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Functional analysis of PAT1

Subject Area Plant Physiology
Term from 2009 to 2014
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 163722927
 
Final Report Year 2014

Final Report Abstract

Phytochrome A and B (phyA, phyB) are red (R)/far-red (FR) light receptors in plants important for the regulation of growth and development as well as for the adaptation to the environment. Nuclear transport of phytochromes is a key step in light signalling, which is tightly regulated. Translocation of phyA from the cytosol into the nucleus requires FHY1, a small protein containing a nuclear localisation signal (NLS) and a phyA binding site. Preliminary work suggested that PAT1 interacts with FHY1 to regulate phyA nuclear transport and that this regulation is perturbed in the pat1-1 mutant, which is hyposensitive to FR due to strongly reduced phyA nuclear accumulation. The hypothesis was that environmental cues, such as light and temperature, or internal signals like hormones might regulate PAT1 and thereby phyA nuclear translocation. However, in our studies we could not confirm the interaction of PAT1 and FHY1 and in a yeast two hybrid screen we did not find any proteins related to light signalling that interact with PAT1. Unexpectedly, however, we found that the pat1-1 mutant, which has been characterised before, contains a second site mutation in FHY1. This mutation disrupts the phyA binding site and results in a non-functional FHY1 protein in pat1-1 leading to seedlings that are hyposensitive to far-red light. Based on these findings we conclude that PAT1 is not involved in far-red light signalling. The COP1/SPA E3-ubitquitin ligase complex inhibits light responses in dark-grown seedlings by targeting positive regulators of photomorphogenesis for degradation by the proteasome. These positively acting transcription factors are stabilised in light-grown seedlings, where phytochromes inactivate COP1/SPA by a yet unknown mechanism. To identify proteins involved in phytochrome-mediated inactivation of COP1/SPA we performed a yeast two hybrid screen for proteins binding to light-activated phyA. In this screen we found SPA1, a member of the SPA protein family, suggesting that phyA directly regulates COP1/SPA. We confirmed the interaction of phyA and SPA1 in plants and could show that also light-activated phyB binds to SPA1. Moreover, competition assays in yeast and plants demonstrate that phyA and phyB inhibit binding of COP1 to SPAs. The direct interaction between COP1 and SPAs is required for the E3-ubiquitin ligase activtiy of the COP1/SPA complex, suggesting that phytochromes promote the accumulation of positive regulators of photomorphogenesis by disrupting the COP1/SPA interaction. However, binding of phytochromes to COP1 prevents dissociation of the COP1/SPA complex even without COP1 and SPA directly interacting with each other. Thus, light-activated phytochromes trigger the remodelling of protein-protein interactions within the COP1-SPA complex leading to its inactivation in light-grown plants. These findings provide insight into how phytochormes and COP1/SPA regulate photomorphogenesis.

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