Die Rolle von AGD12 bei phototroper und gravitroper Signaltransduktion in Arabidopsis thaliana

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. On the one hand, AGD12 represents the actual link between NPH3 and various ARFs by enabling the cyclic GTP/GDP exchange to ARFs and thereby the formation of transport vesicles. Binding with EHB1 on the other hand, prevents this GTP/GDP exchange due to the lack of an Arf-GAP domain, which explains the role of EHB1 as a negative effector. The fact that also homologous proteins such as AGD11 and AGD13 show a positive influence on photo- and gravitropism suggests that far more proteins are involved in these and other physiological reactions. Therefore, probably the ratio between AGD proteins and EHB1-related CAR proteins determines the intensity of a reaction to various environmental stimuli such as light and gravity.