Physiological and Microscopical Evidences for the Contribution of Abscisic Acid and Reactive Oxygen Species on Arabidopsis Thaliana’s Root Gravitropism

This thesis aimed to confirm the hypotheses of light exposition and ABA signalling to modulate the gravitational response of Arabidopsis thaliana roots. Light is known to induce photophobic behaviour in roots and to strengthen the gravitational response of root tips. Therefore light’s influence on the root curvature of a Col-0 wildtype and a 6tuple ABAreceptor- mutant, which is extremely insensitive to abscisic acid, was investigated. Roots were either exposed to light or darkness and gravistimulated for one to four hours by 90 or 180°. The usage of an ABA-signalling-mutant further allowed to draw conclusions on the effect of ABA in gravitropism, which is a controversial question. Furthermore it was investigated, if there is an interaction of the downstream signal transduction chains of ABA and reactive oxygen species (ROS) in gravitropism. From literature it is already known that a lack of ROS results in a loss of root gravitropism and an interaction of both substances is already known for drought induced stomata closure. To specify the particular ROS involved in gravitropism and if gradients between lower and upper halves of horizontal growing roots support the gravitational response, hydrogen peroxide and superoxide - concentration alterations were analysed after one to four hours during 90 or 180° gravistimulation. To confirm the hypotheses that light and ABA signalling modulate the gravitational response of Arabidopsis thaliana roots, gravistimulation was performed by displacement of the roots to 90 or 180° from the vertical position. The influence of light was investigated by exposing roots to light or darkness. Thereby induced gravitropic bending responses were studied for four hours under different light conditions. The response of wildtype plant roots to the 90° treatment seems to be light modulated. While darkness inhibits root curvature, light promotes the gravitropic bending into the direction of the gravitational vector in wildtype and 6tuple ABA-receptor-mutant roots. In accordance to existing literature, those results clearly demonstrate that Arabidopsis thaliana roots behave photophobic. In contrast, 180° gravistimulation of wildtype roots does not result in any alterations in gravitropic bending of plants subjected to light or dark conditions. The response of the investigated plant roots to a 180° deviation from the gravitational vector seems to be independent from light. This is a currently unknown phenomenon, because we expected phototropism being superior to gravitropism. Compared to the wildtype, the 6tuple ABA-receptor-mutant exhibited a stronger reduced gravitational response after two, three and four hours of 180° stimulation in darkness compared to light. Therefore, a modification of the gravitational response of the mutant obviously depends on light. These results demonstrate that light leads to a partial compensation of the alterations between the Arabidopsis wildtype and the 6tuple ABA-receptor-mutant with respect to their bending behaviours during the gravitational responses after 90 or 180° stimulation. If darkness is combined with 180° stimulation, the inhibition of the curvature, induced by the lack of proper ABA-signalling in the mutant, is stronger as in light. The observed and statistically evaluated reduction in the growth rate of the mutant roots subjected to 180° stimulation supports the resulting phenotype. The second part of experiments conducted in this thesis aimed to clarify if ROS gradients influence the curvature of gravistimulated plant roots and to investigate the general role of these radicals during gravitropic bending. Gravistimulation of the wildtype and the ABAreceptor- mutant was performed by displacement of the roots to 90 or 180° and ROS were visualized with NBT (Superoxide) and DCFH2-DA (Hydrogenperoxide). Alterations in gradients between the upper and lower halves are clearly shown in the wildtype and the mutant subjected to four hours of a 90° stimulus but not for 180°. Interestingly the gradients are not matching between the two plant groups, meaning that there are differences between the concentrations of ROS in the upper and lower halves of horizontal roots at different points of time between the mutant and the wildtype. For 90° stimulation it was shown, that there exists no mutant phenotype due to changes of the gravitational vector, so it is concluded that altered gradients of the reactive oxygen species hydrogen peroxide and superoxide - induced by this treatment do not play an essential role in gravitropism. These results might be partly controversial to already published data, demonstrating that ROS may co localize with increased auxin concentrations on the lower half of the horizontally growing roots during gravistimulation. We conclude another important function of ROS during the gravitational response instead of the establishment of gradients between upper and lower halves of gravistimulated plant roots. Maybe other, not yet analysed, ROS like hyperoxide are more important for the gravitropic bending. In addition, we visualized a translocation of hydrogenperoxide from the gravistimulated root tip to the elongation zone (EZ) of upper and lower halves of the root in the course of time. These changes were not evaluated yet. Maybe this transport of hydrogen peroxide influences the gravitropic response. This should be part of future experiments to understand ROS’ contribution to the gravitropic bending of roots more in detail. Furthermore we can conclude, that other interaction molecules of ABA- signalling have to be responsible for the 6tuple ABA-receptor-mutant’s phenotype during 180° gravistimulation, because no changes in the ROS gradients between upper and lower halves of the root were detected during this four hour treatment. Finally it has to be considered that although 6 ABA receptors are knocked-out, there are still 4 active receptors which perhaps partly compensate the possible reduction of ABA signalling. A 10 tuple ABA-receptor-mutant, if it is not lethal, should clarify the inhibiting effect of ABA plant root gravitropism

ARADISH - Development of a Standardized Plant Growth Chamber for Experiments in Gravitational Biology Using Ground-Based Facilities

Plant development strongly relies on environ- mental conditions. Growth of plants in Biological Life Support Systems (BLSS), which are a necessity to allow human survival during long-term space exploration missions, poses a particular problem for plant growth, as in addition to the traditional environmental factors, microgravity (or reduced gravity such as on Moon or Mars) and limited gas exchange hamper plant growth. Studying the effects of reduced gravity on plants requires real or simulated microgravity experiments under highly standardized conditions, in order to avoid the influence of other environmental factors. Analysis of a large number of biological replicates, which is necessary for the detection of subtle phenotypical differences, can so far only be achieved in Ground-Based Facilities (GBF). Besides different experimental conditions, the usage of a variety of different plant growth chambers was a major factor that led to a lack of reproducibility and comparability in previous studies. We have developed a flexible and customizable plant growth chamber, called ARAbidopsis DISH (ARADISH), which allows plant growth from seed to seedling, being realized in a hydroponic system or on Agar. By developing a special holder, the ARADISH can be used for experiments with Arabidopsis thaliana or a plant with a similar habitus on common GBF hardware, including 2D clinostats and Random Positioning Machines (RPM). The ARADISH growth chamber has a controlled illumination system of red and blue light emitting diodes (LED), which allows the user to apply defined light conditions. As a proof of concept we tested a prototype in a proteomic experiment in which plants were exposed to simulated microgravity or a 90◦ stimulus. We optimized the design and performed viability tests after several days of growth in the hardware that underline the utility of ARADISH in microgravity research

The fungal-specific beta-glucan-binding lectin FGB1 alters cell-wall composition and suppresses glucan-triggered immunity in plants

beta-glucans are well-known modulators of the immune system in mammals but little is known about beta-glucan triggered immunity in planta. Here we show by isothermal titration calorimetry, circular dichroism spectroscopy and nuclear magnetic resonance spectroscopy that the FGB1 gene from the root endophyte Piriformospora indica encodes for a secreted fungal-specific beta-glucan-binding lectin with dual function. This lectin has the potential to both alter fungal cell wall composition and properties, and to efficiently suppress beta-glucan-triggered immunity in different plant hosts, such as Arabidopsis, barley and Nicotiana benthamiana. Our results hint at the existence of fungal effectors that deregulate innate sensing of beta-glucan in plants