of this work, these findings were confirmed by confocal microscopy studies on seedlings expressing AGD12:GFP and EHB1:GFP consti-tutively. Both fusion proteins localized as a result of treatment with Brefeldin A in so-called BFA compartments, suggesting an association of AGD12 and EHB1 within the trans-Golgi network. Thus the demonstrated interaction with NPH3 and various ARFs is supported by a common localization with AGD12 and EHB1. In silico modeled Ca2+ binding sites within AGD12 and EHB1 suggest that their function can be modulated by Ca2+. In parallel, the addition of Ca2+ into the growth medium showed that the gravitropism of plants without AGD12 and EHB1 were far less sensitive to fluctuations in the external Ca2+ concentration. Therefore, both proteins are likely involved in the early gravitropic signal transduction via Ca2+ signals. The instantaneous increase of the cytosolic Ca2+ concentration after gravity stimulation could lead to Ca2+ binding to AGD12 and EHB1, which gets functionally modulated and initiate further signal transduction. It is to be assumed that both proteins are in competition with the respective binding to NPH3 and various ARFs, resulting in their opposing effect on photo- and gravitropisms. 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Philipps-Universität Marburg https://archiv.ub.uni-marburg.de/diss/z2017/0667/cover.png Life sciences Biowissenschaften, Biologie 2017-10-09 Die Rolle von AGD12 bei phototroper und gravitroper Signaltransduktion in Arabidopsis thaliana doctoralThesis The role of AGD12 in phototrope and gravitrope signal tranduction in Arabidopsis thaliana Durch Licht oder Schwerkraft induziertes gerichtetes Wachstum von Pflanzen wird als Phototro-pismus bzw. Gravitropismus bezeichnet. Diese werden unterteilt in Reizwahrnehmung, Signalweiter-leitung und einer abschließenden photo- bzw. gravitropen Reaktion. In höheren Pflanzen wie Arabidopsis thaliana erfolgt die Wahrnehmung von Licht über den Blaulicht-Photorezeptor Phototro-pin. Die Wahrnehmung von Schwerkraft wird hingegen durch die Sedimentation stärkehaltiger Statolithen unterstützt, wobei der tatsächliche Schwerkraftrezeptor gegenwärtig unbekannt ist. Wei-tere Details zur daran anschließenden frühen Signalweiterleitung von Photo- und Gravitropismus sind ebenso unbekannt. Erst die durch ARFs gesteuerte Umverteilung von PIN-Proteinen über das Trans-Golgi-Netzwerk bildet den nächsten bekannten Zwischenschritt in der gemeinsamen Signaltransduk-tion von Photo- und Gravitropismus. Das Resultat ist eine Verlagerung des Auxintransports, Aufbau eines Auxingradienten und schließlich asymmetrisches Wachstum. Die Resultate dieser Arbeit verdeutlichen, dass AGD12 und EHB1 als positiver bzw. negativer Effektor der photo- und gravitropen Signaltransduktion fungieren. So konnte gezeigt werden, dass eine agd12 loss-of-function Mutante im Vergleich zum Wildtyp einen verringerten Photo- und Gravitropismus aufweist. Ein Phänotyp der diametral zur hypergravitropen ehb1-2-Mutante ist. Zudem konnte ge-zeigt werden, dass beide Proteine in vivo mit dem für Phototropismus essentiellen NPH3-Protein interagieren. Ebenfalls wurden mittels Hefe-Zwei-Hybrid-System Interaktionen von AGD12 und EHB1 mit diversen ARF Proteinen in vivo nachgewiesen, welche essentiell für die gemeinsame Signalweiter-leitung von Photo- und Gravitropismus sind. Im Rahmen dieser Arbeit wurden diese Befunde durch konfokalmikroskopische Untersuchungen an Keimlingen untermauert, die AGD12:GFP und EHB1:GFP konstitutiv exprimieren. So lokalisierten beide Fusionsproteine als Folge einer Behandlung mit Bre-feldin A in sogenannten BFA-Kompartimenten, was auf eine Assoziation von AGD12 und EHB1 mit dem Trans-Golgi-Netzwerk schließen lässt. Damit wird die nachgewiesene Interaktion mit NPH3 und diversen ARFs durch eine gemeinsame Lokalisation mit AGD12 und EHB1 gestützt. In silico modellier-te Ca2+-Bindestellen bei AGD12 und EHB1 legen nahe, dass ihre Funktion durch Ca2+ moduliert werden kann. Parallel dazu konnte durch Zugabe von Ca2+ in das Wachstumsmedium gezeigt werden, dass der Gravitropismus von Pflanzen ohne AGD12 und EHB1 weit geringer durch Schwankungen der externen Ca2+-Konzentration beeinflusst wird. Daher ist zu vermuten, dass beide Proteine über Ca2+-Signale an der frühen gravitropen Signaltransduktion beteiligt sind. Dabei könnte die unmittelbare Erhöhung der cytosolischen Ca+2-Konzentration nach Schwerkraftstimulus zu einer Ca2+-Bindung an AGD12 und EHB1 führen, deren Funktionalität modulieren und so die Signaltransduktion fortgeführt werden. Es ist anzunehmen, dass beide Proteine um die jeweilige Bindung an NPH3 und diversen ARFs in Kon-kurrenz stehen und sich daraus ihre gegensätzliche Wirkung auf die Tropismen ergibt. So stellt AGD12 die eigentliche Verbindung zwischen NPH3 und diversen ARFs dar, indem es den zyklischen GTP/GDP-Austausch an ARFs und somit die Bildung von Transportvesikeln ermöglicht. Eine Bindung mit EHB1 hingegen unterbindet diesen GTP/GDP-Austausch aufgrund der fehlenden Arf-GAP-Domäne, was die Rolle von EHB1 als negativen Effektor erklärt. Der Umstand, dass homologe Protei-ne wie AGD11 und AGD13 ebenso positiven Einfluss auf Photo- und Gravitropismus ausüben, lässt vermuten, dass weit mehr Proteine an diesen und anderen physiologischen Reaktionen beteiligt sind. Demnach bestimmt das Verhältnis zwischen AGD-Proteinen und den EHB1-verwandten CAR-Proteinen die Intensität einer Reaktion auf diverse Umweltreize wie Licht und Schwerkraft. 2017-10-04 Phototrapismus https://doi.org/10.17192/z2017.0667 Biologie opus:7743 2017-09-21 Fachbereich Biologie 172 application/pdf Michalski, Christian Michalski Christian urn:nbn:de:hebis:04-z2017-06673 monograph German Gravitropismus