Biomedical subjects

Eberhard Schäfer

Publications and source records attributed to Eberhard Schäfer.

At least 19 recordsLinked to original sources

PubMed2006

bZIP10-LSD1 antagonism modulates basal defense and cell death in Arabidopsis following infection.

Plants use sophisticated strategies to balance responses to oxidative stress. Programmed cell death, including the hypersensitive response (HR) associated with successful pathogen recognition, is one cellular response regulated by reactive oxygen in various cellular contexts. The Arabidopsis basic leucine zipper (bZIP) transcription factor AtbZIP10 shuttles between the nucleus and the cytoplasm and binds consensus G- and C-box DNA sequences. Surprisingly, AtbZIP10 can be retained outside the nucleus by LSD1, a protein that protects Arabidopsis cells from death in the face of oxidative stress signals. We demonstrate that AtbZIP10 is a positive mediator of the uncontrolled cell death observed in lsd1 mutants. AtbZIP10 and LSD1 act antagonistically in both pathogen-induced HR and basal defense responses. LSD1 likely functions as a cellular hub, where its interaction with AtbZIP10 and additional, as yet unidentified, proteins contributes significantly to plant oxidative stress responses.

Apoptosis↗

PubMed2006

Photoactivated phytochrome induces rapid PIF3 phosphorylation prior to proteasome-mediated degradation.

Following light-induced nuclear translocation, specific members of the phytochrome (phy) photoreceptor family (phyA to phyE) interact with bHLH transcription factors, such as PIF3, and induce changes in target-gene expression. The biochemical mechanism comprising signal transfer from phy to PIF3 has remained undefined but results in rapid degradation of PIF3. We provide evidence that photoactivation of phy induces rapid in vivo phosphorylation of PIF3 preceding degradation. Both phyA and phyB redundantly induce this PIF3 phosphorylation, as well as nuclear speckle formation and degradation, by direct interaction with PIF3 via separate binding sites. These data suggest that phy-induced phosphorylation of proteins such as PIF3 may represent the primary intermolecular signaling transaction of the activated photoreceptor, tagging the target protein for proteosomal degradation, possibly in nuclear speckles.

Active Transport, Cell Nucleus↗

PubMed2006

FHY1 and FHL act together to mediate nuclear accumulation of the phytochrome A photoreceptor.

The phytochrome family of red/far-red photoreceptors is involved in the regulation of a wide range of developmental responses in plants. The Arabidopsis genome contains five phytochromes (phyA-E), among which phyA and phyB play the most important roles. Phytochromes localize to the cytosol in the dark and accumulate in the nucleus under light conditions, inducing specific phytochrome-mediated responses. Light-regulated nuclear accumulation of the phytochrome photoreceptors is therefore considered a key regulatory step of these pathways. In fact, one of the most severe phyA signaling mutants, fhy1 (far red elongated hypocotyl 1), is strongly affected in nuclear accumulation of phyA. The fhy1 fhl (fhy1 like) double mutant, lacking both FHY1 and its only close homolog FHL, is virtually blind to far-red light like phyA null seedlings. Here we show that FHL accounts for residual amounts of phyA in the nucleus in a fhy1 background and that nuclear accumulation of phyA is completely inhibited in an fhy1 FHL RNAi knock-down line. Moreover, we demonstrate that FHL and phyA interact with each other in a light-dependent manner and that they co-localize in light-induced nuclear speckles. We also identify a phyA-binding site at the C-terminus of FHY1 and FHL, and show that the N-terminal 406 amino acids of phyA are sufficient for the interaction with FHY1/FHL.

Arabidopsis↗

PubMed2006

CONSTITUTIVELY PHOTOMORPHOGENIC1 is required for the UV-B response in Arabidopsis.

CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) is a negative regulator of photomorphogenesis in Arabidopsis thaliana. COP1 functions as an E3 ubiquitin ligase, targeting select proteins for proteasomal degradation in plants as well as in mammals. Among its substrates is the basic domain/leucine zipper (bZIP) transcription factor ELONGATED HYPOCOTYL5 (HY5), one of the key regulators of photomorphogenesis under all light qualities, including UV-B responses required for tolerance to this environmental threat. Here, we report that, in contrast with the situation in visible light, COP1 is a critical positive regulator of responses to low levels of UV-B. We show that in the cop1-4 mutant, flavonoid accumulation and genome-wide expression changes in response to UV-B are blocked to a large extent. COP1 is required for HY5 gene activation, and both COP1 and HY5 proteins accumulate in the nucleus under supplementary UV-B. SUPPRESSOR OF PHYTOCHROME A-105 family proteins (SPA1 to SPA4) that are required for COP1 function in dark and visible light are not essential in the response to UV-B. We conclude that COP1 performs a specific and novel role in the plants' photomorphogenic response to UV-B, coordinating HY5-dependent and -independent pathways, which eventually results in UV-B tolerance.

Arabidopsis↗

PubMed2005

Nuclear accumulation of the phytochrome A photoreceptor requires FHY1.

The phytochrome family of red/far-red (R/FR)-responsive photoreceptors plays a key role throughout the life cycle of plants . Arabidopsis has five phytochromes, phyA-phyE, among which phyA and phyB play the most predominant functions . Light-regulated nuclear accumulation of the phytochromes is an important regulatory step of this pathway, but to this date no factor specifically required for this event has been identified . Among all phyA signaling mutants, fhy1 and fhy3 (far-red elongated hypocotyl 1 and 3) have the most severe hyposensitive phenotype, indicating that they play particularly important roles . FHY1 is a small plant-specific protein of unknown function localized both in the nucleus and the cytoplasm . Here we show that FHY1 is specifically required for the light-regulated nuclear accumulation of phyA but not phyB. Moreover, phyA accumulation is only slightly affected in fhy3, indicating that the diminished nuclear accumulation of phyA observed in fhy1 seedlings is not simply a general consequence of reduced phyA signaling. By in vitro pull-down and yeast two-hybrid analyses, we demonstrate that FHY1 physically interacts with phyA, preferentially in its active Pfr form. Furthermore, FHY1 and phyA colocalize in planta. We therefore identify the first component required for light-regulated phytochrome nuclear accumulation.

Arabidopsis↗

PubMed2005

Functional characterization of phytochrome interacting factor 3 for the Arabidopsis thaliana circadian clockwork.

Light, in a quality- and quantity-dependent fashion, induces nuclear import of the plant photoreceptors phytochromes and promotes interaction of these receptors with transcription factors including PHYTOCHROME INTERACTING FACTOR 3 (PIF3). PIF3 was shown to form in vitro a ternary complex with the G-box element of the promoters of LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and the Pfr conformer of phytochromes. CCA1 and LHY together with TIMING OF CAB EXPRESSION 1 (TOC1) constitute a transcriptional feed-back loop that is essential for a functional circadian clock in Arabidopsis. These findings led to the hypothesis that the PIF3-containing ternary complex regulates transcription of light-responsive genes and is involved in phototransduction to the central circadian clockwork. Here we report that (i) overexpression or lack of biologically functional PIF3 does not affect period length of rhythmic gene expression or red-light-induced resetting of the circadian clock and (ii) the transcription of PIF3 displays a low-amplitude circadian rhythm. We demonstrated previously that irradiation of etiolated seedlings induces rapid, phytochrome-controlled degradation of PIF3. Here we show that nuclear-localized PIF3 accumulates to relatively high levels by the end of the light phase in seedlings grown under diurnal conditions. Taken together, we show that (i) PIF3 does not play a significant role in controlling light input to and function of the circadian clockwork and (ii) a yet unknown mechanism limits phytochrome-induced degradation of PIF3 at the end of the day under diurnal conditions.

Arabidopsis↗

PubMed2005

Analysis of the function of the photoreceptors phytochrome B and phytochrome D in Nicotiana plumbaginifolia and Arabidopsis thaliana.

To investigate the mechanism of phytochrome action in vivo, NtPHYB, AtPHYB and phyD:green fluorescent protein (GFP) were overexpressed in Nicotiana plumbaginifolia and Arabidopsis thaliana. The expression of 35S:NtPHYB:GFP and 35S:AtPHYB:GFP complemented the tobacco hgl2 and Arabidopsis phyB-9 mutations, whereas the 35S:AtPHYD:GFP only rescued the hgl2 mutant. All three fusion proteins are transported into the nucleus in all genetic backgrounds. These data indicate that AtPHYD:GFP is biologically active and functions as the main red light receptor in transgenic tobacco, and establish an experimental system for the functional analysis of this elusive photoreceptor in vivo.

Active Transport, Cell Nucleus↗

PubMed2005

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.

Arabidopsis↗

PubMed2005

A new type of mutation in phytochrome A causes enhanced light sensitivity and alters the degradation and subcellular partitioning of the photoreceptor.

A specific light program consisting of multiple treatments with alternating red and far-red light pulses was used to isolate mutants in phytochrome A-dependent signal transduction pathways in Arabidopsis. Because of their phenotype, the mutants were called eid for empfindlicher im dunkelroten Licht, which means hypersensitive in far-red light. One of the isolated mutants, eid4, is a novel semi-dominant allele of the phytochrome A gene that carries a missense mutation in the chromophore-binding domain. The mutation did not change the photochemical properties of the photoreceptor, but it leads to an increased stability under light conditions that induce its rapid degradation. Fusion proteins with the green fluorescent protein exhibited clear alterations in subcellular localization of the mutated photoreceptor: The fusion protein was impaired in the formation of sequestered areas of phytochrome in the cytosol, which can explain its reduced light-dependent degradation. In contrast, the mutation stabilizes nuclear speckles (NUS) that appear late under continuous far-red light, whereas the formation of early, transiently appearing NUS remained more or less unaltered.

Amino Acid Sequence↗

PubMed2004

Phytochrome phosphorylation modulates light signaling by influencing the protein-protein interaction.

Plant photoreceptor phytochromes are phosphoproteins, but the question as to the functional role of phytochrome phosphorylation has remained to be elucidated. We investigated the functional role of phytochrome phosphorylation in plant light signaling using a Pfr-specific phosphorylation site mutant, Ser598Ala of oat (Avena sativa) phytochrome A (phyA). The transgenic Arabidopsis thaliana (phyA-201 background) plants with this mutant phyA showed hypersensitivity to light, suggesting that phytochrome phosphorylation at Serine-598 (Ser598) in the hinge region is involved in an inhibitory mechanism. The phosphorylation at Ser598 prevented its interaction with putative signal transducers, Nucleoside Diphosphate Kinase-2 and Phytochrome-Interacting Factor-3. These results suggest that phosphorylation in the hinge region of phytochromes serves as a signal-modulating site through the protein-protein interaction between phytochrome and its putative signal transducer proteins.

Avena↗

PubMed2004

The response regulator 2 mediates ethylene signalling and hormone signal integration in Arabidopsis.

Hormones are important regulators of plant growth and development. In Arabidopsis, perception of the phytohormones ethylene and cytokinin is accomplished by a family of sensor histidine kinases including ethylene-resistant (ETR) 1 and cytokinin-response (CRE) 1. We identified the Arabidopsis response regulator 2 (ARR2) as a signalling component functioning downstream of ETR1 in ethylene signal transduction. Analyses of loss-of-function and ARR2-overexpressing lines as well as functional assays in protoplasts indicate an important role of ARR2 in mediating ethylene responses. Additional investigations indicate that an ETR1-initiated phosphorelay regulates the transcription factor activity of ARR2. This mechanism may create a novel signal transfer from endoplasmic reticulum-associated ETR1 to the nucleus for the regulation of ethylene-response genes. Furthermore, global expression profiling revealed a complex ARR2-involving two-component network that interferes with a multitude of different signalling pathways and thereby contributes to the highly integrated signal processing machinery in higher plants.

Arabidopsis↗

PubMed2004

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 approximately 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.

Arabidopsis↗

PubMed2004

Genome-wide analysis of gene expression reveals function of the bZIP transcription factor HY5 in the UV-B response of Arabidopsis.

The light environment is a key factor that governs a multitude of developmental processes during the entire life cycle of plants. An important and increasing part of the incident sunlight encompasses a segment of the UV-B region (280-320 nm) that is not entirely absorbed by the ozone layer in the stratosphere of the earth. This portion of the solar radiation, which inevitably reaches the sessile plants, can act both as an environmental stress factor and an informational signal. To identify Arabidopsis genes involved in the UV response, we monitored the gene expression profile of UV-B-irradiated seedlings by using high-density oligonucleotide microarrays comprising almost the full Arabidopsis genome (>24,000 genes). A robust set of early low-level UV-B-responsive genes, 100 activated and 7 repressed, was identified. In all cases analyzed, UV-B induction was found to be independent of known photoreceptors. This group of genes is suggested to represent the molecular readout of the signaling cascade triggered by the elusive UV-B photoreceptor(s). Moreover, our analysis identified interactions between cellular responses to different UV-B ranges that led us to postulate the presence of partially distinct but interacting UV-B perception and signaling mechanisms. Finally, we demonstrate that the bZIP transcription factor HY5 is required for UV-B-mediated regulation of a subset of genes.

Arabidopsis↗

PubMed2003

Light perception and signalling in higher plants.

Plants monitor changes in the ambient light environment using sensory photoreceptor families: the phototropins and cryptochromes, which absorb UV-A or blue light; the phytochromes, which sense red/far-red light; and the UV-B photoreceptors, which have not yet been identified. Recent advances suggest that photoreceptor-induced signalling cascades regulate light-modulated gene expression. These regulatory networks interact at the levels of transcription, posttranslational modification and nucleo-cytoplasmic compartmentalisation.

Cryptochromes↗

PubMed2002

Inactivation of the phloem-specific Dof zinc finger gene DAG1 affects response to light and integrity of the testa of Arabidopsis seeds.

We show here that seeds from the knockout mutant of the Arabidopsis DAG1 gene encoding a Dof zinc finger transcription factor have an altered response to red and far-red light. Mutant dag1 seeds are induced to germinate by much lower red light fluence rates, and germination reaches more quickly a point where it is independent of phytochrome signaling. Moreover, although microscopic analysis reveals no obvious structural alterations in the seed coat (testa) of dag1 seeds, staining assays with different dyes point to an abnormal fragility of the testa. By extensive in situ mRNA hybridization analysis we show here that the gene, which is not expressed in the embryo, is specifically expressed in the phloem of all organs of the mother plant.

Arabidopsis↗

PubMed2002

The serine-rich N-terminal domain of oat phytochrome a helps regulate light responses and subnuclear localization of the photoreceptor.

Phytochrome (phy) A mediates two distinct photobiological responses in plants: the very-low-fluence response (VLFR), which can be saturated by short pulses of very-low-fluence light, and the high-irradiance response (HIR), which requires prolonged irradiation with higher fluences of far-red light (FR). To investigate whether the VLFR and HIR involve different domains within the phyA molecule, transgenic tobacco (Nicotiana tabacum cv Xanthi) and Arabidopsis seedlings expressing full-length (FL) and various deletion mutants of oat (Avena sativa) phyA were examined for their light sensitivity. Although most mutants were either partially active or inactive, a strong differential effect was observed for the Delta6-12 phyA mutant missing the serine-rich domain between amino acids 6 and 12. Delta6-12 phyA was as active as FL phyA for the VLFR of hypocotyl growth and cotyledon unfolding in Arabidopsis, and was hyperactive in the VLFR of hypocotyl growth and cotyledon unfolding in tobacco, and the VLFR blocking subsequent greening under white light in Arabidopsis. In contrast, Delta6-12 phyA showed a dominant-negative suppression of HIR in both species. In hypocotyl cells of Arabidopsis irradiated with FR phyA:green fluorescent protein (GFP) and Delta6-12 phyA:GFP fusions localized to the nucleus and coalesced into foci. The proportion of nuclei with abundant foci was enhanced by continuous compared with hourly FR provided at equal total fluence in FL phyA:GFP, and by Delta6-12 phyA mutation under hourly FR. We propose that the N-terminal serine-rich domain of phyA is involved in channeling downstream signaling via the VLFR or HIR pathways in different cellular contexts.

Anthocyanins↗

PubMed2002

Missense mutation in the PAS2 domain of phytochrome A impairs subnuclear localization and a subset of responses.

Phytochrome A signaling shows two photobiologically discrete outputs: so-called very-low-fluence responses (VLFR) and high-irradiance responses (HIR). By modifying previous screening protocols, we isolated two Arabidopsis mutants retaining VLFR and lacking HIR. Phytochrome A negatively or positively regulates phytochrome B signaling, depending on light conditions. These mutants retained the negative but lacked the positive regulation. Both mutants carry the novel phyA-302 allele, in which Glu-777 (a residue conserved in angiosperm phytochromes) changed to Lys in the PAS2 motif of the C-terminal domain. The phyA-302 mutants showed a 50% reduction in phytochrome A levels in darkness, but this difference was compensated for by greater stability under continuous far-red light. phyA-302:green fluorescent protein fusion proteins showed normal translocation from the cytosol to the nucleus under continuous far-red light but failed to produce nuclear spots, suggesting that nuclear speckles could be involved in HIR signaling and phytochrome A degradation. We propose that the PAS2 domain of phytochrome A is necessary to initiate signaling in HIR but not in VLFR, likely via interaction with a specific partner.

Alleles↗

PubMed2002

Nucleocytoplasmic partitioning of the plant photoreceptors phytochrome A, B, C, D, and E is regulated differentially by light and exhibits a diurnal rhythm.

The phytochrome family of plant photoreceptors has a central role in the adaptation of plant development to changes in ambient light conditions. The individual phytochrome species regulate different or partly overlapping physiological responses. We generated transgenic Arabidopsis plants expressing phytochrome A to E:green fluorescent protein (GFP) fusion proteins to assess the biological role of intracellular compartmentation of these photoreceptors in light-regulated signaling. We show that all phytochrome:GFP fusion proteins were imported into the nuclei. Translocation of these photoreceptors into the nuclei was regulated differentially by light. Light-induced accumulation of phytochrome species in the nuclei resulted in the formation of speckles. The appearance of these nuclear structures exhibited distinctly different kinetics, wavelengths, and fluence dependence and was regulated by a diurnal rhythm. Furthermore, we demonstrate that the import of mutant phytochrome B:GFP and phytochrome A:GFP fusion proteins, shown to be defective in signaling in vivo, is regulated by light but is not accompanied by the formation of speckles. These results suggest that (1) the differential regulation of the translocation of phytochrome A to E into nuclei plays a role in the specification of functions, and (2) the appearance of speckles is a functional feature of phytochrome-regulated signaling.

Active Transport, Cell Nucleus↗