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Publications - Molecular Signal Processing

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Publications

Bagchi, R.; Melnyk, C. W.; Christ, G.; Winkler, M.; Kirchsteiner, K.; Salehin, M.; Mergner, J.; Niemeyer, M.; Schwechheimer, C.; Calderón Villalobos, L. I. A.; Estelle, M.; The Arabidopsis ALF4 protein is a regulator of SCF E3 ligases EMBO J. 37, 255-268, (2018) DOI: 10.15252/embj.201797159

The cullin‐RING E3 ligases (CRLs) regulate diverse cellular processes in all eukaryotes. CRL activity is controlled by several proteins or protein complexes, including NEDD8, CAND1, and the CSN. Recently, a mammalian protein called Glomulin (GLMN) was shown to inhibit CRLs by binding to the RING BOX (RBX1) subunit and preventing binding to the ubiquitin‐conjugating enzyme. Here, we show that Arabidopsis ABERRANT LATERAL ROOT FORMATION4 (ALF4) is an ortholog of GLMN. The alf4 mutant exhibits a phenotype that suggests defects in plant hormone response. We show that ALF4 binds to RBX1 and inhibits the activity of SCFTIR1, an E3 ligase responsible for degradation of the Aux/IAA transcriptional repressors. In vivo, the alf4 mutation destabilizes the CUL1 subunit of the SCF. Reduced CUL1 levels are associated with increased levels of the Aux/IAA proteins as well as the DELLA repressors, substrate of SCFSLY1. We propose that the alf4 phenotype is partly due to increased levels of the Aux/IAA and DELLA proteins.
Publications

Ludwig-Müller, J.; Denk, K.; Cohen, J. D.; Quint, M.; An Inhibitor of Tryptophan-Dependent Biosynthesis of Indole-3-Acetic Acid Alters Seedling Development in Arabidopsis J. Plant Growth Regul. 29, 242-248, (2010) DOI: 10.1007/s00344-009-9128-1

Although polar transport and the TIR1-dependent signaling pathway of the plant hormone auxin/indole-3-acetic acid (IAA) are well characterized, understanding of the biosynthetic pathway(s) leading to the production of IAA is still limited. Genetic dissection of IAA biosynthetic pathways has been complicated by the metabolic redundancy caused by the apparent existence of several parallel biosynthetic routes leading to IAA production. Valuable complementary tools for genetic as well as biochemical analysis of auxin biosynthesis would be molecular inhibitors capable of acting in vivo on specific or general components of the pathway(s), which unfortunately have been lacking. Several indole derivatives have been previously identified to inhibit tryptophan-dependent IAA biosynthesis in an in vitro system from maize endosperm. We examined the effect of one of them, 6-fluoroindole, on seedling development of Arabidopsis thaliana and tested its ability to inhibit IAA biosynthesis in feeding experiments in vivo. We demonstrated a correlation of severe developmental defects or growth retardation caused by 6-fluoroindole with significant downregulation of de novo synthesized IAA levels, derived from the stable isotope-labeled tryptophan pool, upon treatment. Hence, 6-fluoroindole shows important features of an inhibitor of tryptophan-dependent IAA biosynthesis both in vitro and in vivo and thus may find use as a promising molecular tool for the identification of novel components of the auxin biosynthetic pathway(s).
Publications

Lee, C.-W.; Efetova, M.; Engelmann, J. C.; Kramell, R.; Wasternack, C.; Ludwig-Müller, J.; Hedrich, R.; Deeken, R.; Agrobacterium tumefaciens Promotes Tumor Induction by Modulating Pathogen Defense in Arabidopsis thaliana Plant Cell 21, 2948-2962, (2009) DOI: 10.1105/tpc.108.064576

Agrobacterium tumefaciens causes crown gall disease by transferring and integrating bacterial DNA (T-DNA) into the plant genome. To examine the physiological changes and adaptations during Agrobacterium-induced tumor development, we compared the profiles of salicylic acid (SA), ethylene (ET), jasmonic acid (JA), and auxin (indole-3-acetic acid [IAA]) with changes in the Arabidopsis thaliana transcriptome. Our data indicate that host responses were much stronger toward the oncogenic strain C58 than to the disarmed strain GV3101 and that auxin acts as a key modulator of the Arabidopsis–Agrobacterium interaction. At initiation of infection, elevated levels of IAA and ET were associated with the induction of host genes involved in IAA, but not ET signaling. After T-DNA integration, SA as well as IAA and ET accumulated, but JA did not. This did not correlate with SA-controlled pathogenesis-related gene expression in the host, although high SA levels in mutant plants prevented tumor development, while low levels promoted it. Our data are consistent with a scenario in which ET and later on SA control virulence of agrobacteria, whereas ET and auxin stimulate neovascularization during tumor formation. We suggest that crosstalk among IAA, ET, and SA balances pathogen defense launched by the host and tumor growth initiated by agrobacteria.
Publications

Calderon-Villalobos, L. I. A.; Nill, C.; Marrocco, K.; Kretsch, T.; Schwechheimer, C.; The evolutionarily conserved Arabidopsis thaliana F-box protein AtFBP7 is required for efficient translation during temperature stress Gene 392, 106-116, (2007) DOI: 10.1016/j.gene.2006.11.016

In eukaryotes, E3 ubiquitin ligases (E3s) mediate the ubiquitylation of proteins that are destined for degradation by the ubiquitin–proteasome system. In SKP1/CDC53/F-box protein (SCF)-type E3 complexes, the interchangeable F-box protein confers specificity to the E3 ligase through direct physical interactions with the degradation substrate. The vast majority of the approximately 700 F-box proteins from the plant model organism Arabidopsis thaliana remain to be characterized. Here, we investigate the previously uncharacterized and evolutionarily conserved Arabidopsis F-box protein 7 (AtFBP7), which is encoded by a unique gene in Arabidopsis (At1g21760). Several apparent fbp7 loss-of-function alleles do not have an obvious phenotype. AtFBP7 is ubiquitously expressed and its expression is induced after cold and heat stress. When following up on a reported co-purification of the eukaryotic elongation factor-2 (eEF-2) with YLR097c, the apparent budding yeast orthologue of AtFBP7, we discovered a general defect in protein biosynthesis after cold and heat stress in fbp7 mutants. Thus, our findings suggest that AtFBP7 is required for protein synthesis during temperature stress.
Publications

Schwager, K. M.; Calderon-Villalobos, L. I. A.; Dohmann, E. M.; Willige, B. C.; Knierer, S.; Nill, C.; Schwechheimer, C.; Characterization of the VIER F-BOX PROTEINE Genes from Arabidopsis Reveals Their Importance for Plant Growth and Development Plant Cell 19, 1163-1178, (2007) DOI: 10.1105/tpc.105.040675

E3 ubiquitin ligases (E3s) target proteins for degradation by the 26S proteasome. In SKP1/CDC53/F-box protein–type E3s, substrate specificity is conferred by the interchangeable F-box protein subunit. The vast majority of the 694 F-box proteins encoded by the Arabidopsis thaliana genome remain to be understood. We characterize the VIER F-BOX PROTEINE (VFB; German for FOUR F-BOX PROTEINS) genes from Arabidopsis that belong to subfamily C of the Arabidopsis F-box protein superfamily. This subfamily also includes the F-box proteins TRANSPORT INHIBITOR RESPONSE1 (TIR1)/AUXIN SIGNALING F-BOX (AFB) proteins and EIN3 BINDING F-BOX proteins, which regulate auxin and ethylene responses, respectively. We show that loss of VFB function causes delayed plant growth and reduced lateral root formation. We find that the expression of a number of auxin-responsive genes and the activity of DR5:β-glucuronidase, a reporter for auxin reponse, are reduced in the vfb mutants. This finding correlates with an increase in the abundance of an AUXIN/INDOLE-3-ACETIC ACID repressor. However, we also find that auxin responses are not affected in the vfb mutants and that a representative VFB family member, VFB2, cannot functionally complement the tir1-1 mutant. We therefore exclude the possibility that VFBs are functional orthologs of TIR1/AFB proteins.
Publications

Calderon-Villalobos, L. I. A.; Kuhnle, C.; Li, H.; Rosso, M.; Weisshaar, B.; Schwechheimer, C.; LucTrap Vectors Are Tools to Generate Luciferase Fusions for the Quantification of Transcript and Protein Abundance in Vivo Plant Physiol. 141, 3-14, (2006) DOI: 10.1104/pp.106.078097

Proper plant growth and development strongly rely on the plant's ability to respond dynamically to signals and cues from the intra- and extracellular environment. Whereas many of these responses require specific changes at the level of gene expression, in recent years it has become increasingly clear that many plant responses are at least in part also controlled at the level of protein turnover. It is a challenge for signal transduction research to understand how distinct incoming signals are integrated to generate specific changes at the transcript or protein level. The activity of luciferase (LUC) reporters can be detected in nondestructive qualitative and quantitative assays in vivo. Therefore,z LUC reporters are particularly well suited for the detection of changes at the transcript and protein level. To the best of our knowledge, the number of plant transformation vectors for LUC fusions is very limited. In this article, we describe the LucTrap plant transformation vectors that allow generation of targeted and random transcriptional and translational fusions with the modified firefly LUC reporter LUC+. We demonstrate that LucTrap-based fusions can be used to monitor rapid changes in gene expression and protein abundance in vivo.
Publications

Meixner, C.; Ludwig-Müller, J.; Miersch, O.; Gresshoff, P.; Staehelin, C.; Vierheilig, H.; Lack of mycorrhizal autoregulation and phytohormonal changes in the supernodulating soybean mutant nts1007 Planta 222, 709-715, (2005) DOI: 10.1007/s00425-005-0003-4

Autoregulatory mechanisms have been reported in the rhizobial and the mycorrhizal symbiosis. Autoregulation means that already existing nodules or an existing root colonization by an arbuscular mycorrhizal fungus systemically suppress subsequent nodule formation/root colonization in other parts of the root system. Mutants of some legumes lost their ability to autoregulate the nodule number and thus display a supernodulating phenotype. On studying the effect of pre-inoculation of one side of a split-root system with an arbuscular mycorrhizal fungus on subsequent mycorrhization in the second side of the split-root system of a wild-type soybean (Glycine max L.) cv. Bragg and its supernodulating mutant nts1007, we observed a clear suppressional effect in the wild-type, whereas further root colonization in the split-root system of the mutant nts1007 was not suppressed. These data strongly indicate that the mechanisms involved in supernodulation also affect mycorrhization and support the hypothesis that the autoregulation in the rhizobial and the mycorrhizal symbiosis is controlled in a similar manner. The accumulation patterns of the plant hormones IAA, ABA and Jasmonic acid (JA) in non-inoculated control plants and split-root systems of inoculated plants with one mycorrhizal side of the split-root system and one non-mycorrhizal side, indicate an involvement of IAA in the autoregulation of mycorrhization. Mycorrhizal colonization of soybeans also resulted in a strong induction of ABA and JA levels, but on the basis of our data the role of these two phytohormones in mycorrhizal autoregulation is questionable.
Publications

Calderon-Villalobos, L. I.; Kuhnle, C.; Dohmann, E. M.; Li, H.; Bevan, M.; Schwechheimer, C.; The Evolutionarily Conserved TOUGH Protein Is Required for Proper Development of Arabidopsis thaliana Plant Cell 17, 2473-2485, (2005) DOI: 10.1105/tpc.105.031302

In this study, we characterize the evolutionarily conserved TOUGH (TGH) protein as a novel regulator required for Arabidopsis thaliana development. We initially identified TGH as a yeast two-hybrid system interactor of the transcription initiation factor TATA-box binding protein 2. TGH has apparent orthologs in all eukaryotic model organisms with the exception of the budding yeast Saccharomyces cerevisiae. TGH contains domains with strong similarity to G-patch and SWAP domains, protein domains that are characteristic of RNA binding and processing proteins. Furthermore, TGH colocalizes with the splicing regulator SRp34 to subnuclear particles. We therefore propose that TGH plays a role in RNA binding or processing. Arabidopsis tgh mutants display developmental defects, including reduced plant height, polycotyly, and reduced vascularization. We found TGH expression to be increased in the amp1-1 mutant, which is similar to tgh mutants with respect to polycotyly and defects in vascular development. Interestingly, we observed a strong genetic interaction between TGH and AMP1 in that tgh-1 amp1-1 double mutants are extremely dwarfed and severely affected in plant development in general and vascular development in particular when compared with the single mutants.
Publications

Schwechheimer, C.; Villalobos, L. I. A. C.; Cullin-containing E3 ubiquitin ligases in plant development Curr. Opin. Plant Biol. 7, 677-686, (2004) DOI: 10.1016/j.pbi.2004.09.009

In eukaryotes, the ubiquitin–proteasome system participates in the control of signal transduction events by selectively eliminating regulatory proteins. E3 ubiquitin ligases specifically bind degradation substrates and mediate their poly-ubiquitylation, a prerequisite for their degradation by the 26S proteasome. On the basis of the analysis of the Arabidopsis genome sequence, it is predicted that there are more than 1000 E3 ubiquitin ligases in plants. Several types of E3 ubiquitin ligases have already been characterized in eukaryotes. Recently, some of these E3 enzymes have been implicated in specific plant signaling pathways.
Publications

Grubb, C. D.; Zipp, B. J.; Ludwig-Müller, J.; Masuno, M. N.; Molinski, T. F.; Abel, S.; Arabidopsis glucosyltransferase UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis Plant J. 40, 893-908, (2004) DOI: 10.1111/j.1365-313X.2004.02261.x

Glucosinolates are a class of secondary metabolites with important roles in plant defense and human nutrition. Here, we characterize a putative UDP‐glucose:thiohydroximate S‐glucosyltransferase, UGT74B1, to determine its role in the Arabidopsis glucosinolate pathway. Biochemical analyses demonstrate that recombinant UGT74B1 specifically glucosylates the thiohydroximate functional group. Low K m values for phenylacetothiohydroximic acid (approximately 6 μ m ) and UDP‐glucose (approximately 50 μm ) strongly suggest that thiohydroximates are in vivo substrates of UGT74B1. Insertional loss‐of‐function ugt74b1 mutants exhibit significantly decreased, but not abolished, glucosinolate accumulation. In addition, ugt74b1 mutants display phenotypes reminiscent of auxin overproduction, such as epinastic cotyledons, elongated hypocotyls in light‐grown plants, excess adventitious rooting and incomplete leaf vascularization. Indeed, during early plant development, mutant ugt74b1 seedlings accumulate nearly threefold more indole‐3‐acetic acid than the wild type. Other phenotypes, however, such as chlorosis along the leaf veins, are likely caused by thiohydroximate toxicity. Analysis of UGT74B1 promoter activity during plant development reveals expression patterns consistent with glucosinolate metabolism and induction by auxin treatment. The results are discussed in the context of known mutations in glucosinolate pathway genes and their effects on auxin homeostasis. Taken together, our work provides complementary in vitro and in vivo evidence for a primary role of UGT74B1 in glucosinolate biosynthesis.
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