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In a very simplified view, the plant leaf growth can be reduced to two processes, cell division and cell expansion, accompanied by expansion of their surrounding cell walls. The vacuole, as being the largest compartment of the plant cell, plays a major role in controlling the water balance of the plant. This is achieved by regulating the osmotic pressure, through import and export of solutes over the vacuolar membrane (the tonoplast) and by controlling the water channels, the aquaporins. Together with the control of cell wall relaxation, vacuolar osmotic pressure regulation is thought to play an important role in cell expansion, directly by providing cell volume and indirectly by providing ion and pH homestasis for the cytosoplasm. In this thesis the role of tonoplast protein coding genes in cell expansion in the model plant Arabidopsis thaliana is studied and genes which play a putative role in growth are identified. Since there is, to date, no clearly identified protein localization signal for the tonoplast, there is no possibility to perform genome-wide prediction of proteins localized to this compartment. Thus, a series of recent proteomic studies of the tonoplast were used to compile a list of cross-membrane tonoplast protein coding genes (117 genes), and other growth-related genes from notably the growth regulating factor (GRF) and expansin families were included (26 genes). For these genes a platform for high-throughput reverse transcription quantitative real time polymerase chain reaction (RT-qPCR) was developed by selecting specific primer pairs. To this end, a software tool (called QuantPrime, see http://www.quantprime.de) was developed that automatically designs such primers and tests their specificity in silico against whole transcriptomes and genomes, to avoid cross-hybridizations causing unspecific amplification. The RT-qPCR platform was used in an expression study in order to identify candidate growth related genes. Here, a growth-associative spatio-temporal leaf sampling strategy was used, targeting growing regions at high expansion developmental stages and comparing them to samples taken from non-expanding regions or stages of low expansion. Candidate growth related genes were identified after applying a template-based scoring analysis on the expression data, ranking the genes according to their association with leaf expansion. To analyze the functional involvement of these genes in leaf growth on a macroscopic scale, knockout mutants of the candidate growth related genes were screened for growth phenotypes. To this end, a system for non-invasive automated leaf growth phenotyping was established, based on a commercially available image capture and analysis system. A software package was developed for detailed developmental stage annotation of the images captured with the system, and an analysis pipeline was constructed for automated data pre-processing and statistical testing, including modeling and graph generation, for various growth-related phenotypes. Using this system, 24 knockout mutant lines were analyzed, and significant growth phenotypes were found for five different genes.
Die vorliegende Dissertation behandelt die Ökologie von Cnidium dubium (Schkuhr) Thell. (Sumpf-Brenndolde), Gratiola officinalis L. (Gottes-Gnadenkraut) und Juncus atratus Krocker (Schwarze Binse), drei gefährdeten Arten, die als sogenannte Stromtalpflanzen in Mitteleuropa in ihrem Vorkommen eng an die Flussauen gebunden sind. Die Arbeit basiert auf verschiedenen Simulationsexperimenten und Feldstudien in der Unteren Havelniederung, einem „Feuchtgebiet von internationaler Bedeutung“. Sie behandelt Themenkomplexe wie das Samenbankverhalten, die Samenkeimung, die Stickstofflimitierung, die Konkurrenzkraft, das Verhalten der Pflanzen nach einer Sommertrockenheit und nach einer Winter/Frühjahrsüberflutung. Ferner widmet sie sich der Populationsbiologie der Arten und dem Verhalten der Pflanzen nach besonderen Störungsereignissen wie Mahd, Herbivorie und der Sommerflut 2002. Der Leser erfährt, wie die Pflanzen in verschiedenen Lebensphasen auf die auentypische Umwelt reagieren und erhält umfassende Einblicke in physiologische Mechanismen, die der Anpassung an die typischen Bedingungen einer mitteleuropäischen Flussaue dienen. Eine Interpretation der Ergebnisse zeigt auf, welche der spezifischen Eigenschaften zur Gefährdung der drei Stromtalarten beitragen. Die Arbeit ist für den Arten-, Biotop- und Landschaftsschutz interessant. Darüber hinaus bietet sie zahlreiche Anknüpfungspunkte zur ökophysiologischen Grundlagenforschung. Die verstärkte Nutzung physiologischer Methoden bei der Klärung ökologischer Fragestellungen wird angeregt.