Final Report Summary - BRXTRAFFIC (Plasma membrane to nucleus trafficking of the transcriptional co-activator BRX - characterization of a novel hormone signaling pathway in plants)
The focus of the proposed research was to unravel how BRX, a highly conserved plant-specific transcriptional regulator, resides at the plasma membrane and how it is subsequently translocated to the nucleus upon auxin treatment. To investigate this potential novel auxin signaling pathway, we elaborated on the hypothesis that PRAF proteins would be instrumental for BRX localisation and translocation. Nevertheless, by microscopically analyzing transgenic lines, that were established during the first term of the fellowship, localisation of PRAF1 could be clearly demonstrated at the plasma-membrane and in the nucleus thus overlapping with BRX localization. In addition, Bimolecular Fluorescence Complementation (BiFC) assays also indicated that interactions occur between some PRAF family members and some BRX family members, mainly at the level of the plasma-membrane, supporting our initial hypothesis. PRAF proteins however did not follow BRX behaviour: attempts to change the localisation by auxin and/or MG132 treatment were unsuccesful. PRAF proteins possess FYVE and PH domains which are presumably important for membrane trafficking as they are lipid binding domains. However, our experiments could not confirm the importance of these domains for membrane binding as a dominant negative approach in which we mutagenised the PH domain did not change the localisation pattern of the PRAF protein: it was still observed at the PM and occasionally in the nucleus as the WT PRAF protein, in the Col-0 background. Interestengly, in the brx2 background however, a minor change in expression window (i.e. expression appears earlier than compared with the WT) and localisation (i.e. more cytosolic) was noted.
