The work presented focussed on the elucidation of cytoplasmic phytochrome function. Physcomitrella patens, a lower plant model system, offered exquisite conditions to access investigation on the cytoplasmic signaling system underlying directional light sensing: a sequenced genome, excellent accessibility of molecular genetics, cellular and microscopic applications and developmental and physiological analysis.Studies using fluorescent fusions proofed phy4 localisation to be sensitive to tag-positioning in Physcomitrella, with predominant cytoplasmic localisation and the ability to enter the nucleus in a light-independent manner. Further localisation studies on higher plant phytochromes phyA and phyB in Physcomitrella and phy4 expression in higher plant cells revealed the existence of a phytochrome nuclear transport machinery in Physcomitrella. As both phyA and phy4 nuclear translocation was affected by tag-positioning this transport mechanism appeared to share features with the fhy1/fhl-transport mechanism acquired by angiosperms and may be considered a ancestor of phytochrome nuclear translocation. In silico analysis of the Physcomitrella genome pointed on a gene probably involved in this mechanism with considerable homology to the C-terminus of FHY1. The assembly of holo-phy4 on PCB-complemented medium resulted in functional phytochrome in yeast cells. Positioning of the BD-tag did not impair phytochrome function but affected binding of putative interactors. Using holo-BD:phy4 four putative interaction partners could be identified from a cDNA library, 2 of which exhibited state-dependent interaction and R/FR-reversibility in Y2H. In silico analysis characterised those putative interactors as (i) a transmembrane protein with ATP / GTP binding function (PLP), (ii) a WD40-domain protein (PRL), (iii) a protein involved in actin filaments binding and cytoskeleton assembly (EF1&
#945;) and (iv) an interactor of the heterotrimeric G-protein s alpha subunit (Pirin). Further analysis by sYFP confirmed in vivo interaction of all putative interactors with phy4 within the cytoplasm. Possible functions in phy4 cytoplasmic signaling, however, can only be deduced from in silico analysis and will need further elucidation on the physiological level by analysis of knockdown or knockout mutants. The notion of exclusively cytoplasmic GFP:phy4 and the establishment of functional holo-BD:phy4 in yeast encouraged the investigation of the connection between R and B in directional light sensing in both Physcomitrella and Arabidopsis by assuming a physical interaction between phytochrome and phototropin. Using Y2H assays an interaction of N-terminally tagged phy4 with any of the four described Physcomitrella phototropins was demonstrated, while the reciprocal C-terminal BD-fusion clearly inhibited direct binding of phy4 to phototropin. phy4-phot interaction was additionally shown to be strengthened in a R dependent manner and was further consolidated to be phytochrome-specific. sYFP methods proofed in vivo interaction and simultaneously revealed plasma membrane association of the phy-phot complex in accordance with phototropin localisation previously shown by fluorescent-fusions. The loss of directional R responses in phot knockout mutants confirmed physiological necessity of phy4-phot interaction at the plasma membrane. These results supported the hypothesis of phy4 s plasma membrane fixation with the help of phototropins, thereby not only fulfilling the requirements of the Jaffe/Etzold/Haupt hypothesis but also explaining the intrinsic connection between R and B signaling in directional light sensing of lower plants. The physical interaction of the two photoreceptors is also reflected by the occurrence of neochrome, a phytochrome - phototropin-chimera, twice in evolution. Since a connection of R and B in directional responses has been reported for higher plants too, consequently a phyA-phot1 interaction was hypothesised. Y2H analysis turned out with no direct phyA - phot1 interaction. Astonishingly, an in vivo interaction could still be demonstrated using sYFP methods. In accordance with the notion of the establishment of a more elaborate phytochrome system, especially in regard of phyA, an angiosperm-specific light-labile phytochrome, an indirect, more complex interaction between phyA and phot1 involving other proteins has to be assumed for higher plants. A possible candidate to mediate this complex formation is PKS1. Taken the results of this work together, phytochromes appear to fulfil different functions: (i) as integrators of light in regulation of gene expression by interaction with transcription factors within the nucleus, (ii) as sensors and mediators of directional responses connected to phototropins and thereby associated with the plasma membrane and a yet uncharacterised cytoplasmic signaling cascade and (iii) as regulators of genproduct abundance by tranlationally control as recently reported.
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