Fitokróm-A irányította jelátviteli hálózatok Arabidopsis thaliana-ban = Phytochrome A controlled signalling networks in Arabidopsis thaliana

Megállapítottuk, hogy rövid PHYA N-terminális fragmentumok képesek az FHY1/FHL fehérjékkel konformációfüggő módon kölcsönhatásba lépni és fényindukált módon a sejtmagba transzportálódni. Kimutattuk, hogy ezek a PHYA N-terminális fragmentumok azonban nem képesek funkcionális fotoreceptorként működni, és hogy a PHYA fényindukált proteolízisét és sejtmagba irányuló transzportját a fehérje különböző doménjei közvetítik. Nevezetesen az első 406 aminosav alkotta fragmentum elégséges a PHYA fotoreceptor fényindukált sejtmagi transzportjához, míg a 406-686 aminosavakat tartalmazó PHYA fragmentum szükséges a fotoreceptor fényindukált proteolíziséhez. A PHYA fotoreceptort tartalmazó sejtmagi komplexek jellemzésére olyan mutánsokat izoláltunk, amelyekben a PHYA komplexek kialakulása markánsan eltér a vad típusban leírttól. Genetikai térképezéssel és szekvenálással bizonyítottuk, hogy a mutánsok egy csoportjában a mutációk nem a PHYA génben lokalizáltak. E mutánsokban a fúziós PHYA fehérje ugyan degradálódik fény hatására, de a mutánsok hiposzenzitívek távoli vörös fényben, és nem mutathatók ki PHYA-tartalmú sejtmagi komplexek. Mostanra befejeztük hat mutáns részletes genetikai térképezést és az egyes mutációkat pozícióját kb. 150-200 kb-nyi pontossággal behatároltuk. A genetikai térképezés alapján biztosak vagyunk abban, hogy ezek a mutációk olyan géneket érintenek, amelyek eddig nem voltak összefüggésbe hozhatók a PHYA-tartalmú sejtmagi komplexek kialakulásával és működésével. | We provided evidence that, although they cannot function as active photoreceptors, short N-terminal fragments of the photoreceptor PHYA interact in a conformation-dependent fashion with the transport proteins FHY1-FHL and translocate into the nucleus in a light-induced manner. In addition we documented that light-induced nuclear import and rapid degradation of these PHYA N-terminal fragments can be separated and mediated by different domains of the molecule. The short, far N-terminal 406 fragment is sufficient for light-induced nuclear import, whereas the domain between 406-686 is required for light-induced rapid degradation of the PHYA protein. We performed a novel genetic screen to isolate mutants displaying aberrant formation of nuclear bodies containing the photoreceptor PHYA. We provided evidence by genetic mapping and DNA sequencing that in a group of mutants the mutations do not localise in the PHYA gene. In these mutants light induces rapid degradation of the PHYA/YFP fusion protein, yet the mutants are hyposensitive to far-red light and we were unable to detect formation of PHYA/YFP containing nuclear bodies. We have completed detailed genetic mapping of these lines and positioned the mutations to 150-200 kb genomic fragments. Based on their map positions we conclude that these mutations will define novel components required for the formation or stabilization of PHYA-containing nuclear bodies

Phosphorylation of phytochrome B inhibits light-induced signaling via accelerated dark reversion in Arabidopsis

The photoreceptor phytochrome B (phyB) interconverts between the biologically active Pfr (lmax = 730 nm) and inactive Pr (lmax = 660 nm) forms in a red/far-red–dependent fashion and regulates, as molecular switch, many aspects of lightdependent development in Arabidopsis thaliana. phyB signaling is launched by the biologically active Pfr conformer and mediated by specific protein–protein interactions between phyB Pfr and its downstream regulatory partners, whereas conversion of Pfr to Pr terminates signaling. Here, we provide evidence that phyB is phosphorylated in planta at Ser-86 located in the N-terminal domain of the photoreceptor. Analysis of phyB-9 transgenic plants expressing phospho-mimic and nonphosphorylatable phyB–yellow fluorescent protein (YFP) fusions demonstrated that phosphorylation of Ser-86 negatively regulates all physiological responses tested. The Ser86Asp and Ser86Ala substitutions do not affect stability, photoconversion, and spectral properties of the photoreceptor, but light-independent relaxation of the phyBSer86Asp Pfr into Pr, also termed dark reversion, is strongly enhanced both in vivo and in vitro. Faster dark reversion attenuates red light–induced nuclear import and interaction of phyBSer86Asp-YFP Pfr with the negative regulator PHYTOCHROME INTERACTING FACTOR3 compared with phyB–green fluorescent protein. These data suggest that accelerated inactivation of the photoreceptor phyB via phosphorylation of Ser-86 represents a new paradigm for modulating phytochrome-controlled signaling

A fitokróm fotoreceptorok indukálta jelátviteli lánc molekuláris vizsgálata Arabidopsis thalianában = Molecular analysis of phytochrome induced signal transduction in Arabidopsis thaliana

Bebizonyítottuk, hogy a fitokróm-A sejtmagba történő transzlokációjában és sejtmagi akkumulációjában, tehát a fitokróm-A szabályozta jelátviteli lánc aktiválásában az FHY1 fehérje meghatározó szerepet játszik. Kimutattuk, hogy a fitokróm-A autofoszforilációja negatívan szabályozza a jelátviteli lánc aktivitását és azonosítottunk egy olyan foszfatázt, ami specifikusan a fitokróm-A fotoreceptor defoszforilációjáért felelős. Bebizonyítottuk, hogy a PIF3 fehérje nem pozitív, hanem negatív regulátora a fotomorfogenezisnek, a PIF3 fehérje nem szükséges a cirkadián óra működéséhez, továbbá, hogy fény hatására a PIF3 fehérje gyorsan degradálódik és hogy a fitokróm- A-B és D fotoreceptorok együttesen szabályozzák a PIF3 transzkripciós faktor fény indukálta degradációját. | We have provided compelling evidence that nuclear translocation and accumulation of phytochrome-A requires the FHY1 protein and that FHY1 interacts with the phyA molecule in a conformation dependent fashion and this interaction is mediated by the very N-terminal domain of the photoreceptor. We demonstrated that PIF3 transcription factor negatively regulates phytochrome controlled photomorphogenesis, its function is not required for light dependent entrainment of the plant circadian clock and that phytochrome-A in harmony with phytochrome-B and D regulates light induced degradation of PIF3. Moreover, we showed that interaction of phytochrome-A with various components of the phytochrome-A dependent signalling cascade is negatively regulated by the autophosphorylation of the photoreceptor and that dephosphorylation of the photoreceptor is mediated by a specific phosphatase

Altered Dark- and Photoconversion of Phytochrome B Mediate Extreme Light Sensitivity and Loss of Photoreversibility of the phyB-401 Mutant

The phyB-401 mutant is 103 fold more sensitive to red light than its wild-type analogue and shows loss of photoreversibility of hypocotyl growth inhibition. The phyB-401 photoreceptor displays normal spectral properties and shows almost no dark reversion when expressed in yeast cells. To gain insight into the molecular mechanism underlying this complex phenotype, we generated transgenic lines expressing the mutant and wild-type phyB in phyB-9 background. Analysis of these transgenic lines demonstrated that the mutant photoreceptor displays a reduced rate of dark-reversion but normal Pfr to Pr photoconversion in vivo and shows an altered pattern of association/dissociation with nuclear bodies compared to wild-type phyB. In addition we show (i) an enhanced responsiveness to far-red light for hypocotyl growth inhibition and CAB2 expression and (ii) that far-red light mediated photoreversibility of red light induced responses, including inhibition of hypocotyl growth, formation of nuclear bodies and induction of CAB2 expression is reduced in these transgenic lines. We hypothesize that the incomplete photoreversibility of signalling is due to the fact that far-red light induced photoconversion of the chromophore is at least partially uncoupled from the Pfr to Pr conformation change of the protein. It follows that the phyB-401 photoreceptor retains a Pfr-like structure (Pr*) for a few hours after the far-red light treatment. The greatly reduced rate of dark reversion and the formation of a biologically active Pr* conformer satisfactorily explain the complex phenotype of the phyB-401 mutant and suggest that amino acid residues surrounding the position 564 G play an important role in fine-tuning phyB signalling