Forschung

Wassermangel, als Folge sich ändernder Umweltparameter, ist eines der immanentesten und schwerwiegendsten Probleme der Landwirtschaft. Dies betrifft natürlich nicht nur für Kulturpflanzen auf dem Ackerland, sondern auch für unsere Gesellschaft und deren wichtigste Ressource - die Nahrungsmittelversorgung. Konventionelle Züchtung die auf die Erzeugung dürre-toleranter und ertragreicher Pflanzen abzielt, hat in den letzten Jahrzehnten erhebliche Fortschritte erzielt. Unglücklicherweise ist diese Art der Züchtung äußerst zeitaufwändig und im Lichte der gegenwärtigen drastischen klimatischen Veränderungen, die wir auch in Mitteleuropa erleben, wären schnellere, innovative Lösungen von großem Vorteil. In einer Reihe von Ansätzen wurden und werden Kulturpflanzen, unter Zuhilfenahme von gentechnologischen Methoden, verändert, um eine verbesserte Anpassung an Umweltstressbedingungen zu ermöglichen. Aufgrund der momentanen Gesetzeslage und der begrenzten Akzeptanz in der Öffentlichkeit erscheint es jedoch unwahrscheinlich, dass GVO-Pflanzen in absehbarer Zeit ihren Weg auf lokales Ackerland finden werden. Aus diesem Grund wollen wir Ergebnisse aus der Grundlagenforschung verwenden, um dürre-verträgliche und GVO-freie Nutzpflanzen zu erzeugen. In einer engen Zusammenarbeit zwischen Kollegen vom BOKU-Campus Tulln und dem IST-Klosterneuburg sollen trockentolerante Sojabohnen erzeugt werden, indem die Aktivität einer pflanzenspezifischen, kleinen Familie von Genen modifiziert wird. Dieser Ansatz basiert auf modernster CRISPR/Cas9 Genom-Editierung Technologie und soll neuartige Sojavarietäten hervorbringen. Diese Linien werden dann sorgfältig analysiert, um jene Genkombination(en) zu ermitteln, die zu verbesserter Trockentoleranz beitragen, ohne weitere, wichtige agronomische Wachstumsparameter zu beeinflussen. Sobald der Nachweis der Trockentoleranz erbracht ist, sollen natürlich vorkommende genetische Soja-Varianten aus Sammlungen von Sojavarietäten identifiziert werden. Dies ermöglicht die Erzeugung von gentechnikfreiem und trockentolerantem Elite-Saatgut, das auch drastischen Veränderungen in unserer Umwelt standhalten sollte.

Wider research context / theoretical framework Climate change and its effects on the adaptability of plants to withstand longer drought periods is a highly topical research field. This project deals with the cellular response of higher plants to drought stress, with a special focus on the downregulation of PM associated key components of the absisic acid (ABA) signaling machinery. We intend to functionally characterize the involvement of the TOL (TOM1-like) protein family in degradative sorting of important ABA signaling components via the vacuolar pathway and the involvement of potential trans acting factors, like E3 ligases capable of the formation of K63-linked poly-ubiquitin chains. TOLs functions in the initial steps of ESCRT (Endosomal Sorting Complex Required for Transport)- dependent protein degradation and are thought to be responsible for initiating the pathway for guiding ubiquitinated PM proteins to the vacuole for their degradation. Hypotheses/research questions /objectives Recently a role for the ESCRT pathway in ABA signaling has been described and higher order tol mutant plant line, which resembles a wild type plant line of the same ecotype under standard conditions, is hypersensitive to the plant hormone ABA. In the next 3 years we intend to assess the crosstalk between the TOLs genes and the ABA signaling pathway. Approach/methods To link the function of TOL genes to the regulation of ABA responses we will assess genetic interaction of the TOLs with ABA-biosynthesis and signaling mutants. Subunits of the ESCRT complex involved in endosomal degradation of ABA receptors will be analyzed for interaction with the TOLs to reveal functional links. ABA transporters and receptors as well as TOL-reporter lines will be assessed with respect to their localization and abundance in different tol mutant backgrounds under drought stress or ABA signaling. The role of TOLs in the downregulation of ABA transporters and ABA receptors will be analyzes as well as indepth systematic localization study of all TOLs. Potential trans acting determinants affecting the role of TOLs in the ABA pathway will include the analysis of an E3 Ligase family capable of catalyzing K63 polyubiquitin chains. Thus, this project will elucidate how the TOL proteins, as initial components of the ESCRT machinery, function in modulating ABA signaling in plants. Level of originality/innovation In summary, this project will help to allow us to understand how TOLs, next to their general role in the endosomal degradation pathway, play a more differentiated role in diverse signaling pathways, allowing plants to fine-tune their responses. It will contribute to the question of how plants manage to adapt to their ever-changing environment, with a special focus on drought tolerance. Primary researchers involved This project will be used to fund two PhD students for three years each as well as a technician, who will mainly be responsible for the extensive plant work.

Rationale and significance Plants, being sessile organisms, have evolved a plethora of mechanisms to be able to respond quickly and accurately to their surroundings. The plasma membrane (PM), which forms the boundary between the extracellular environment and cellular constituents, is densely packed with a vast array of proteins involved in the sensing and transmitting of stimuli as well as transport processes. The regulation of protein abundance and localization at membranes is achieved by trafficking via the endomembrane system. The TOL (TOM1-like) proteins are responsible for initiating a pathway of guiding ubiquitinated PM proteins to the vacuole for their degradation, functioning as ubiquitin receptors in the early steps of the ESCRT (Endosomal Sorting Complex Required for Transport) pathway in plants [1-3]. Nevertheless, the precise function of the individual TOLs as well as how the TOL homologs and other ubiquitin adaptors are coordinated remains undescribed. There are apparent redundancies within the nine TOL family members, as single knock out plant lines show no obvious phenotype, while a higher order mutant exhibits severe pleiotropic defects. Another higher order TOL mutant, lacking apparent phenotypes under standard conditions, is hypersensitive to the plant hormone abscisic acid (ABA) (section 2.4), signifying that TOLs function discretely in separate pathways. Indications that TOLs perform specific, individual functions comes from their distinct C-terminal domains, their diverse expression patters as well as their different subcellular localization [1-4](section 2.1) and a distinctive sensitivity to drugs disrupting endosomal trafficking (section 2.3). TOLs additionally seem to be subjected to dynamic rearrangements, not only upon certain environmental stresses [1] (section 2.2), but also upon alterations in their post translational modifications [3]. We also observed re-localization of cytoplasmic TOLs to the PM in higher order mutants (section 2.1). In this project, we want to assess how the individual TOLs proteins function in the regulation of the abundance of PM proteins. This is signified by observations, indicating that next to a general role in the endosomal degradation pathway, individual TOLs may exhibit a more discriminating role in distinct signaling pathways, allowing them to fine-tune responses of plants to their ever-changing surroundings.