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Zusammenfassung:
The small molecule auxin is a major morphogen in plants. It is indispensable for organ initiation, tropic growth responses and cell-cycle control. Here we report the engineering of a FRET-based biosensor for auxin by redesigning the binding pocket of the E. coli tryptophan repressor. This sensor enables us to visualize auxin directly and with high temporal and spatial resolution.Traditionally auxin localization is inferred from the expression of reporter genes under the control of the auxin-inducible promoter DR5. The presence of reporter signal is usually referred to as an auxin-response maximum, and it is assumed that each reporter-signal maximum represents a local auxin maximum. Quantitative measurements indicate that this is not necessarily the case. We employed the similarities between auxin and tryptophan to develop an auxin sensor based on Förster resonance energy transfer (FRET) by semi-rational redesign of an established tryptophan sensor. We improved the IAA binding, diminished the binding to IAA-related molecules, optimized the fluorophore pair, and improved the linkers between the sensory domains and the fluorophores. The final version exhibits a 3-fold change of the FRET ratio upon treatment with 50 μM IAA, which was shown to be the upper limit of the IAA concentration in roots inferred from root protoplasting.As a first step to confirm the functionality of the IAA sensor in planta, we expressed the sensor transiently in Arabidopsis protoplasts and showed that the sensor is functional in planta. With plants stably expressing the sensor we monitor the fast uptake and clearance of auxin by the plant at an unprecedented timescale. The sensor allows the dynamic visualization of auxin distribution patterns previously inaccessible for genetics and biochemistry thus increasing our understanding of a major developmental regulator during the life-cycle of the plant.
