Developmentally regulated expression of the BRI1 brassinosteroid receptor in Arabidopsis thaliana

Brassinosteroids (BRs) are important regulators of morphogenic events during plant development. The lack of active transport and well-characterized biosynthesis offer ideal conditions for studying the local and temporal effects of this hormone group. While recent studies have found clear coincidence between the sites of BR accumulation and organ differentiation, they have also provided evidence for developmental changes in hormone susceptibility. In order to investigate the role of the BR receptor BRI1 in the modulation of hormone sensitivity, we studied the time course and localization of BRI1 gene activity in Arabidopsis seedlings. To this end, we generated transgenic lines carrying BRI1 promoter-driven luciferase or GUS reporter genes and characterized the expression patterns of these chimeric genes. Our results showed increased BRI1 expression in dark grown seedlings, particularly in the elongation zone of the hypocotyl, and also at the sites of organ development in green seedlings. These data indicate that, in addition to local increases in the hormone level, the abundance of the receptor can also be instrumental in eliciting the BR response

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

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

A fitokrómok N-terminális régiójának szerepe a vörös és távoli vörös fény érzékelésében

Munkánk céljául a fitokróm fotoreceptorok (PHY) N-terminális NTE régiójának Arabidopsis thaliana növényben betöltött szerepének tanulmányozását tűztük ki. A fitokrómok vörös és távoli vörös fényt érzékelő fotoreceptorok, melyek a központi szerepet játszanak a növények egész életében a fényfüggő fejlődési folyamatok szabályozásában: a csírázástól egészen a virágzásig. Arabidopsis-ban a fitokrómok egy kis, öttagú géncsaládot alkotnak (PHYA - PHYE), melyek közül PHYA és a PHYB rendelkeznek kiemelkedő szereppel. Az elmúlt két évtizedben mutagenizált magpopulációk vizsgálatával próbálták azonosítani a fitokrómokról kiinduló jelátviteli lánc elemeit. Meglepő módon ezeknek a munkáknak nem sikerült azonosítani egy hosszú sok elemből álló, állati rendszerekre jellemző jelátviteli hálózatot, melynek létét a fitokrómok szerteágazó hatásai alapján feltételezték. Ezzel szemben nagy számú mutáns fitokóm allélt (SNP) azonosítottak. Ezeknek a megváltozott jelátvitellel rendelkező fitokróm változatoknak a vizsgálata arra utalt, hogy a fitokrómok jelátvitelének szabályozása nagyrészt a magán a molekulán történik. Ennek eredményeként a fitokrómok poszttranszlációs módosításai (pl.: foszforiláció, szumoiláció) és sejtmagi importjuk szabályozása váltak a fitokrómok közvetítette fényérzékelés kutatásának központi kérdéseivé. Az tézisemben bemutatott munka első felében karakterizáltuk a phyA-5 hiposzenzitív PHYA mutánst, melynek fiziológiai és molekuláris vizsgálatának segítségével bizonyítottuk, hogy az NTE régióban elhelyezkedő, evolúciósan konzervált Ala30 aminosav oldallánc fontos szerepet játszik a PHYA sejtmagi importját végző ― FHY1 és FHL ― molekulákkal való kölcsönhatás létrehozásában. Az Ala30 és környezete nagy valószínűséggel része annak a fehérjefelszínnek, mely a PHYA sejtmagi transzporterekkel való kölcsönhatás fizikai felszínét biztosítja és a PHYA fényaktivációjával, ― a Pfr forma kialakulásával ― válik funkcionálissá. A munka második felében a PHYB NTE régiójában elhelyezkedő, kétszikűek körében konzervált Ser86 aminosav oldallánc foszforilálciójának vörös fényérzékelésre gyakorolt hatását vizsgáltuk. A fiziológiai és molekuláris vizsgálatokhoz célzott mutagenezissel előállított, a Ser86 foszforilált (PHYB(Ser86Asp)) és defoszforilált (PHYB(Ser86Ala)) állapotait utánzó molekulákat fejeztettünk phyB null mutáns növényekben. A vizsgálatok meggyőzően bizonyították, hogy az aktív PHYB (Pfr) molekulák inaktivációjának sebessége erősen függ a Ser86 oldallánc foszforiláltságának állapotától. A PHYB(Ser86Asp) esetében a relaxáció olyan mértékben felgyorsul, ami már összemérhető a fotokonverzió ― Pfr molekulák keletkezésének ― sebességével ezzel hiposzenzitív viselkedést idézve elő. A PHYB(Ser86Ala) változat ezzel ellentétben hiperszenzitivitást idéz elő a relaxáció drasztikus csökkenésén keresztül. Élesztőben termeltetett PHYB változatok segítségével bizonyítottuk, hogy a Ser86 aminosav foszforilációs állapota valóban megváltoztatja a PHYB inaktivációjának sebességét

A short amino-terminal part of Arabidopsis phytochrome A induces constitutive photomorphogenic response

Phytochrome A (phyA) is the dominant photoreceptor of far-red light sensing in Arabidopsis thaliana. phyA accumulates at high levels in the cytoplasm of etiolated seedlings, and light-induced phyA signaling is mediated by a complex regulatory network. This includes light- and FHY1/FHL protein-dependent translocation of native phyA into the nucleus in vivo. It has also been shown that a short N-terminal fragment of phyA (PHYA406) is sufficient to phenocopy this highly regulated cellular process in vitro. To test the biological activity of this N-terminal fragment of phyA in planta, we produced transgenic phyA-201 plants expressing the PHYA406-YFP (YELLOW FLUORESCENT PROTEIN)-DD, PHYA406-YFP-DD-NLS (nuclear localization signal), and PHYA406-YFP-DD-NES (nuclear export signal) fusion proteins. Here, we report that PHYA406-YFP-DD is imported into the nucleus and this process is partially light-dependent whereas PHYA406-YFP-DD-NLS and PHYA406-YFP-DD-NES display the expected constitutive localization patterns. Our results show that these truncated phyA proteins are light-stable, they trigger a constitutive photomorphogenic-like response when localized in the nuclei, and neither of them induces proper phyA signaling. We demonstrate that in vitro and in vivo PHYA406 Pfr and Pr bind COP1, a general repressor of photomorphogenesis, and co-localize with it in nuclear bodies. Thus, we conclude that, in planta, the truncated PHYA406 proteins inactivate COP1 in the nuclei in a light-independent fashion

The AAA-ATPase molecular chaperone Cdc48/p97 disassembles sumoylated centromeres, decondenses heterochromatin, and activates ribosomal RNA genes

Centromeres mediate chromosome segregation and are defined by the centromere-specific histone H3 variant (CenH3)/centromere protein A (CENP-A). Removal of CenH3 from centromeres is a general property of terminally differentiated cells, and the persistence of CenH3 increases the risk of diseases such as cancer. However, active mechanisms of centromere disassembly are unknown. Nondividing Arabidopsis pollen vegetative cells, which transport engulfed sperm by extended tip growth, undergo loss of CenH3; centromeric heterochromatin decondensation; and bulk activation of silent rRNA genes, accompanied by their translocation into the nucleolus. Here, we show that these processes are blocked by mutations in the evolutionarily conserved AAA-ATPase molecular chaperone, CDC48A, homologous to yeast Cdc48 and human p97 proteins, both of which are implicated in ubiquitin/small ubiquitin-like modifier (SUMO)-targeted protein degradation. We demonstrate that CDC48A physically associates with its heterodimeric cofactor UFD1-NPL4, known to bind ubiquitin and SUMO, as well as with SUMO1-modified CenH3 and mutations in NPL4 phenocopy cdc48a mutations. In WT vegetative cell nuclei, genetically unlinked ribosomal DNA (rDNA) loci are uniquely clustered together within the nucleolus and all major rRNA gene variants, including those rDNA variants silenced in leaves, are transcribed. In cdc48a mutant vegetative cell nuclei, however, these rDNA loci frequently colocalized with condensed centromeric heterochromatin at the external periphery of the nucleolus. Our results indicate that the CDC48ANPL4 complex actively removes sumoylated CenH3 from centromeres and disrupts centromeric heterochromatin to release bulk rRNA genes into the nucleolus for ribosome production, which fuels single nucleus-driven pollen tube growth and is essential for plant reproduction