Biomedical subjects

Monika Dieterle

Publications and source records attributed to Monika Dieterle.

12 recordsLinked to original sources

PubMed2006

Tobacco WLIM1 is a novel F-actin binding protein involved in actin cytoskeleton remodeling.

We used confocal microscopy and in vitro analyses to show that Nicotiana tabacum WLIM1, a LIM domain protein related to animal Cys-rich proteins, is a novel actin binding protein in plants. Green fluorescent protein (GFP)-tagged WLIM1 protein accumulated in the nucleus and cytoplasm of tobacco BY2 cells. It associated predominantly with actin cytoskeleton, as demonstrated by colabeling and treatment with actin-depolymerizing latrunculin B. High-speed cosedimentation assays revealed the ability of WLIM1 to bind directly to actin filaments with high affinity. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed a highly dynamic in vivo interaction of WLIM1-GFP with actin filaments. Expression of WLIM1-GFP in BY2 cells significantly delayed depolymerization of the actin cytoskeleton induced by latrunculin B treatment. WLIM1 also stabilized actin filaments in vitro. Importantly, expression of WLIM1-GFP in Nicotiana benthamiana leaves induces significant changes in actin cytoskeleton organization, specifically, fewer and thicker actin bundles than in control cells, suggesting that WLIM1 functions as an actin bundling protein. This hypothesis was confirmed by low-speed cosedimentation assays and direct observation of F-actin bundles that formed in vitro in the presence of WLIM1. Taken together, these data identify WLIM1 as a novel actin binding protein that increases actin cytoskeleton stability by promoting bundling of actin filaments.

Actin Cytoskeleton↗

PubMed2006

CUL4 associates with DDB1 and DET1 and its downregulation affects diverse aspects of development in Arabidopsis thaliana.

Cullins are central scaffolding subunits in eukaryotic E3 ligases that facilitate the ubiquitination of target proteins. Arabidopsis contains at least 11 cullin proteins but only a few of them have been assigned biological roles. In this work Arabidopsis cullin 4 is shown to assemble with DDB1, RBX1, DET1 and DDB2 in vitro and in planta. In addition, by using T-DNA insertion and CUL4 antisense lines we demonstrate that corresponding mutants are severely affected in different aspects of development. Reduced CUL4 expression leads to a reduced number of lateral roots, and to abnormal vascular tissue and stomatal development. Furthermore, cul4 mutants display a weak constitutive photomorphogenic phenotype. These results therefore assign an important function to CUL4 during plant development and provide strong evidence that CUL4 assembles together with RBX1 and DDB1 proteins to form a functional E3 ligase in Arabidopsis.

Arabidopsis↗

PubMed2006

F-box-like domain in the polerovirus protein P0 is required for silencing suppressor function.

Plants employ small RNA-mediated posttranscriptional gene silencing as a virus defense mechanism. In response, plant viruses encode proteins that can suppress RNA silencing, but the mode of action of most such proteins is poorly understood. Here, we show that the silencing suppressor protein P0 of two Arabidopsis-infecting poleroviruses interacts by means of a conserved minimal F-box motif with Arabidopsis thaliana orthologs of S-phase kinase-related protein 1 (SKP1), a component of the SCF family of ubiquitin E3 ligases. Point mutations in the F-box-like motif abolished the P0-SKP1 ortholog interaction, diminished virus pathogenicity, and inhibited the silencing suppressor activity of P0. Knockdown of expression of a SKP1 ortholog in Nicotiana benthamiana rendered the plants resistant to polerovirus infection. Together, the results support a model in which P0 acts as an F-box protein that targets an essential component of the host posttranscriptional gene silencing machinery.

Amino Acid Sequence↗

PubMed2006

Functional analysis of EID1, an F-box protein involved in phytochrome A-dependent light signal transduction.

Empfindlicher im Dunkelroten Licht 1 (EID1) is an F-box protein that functions as a negative regulator in phytochrome A (phyA)-specific light signalling. F-box proteins are components of SCF ubiquitin ligase complexes that target proteins for degradation in the proteasome. Here we present further characterization of EID1 at the expression level, and show that it regulates photomorphogenesis in seedlings, rosette leaf development and flowering. Data on transcript expression patterns indicate that EID1 is expressed during all stages of Arabidopsis development and exhibits no light response. Microscope studies demonstrate that EID1 is localized to the nucleus, where it can form speckles under continuous far-red light that resemble clastosomes. To characterize the composition and formation of SCF(EID1) complexes further, we used two-hybrid and bridge assays in yeast and in planta. EID1 interacts specifically with several Arabidopsis Skp1-like (ASK) proteins and Cullin1 to form stable dimeric and trimeric complexes. Our results support a two-step association process in which the F-box protein binds first to the ASK adaptor, forming a unit which then associates with the catalytic core of the SCF complex. Finally, our data indicate that the EID1 target interaction domain is composed of two independent modules.

Alleles↗

PubMed2005

Plant CULLIN-based E3s: phytohormones come first.

CULLIN (CUL)-dependent ubiquitin ligases form a class of structurally related multi-subunit enzymes that control the rapid and selective degradation of important regulatory proteins involved in cell cycle progression and development, among others. Several classes of these E3s are also conserved in plants and genetic analyses, using Arabidopsis thaliana, indicate that they play an important function during plant development and responses to the environment. In this review, we will discuss the molecular composition and function of these enzymes in plants with a major emphasis on phytohormone signal transduction pathways.

Cell Cycle↗

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↗

PubMed2005

Molecular and functional characterization of Arabidopsis Cullin 3A.

Cullin proteins, which belong to multigenic families in all eukaryotes, associate with other proteins to form ubiquitin protein ligases (E3s) that target substrates for proteolysis by the 26S proteasome. Here, we present the molecular and genetic characterization of a plant Cullin3. In contrast to fungi and animals, the genome of the model plant Arabidopsis thaliana contains two related CUL3 genes, called CUL3A and CUL3B. We found that CUL3A is ubiquitously expressed in plants and is able to interact with the ring-finger protein RBX1. A genomic search revealed the existence of at least 76 BTB-domain proteins in Arabidopsis belonging to 11 major families. Yeast two-hybrid experiments indicate that representative members of certain families are able to physically interact with both CUL3A and CUL3B, suggesting that Arabidopsis CUL3 forms E3 protein complexes with certain BTB domain proteins. In order to determine the function of CUL3A, we used a reverse genetic approach. The cul3a null mutant flowers slightly later than the control plants. Furthermore, this mutant exhibits a reduced sensitivity of the inhibition of hypocotyl growth in far-red light and miss-expresses COP1. The viability of the mutant plants suggests functional redundancy between the two CUL3 genes in Arabidopsis.

Arabidopsis↗

PubMed2005

Arabidopsis CUL3A and CUL3B genes are essential for normal embryogenesis.

Cullin (CUL)-dependent ubiquitin ligases form a class of structurally related multisubunit enzymes that control the rapid and selective degradation of important regulatory proteins involved in cell cycle progression and development, among others. The CUL3-BTB ligases belong to this class of enzymes and despite recent findings on their molecular composition, our knowledge on their functions and substrates remains still very limited. In contrast to budding and fission yeast, CUL3 is an essential gene in metazoans. The model plant Arabidopsis thaliana encodes two related CUL3 genes, called CUL3A and CUL3B. We recently reported that cul3a loss-of-function mutants are viable but exhibit a mild flowering and light sensitivity phenotype. We investigated the spatial and temporal expression of the two CUL3 genes in reproductive tissues and found that their expression patterns are largely overlapping suggesting possible functional redundancy. Thus, we investigated the consequences on plant development of combined Arabidopsis cul3a cul3b loss-of-function mutations. Homozygous cul3b mutant plants developed normally and were fully fertile. However, the disruption of both the CUL3A and CUL3B genes reduced gametophytic transmission and caused embryo lethality. The observed embryo abortion was found to be under maternal control. Arrest of embryogenesis occurred at multiple stages of embryo development, but predominantly at the heart stage. At the cytological level, CUL3 loss-of-function mutations affected both embryo pattern formation and endosperm development.

Amino Acid Sequence↗

PubMed2004

Arabidopsis AtCUL3a and AtCUL3b form complexes with members of the BTB/POZ-MATH protein family.

The ubiquitin proteasome pathway in plants has been shown to be important for many developmental processes. The E3 ubiquitin-protein ligases facilitate transfer of the ubiquitin moiety to substrate proteins. Many E3 ligases contain cullin proteins as core subunits. Here, we show that Arabidopsis (Arabidopsis thaliana) AtCUL3 proteins interact in yeast two-hybrid and in vitro pull-down assays with proteins containing a BTB/POZ (broad complex, tramtrack, bric-a-brac/pox virus and zinc finger) motif. By changing specific amino acid residues within the proteins, critical parts of the cullin and BTB/POZ proteins are defined that are required for these kinds of interactions. In addition, we show that AtCUL3 proteins assemble with the RING-finger protein AtRBX1 and are targets for the RUB-conjugation pathway. The analysis of AtCUL3a and AtCUL3b expression as well as several BTB/POZ-MATH genes indicates that these genes are expressed in all parts of the plant. The results presented here provide strong evidence that AtCUL3a and AtCUL3b can assemble in Arabidopsis with BTB/POZ-MATH and AtRBX1 proteins to form functional E3 ligases.

Amino Acid Sequence↗

PubMed2003

Characterization of a novel non-constitutive photomorphogenic cop1 allele.

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 in Arabidopsis seedlings. Because of their phenotype, the mutants were called eid (empfindlicher im dunkelroten Licht, which means hypersensitive in far-red light). One of the isolated mutants, eid6, is a novel recessive allele of the COP1 gene (constitutive photomorphogenic 1) that carries an amino acid transition in a conserved histidine residue of the RING finger domain. Mutant seedlings exhibited an extreme hypersensitivity towards all tested light qualities, but in contrast to known cop1 alleles, no constitutive photomorphogenic phenotype was detectable in darkness. Thus, the novel cop1eid6 allele seems to encode for a protein whose remaining activity is sufficient for the suppression of photomorphogenesis in dark-grown plants. In adult cop1eid6 plants, the development of the Cop1 phenotype is dominated by phytochrome B. Comparison of the phenotype of the novel cop1eid6 and the weak cop1-4 allele under continuous far-red light indicates that the RING finger and coiled-coil domains of COP1 are sufficient for some specific regulatory function in phytochrome A-dependent high irradiance responses.

Amino Acid Sequence↗

PubMed2002

The negatively acting factors EID1 and SPA1 have distinct functions in phytochrome A-specific light signaling.

EID1 (empfindlicher im dunkelroten Licht) and SPA1 (suppressor of phytochrome A[phyA]-105) function as negatively acting components in phyA-specific light signaling. Mutants in the respective genes led to very similar phenotypes under weak-light conditions. To examine whether both genes are functionally redundant, detailed physiological and genetic analyses were performed with eid1 and spa1 mutants isolated from the same wild-type background. Measurements of hypocotyl elongation, anthocyanin accumulation, and Lhcb1-transcript accumulation under different light treatments demonstrated that SPA1 has a strong influence on the regulation of very low fluence responses and a weaker influence on high-irradiance responses. In contrast, EID1 severely altered high-irradiance responses and caused almost no change on very low fluence responses. Analyses on eid1 phyA-105 double mutants demonstrated that EID1 could not suppress the phenotype of the weak phyA allele under continuous far-red light. Measurements on eid1 spa1 double mutants exhibited a strong interference of both genes in the regulation of hypocotyl elongation. These results indicate that EID1 and SPA1 are involved in different but interacting phyA-dependent signal transduction chains.

Alleles↗

PubMed2002

Phytochrome E controls light-induced germination of Arabidopsis.

Germination of Arabidopsis seeds is light dependent and under phytochrome control. Previously, phytochromes A and B and at least one additional, unspecified phytochrome were shown to be involved in this process. Here, we used a set of photoreceptor mutants to test whether phytochrome D and/or phytochrome E can control germination of Arabidopsis. The results show that only phytochromes B and E, but not phytochrome D, participate directly in red/far-red light (FR)-reversible germination. Unlike phytochromes B and D, phytochrome E did not inhibit phytochrome A-mediated germination. Surprisingly, phytochrome E was required for germination of Arabidopsis seeds in continuous FR. However, inhibition of hypocotyl elongation by FR, induction of cotyledon unfolding, and induction of agravitropic growth were not affected by loss of phytochrome E. Therefore, phytochrome E is not required per se for phytochrome A-mediated very low fluence responses and the high irradiance response. Immunoblotting revealed that the need of phytochrome E for germination in FR was not caused by altered phytochrome A levels. These results uncover a novel role of phytochrome E in plant development and demonstrate the considerable functional diversification of the closely related phytochromes B, D, and E.

Apoproteins↗