Tim Kunkel

Constitutive Photomorphogenesis 1 and Multiple Photoreceptors Control Degradation of Phytochrome Interacting Factor 3, a Transcription Factor Required for Light Signaling in Arabidopsis

Light, in a quality- and quantity-dependent fashion, induces nuclear import of the plant photoreceptors phytochrome, promotes interaction of phytochrome A (phyA) and phyB with transcription factors including phytochrome interacting factor 3 (PIF3), and is thought to trigger a transcriptional cascade to regulate the expression of ∼2500 genes in Arabidopsis thaliana. Here, we show that controlled degradation of the transcription factor PIF3 is a major regulatory step in light signaling. We demonstrate that accumulation of PIF3 in the nucleus in dark requires constitutive photomorphogenesis 1 (COP1), a negative regulator of photomorphogenesis, and show that red (R) and far-red light (FR) induce rapid degradation of the PIF3 protein. This process is controlled by the concerted action of the R/FR absorbing phyA, phyB, and phyD photoreceptors, and it is not affected by COP1. Rapid light-induced degradation of PIF3 indicates that interaction of PIF3 with these phytochrome species is transient. In addition, we provide evidence that the poc1 mutant, a postulated PIF3 overexpressor that displays hypersensitivity to R but not to FR, lacks detectable amounts of the PIF3 protein. Thus, we propose that PIF3 acts transiently, and its major function is to mediate phytochrome-induced signaling during the developmental switch from skotomorphogenesis to photomorphogenesis and/or dark to light transitions.

Phytochrome-Specific Type 5 Phosphatase Controls Light Signal Flux by Enhancing Phytochrome Stability and Affinity for a Signal Transducer

Environmental light information such as quality, intensity, and duration in red (approximately 660 nm) and far-red (approximately 730 nm) wavelengths is perceived by phytochrome photoreceptors in plants, critically influencing almost all developmental strategies from germination to flowering. Phytochromes interconvert between red light-absorbing Pr and biologically functional far-red light-absorbing Pfr forms. To ensure optimal photoresponses in plants, the flux of light signal from Pfr-phytochromes should be tightly controlled. Phytochromes are phosphorylated at specific serine residues. We found that a type 5 protein phosphatase (PAPP5) specifically dephosphorylates biologically active Pfr-phytochromes and enhances phytochrome-mediated photoresponses. Depending on the specific serine residues dephosphorylated by PAPP5, phytochrome stability and affinity for a downstream signal transducer, NDPK2, were enhanced. Thus, phytochrome photoreceptors have developed an elaborate biochemical tuning mechanism for modulating the flux of light signal, employing variable phosphorylation states controlled by phosphorylation and PAPP5-mediated dephosphorylation as a mean to control phytochrome stability and affinity for downstream transducers.

Phosphorylation of Phytochrome B Inhibits Light-Induced Signaling via Accelerated Dark Reversion in Arabidopsis

This work shows that the photoreceptor phytochrome B is phosphorylated in vivo and demonstrates that this posttranslational modification inhibits red light–induced photomorphogenesis under nonsaturating light conditions by accelerating light-independent inactivation and dark reversion of the biologically active phyB conformer. The photoreceptor phytochrome B (phyB) interconverts between the biologically active Pfr (λmax = 730 nm) and inactive Pr (λmax = 660 nm) forms in a red/far-red–dependent fashion and regulates, as molecular switch, many aspects of light-dependent 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.

Interaction with plant transcription factors can mediate nuclear import of phytochrome B

Phytochromes (phy) are red/far-red–absorbing photoreceptors that regulate the adaption of plant growth and development to changes in ambient light conditions. The nuclear transport of the phytochromes upon light activation is regarded as a key step in phytochrome signaling. Although nuclear import of phyA is regulated by the transport facilitators far red elongated hypocotyl 1 (FHY1) and fhy1-like, an intrinsic nuclear localization signal was proposed to be involved in the nuclear accumulation of phyB. We recently showed that nuclear import of phytochromes can be analyzed in a cell-free system consisting of isolated nuclei of the unicellular green algae Acetabularia acetabulum. We now show that this system is also versatile to elucidate the mechanism of the nuclear transport of phyB. We tested the nuclear transport characteristics of full-length phyB as well as N- and C-terminal phyB fragments in vitro and showed that the nuclear import of phyB can be facilitated by phytochrome-interacting factor 3 (PIF3). In vivo measurements of phyB nuclear accumulation in the absence of PIF1, -3, -4, and -5 indicate that these PIFs are the major transport facilitators during the first hours of deetiolation. Under prolonged irradiations additional factors might be responsible for phyB nuclear transport in the plant.

A cell-free system for light-dependent nuclear import of phytochrome

Translocation from the cytosol to the nucleus is an essential step in phytochrome (phy) signal transduction. In the case of phytochrome A (phyA), this step occurs with the help of FHY1 (far-red-elongated hypocotyl 1), a specific transport protein. To investigate the components involved in phyA transport, we used a cell-free system that facilitates the controlled addition of transport factors. For this purpose, we isolated nuclei from the unicellular green algae Acetabularia acetabulum. These nuclei are up to 100 mum in diameter and allow easy detection of imported proteins. Experiments with isolated nuclei of Acetabularia showed that FHY1 is sufficient for phyA transport. The reconstituted system demonstrates all the characteristics of phytochrome transport in Arabidopsis thaliana. In addition, FHY1 was also actively exported from the nucleus, consistent with its role as a shuttle protein in plants. Therefore, we believe that isolated Acetabularia nuclei may be used as a general tool to study nuclear transport of plant proteins.

In Vivo Characterization of Chimeric Phytochromes in Yeast

Phytochromes are plant photoreceptors that play a major role in photomorphogenesis. Two members of the phytochrome family have been characterized in some detail. Phytochrome A, which controls very low fluence and high irradiance responses, is rapidly degraded in the light, forms sequestered areas of phytochrome (SAPs), and does not exhibit dark reversion in monocotyledonous seedlings. Phytochrome B mediates red/far-red reversible responses, is stable in the light, and does not form SAPs. We report on the behavior in yeast of the phytochrome apoproteins of rice PHYA, tobacco PHYB, and chimeric PHYAB and PHYBA and on the behavior of the respective holoprotein adducts after assembly with phycocyanobilin chromophore (PHY*). SAP-like formation in yeast was not observed for PHYB, but was detectable for PHYA, PHYAB, and PHYBA. Rice PHYA* did not undergo dark reversion in yeast. Surprisingly, all other tested phytochrome constructs did exhibit dark reversion, including chimeric phytochromes with a short N-terminal part of tobacco PHYB or parsley PHYA fused to rice PHYA. Furthermore, the proportion of phytochrome undergoing dark reversion and the rate of reversion were increased for both the N terminus-swapped constructs and PHYBA*. These results are discussed with respect to structure/function analysis of phytochromes A and B.

Optogenetic control shows that kinetic proofreading regulates the activity of the T cell receptor

The pivotal task of the immune system is to distinguish between self and foreign antigens. The kinetic proofreading model (KPR) proposes that T cells discriminate self from foreign ligands by the different ligand binding half-lives to the T cell receptor (TCR). It is challenging to test KPR as the available experimental systems fall short of only altering the binding half-lives and keeping other parameters of the ligand-TCR interaction unchanged. We engineered an optogenetic system using the plant photoreceptor phytochrome B to selectively control the dynamics of ligand binding to the TCR by light. Combining experiments with mathematical modeling we find that the ligand-TCR interaction half-life is the decisive factor for activating downstream TCR signaling, substantiating the KPR hypothesis. One Sentence Summary The half-life of the ligand-T cell receptor complex determines T cell activation.

Photo Quiz: Mysterious Objects in a Pleural Biopsy Sample from a Patient with Recurrent Pleural Empyema

A 66-year-old woman with a history of mitral valve disease was hospitalized because of pneumonia of the right lung with bilateral pleural effusions. Antibiotic therapy was initiated, and a chest tube was placed into the right pleural cavity. The patient responded well to the treatment and was discharged 3 weeks later. Echocardiography performed during hospitalization showed that mitral valve replacement was indicated, and 7 weeks later, the patient underwent minimally invasive surgery with implantation of an artificial heart valve. The operation was successful, but due to postoperative bleeding, an emergency thoracotomy with medial sternotomy became necessary. On the following day, the patient developed septic shock. An empyema of the left pleural cavity was identified as the most likely focus of infection, and pleural aspirates showed growth of Enterobacter cloacae, Enterococcus avium, Pseudomonas aeruginosa, and Candida albicans. Surgical revision including the placement of additional chest tubes and multiple lavages of the pleural cavity led to a clinical and radiological improvement. However, during the following weeks, the condition of the sternotomy wound deteriorated. A computed tomography (CT) scan showed signs of sternum osteomyelitis, and again, drainage of a distinct pleural empyema was required. Sternal swabs grew Candida albicans in large numbers. Because conservative therapy with fluconazole and vacuum-assisted closure systems failed to improve the osteomyelitis and the empyema could not be removed by drainage alone, rethoracotomy was performed on days 91 and 105, respectively. Biopsy specimens were obtained during the surgical removal of pleural fibrosis, and histological examination revealed several egg-like objects (Fig. 1). The patient had lived all his life in Germany and had traveled once to South Africa, 10 years previously. The differential blood count showed no eosinophilia, and IgE levels were normal. Sonography of the abdomen showed a slight hepatomegaly and normal findings for spleen and kidneys.