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Publikationen - Molekulare Signalverarbeitung

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Publikation

Bürstenbinder, K.; Savchenko, T.; Müller, J.; Adamson, A. W.; Stamm, G.; Kwong, R.; Zipp, B. J.; Dinesh, D. C.; Abel, S.; Arabidopsis Calmodulin-binding Protein IQ67-Domain 1 Localizes to Microtubules and Interacts with Kinesin Light Chain-related Protein-1 J. Biol. Chem. 288, 1871-1882, (2013) DOI: 10.1074/jbc.M112.396200

Calcium (Ca2+) is a key second messenger in eukaryotes and regulates diverse cellular processes, most notably via calmodulin (CaM). In Arabidopsis thaliana, IQD1 (IQ67 domain 1) is the founding member of the IQD family of putative CaM targets. The 33 predicted IQD proteins share a conserved domain of 67 amino acids that is characterized by a unique arrangement of multiple CaM recruitment motifs, including so-called IQ motifs. Whereas IQD1 has been implicated in the regulation of defense metabolism, the biochemical functions of IQD proteins remain to be elucidated. In this study we show that IQD1 binds to multiple Arabidopsis CaM and CaM-like (CML) proteins in vitro and in yeast two-hybrid interaction assays. CaM overlay assays revealed moderate affinity of IQD1 to CaM2 (Kd ∼ 0.6 μm). Deletion mapping of IQD1 demonstrated the importance of the IQ67 domain for CaM2 binding in vitro, which is corroborated by interaction of the shortest IQD member, IQD20, with Arabidopsis CaM/CMLs in yeast. A genetic screen of a cDNA library identified Arabidopsis kinesin light chain-related protein-1 (KLCR1) as an IQD1 interactor. The subcellular localization of GFP-tagged IQD1 proteins to microtubules and the cell nucleus in transiently and stably transformed plant tissues (tobacco leaves and Arabidopsis seedlings) suggests direct interaction of IQD1 and KLCR1 in planta that is supported by GFP∼IQD1-dependent recruitment of RFP∼KLCR1 and RFP∼CaM2 to microtubules. Collectively, the prospect arises that IQD1 and related proteins provide Ca2+/CaM-regulated scaffolds for facilitating cellular transport of specific cargo along microtubular tracks via kinesin motor proteins.
Publikation

Abel, S.; Bürstenbinder, K.; Müller, J.; The emerging function of IQD proteins as scaffolds in cellular signaling and trafficking Plant Signal Behav. 8, e24369, (2013) DOI: 10.4161/psb.24369

Calcium (Ca2+) signaling modules are essential for adjusting plant growth and performance to environmental constraints. Differential interactions between sensors of Ca2+ dynamics and their molecular targets are at the center of the transduction process. Calmodulin (CaM) and CaM-like (CML) proteins are principal Ca2+-sensors in plants that govern the activities of numerous downstream proteins with regulatory properties. The families of IQ67-Domain (IQD) proteins are a large class of plant-specific CaM/CML-targets (e.g., 33 members in A. thaliana) which share a unique domain of multiple varied CaM retention motifs in tandem orientation. Genetic studies in Arabidopsis and tomato revealed first roles for IQD proteins related to basal defense response and plant development. Molecular, biochemical and histochemical analysis of Arabidopsis IQD1 demonstrated association with microtubules as well as targeting to the cell nucleus and nucleolus. In vivo binding to CaM and kinesin light chain-related protein-1 (KLCR1) suggests a Ca2+-regulated scaffolding function of IQD1 in kinesin motor-dependent transport of multiprotein complexes. Furthermore, because IQD1 interacts in vitro with single-stranded nucleic acids, the prospect arises that IQD1 and other IQD family members facilitate cellular RNA localization as one mechanism to control and fine-tune gene expression and protein sorting.
Publikation

Kopycki, J.; Wieduwild, E.; Kohlschmidt, J.; Brandt, W.; Stepanova, A.; Alonso, J.; Pedras, M. S.; Abel, S.; Grubb, C. D.; Kinetic analysis of Arabidopsis glucosyltransferase UGT74B1 illustrates a general mechanism by which enzymes can escape product inhibition Biochem. J. 450, 37-46, (2013) DOI: 10.1042/BJ20121403

Plant genomes encode numerous small molecule glycosyltransferases which modulate the solubility, activity, immunogenicity and/or reactivity of hormones, xenobiotics and natural products. The products of these enzymes can accumulate to very high concentrations, yet somehow avoid inhibiting their own biosynthesis. Glucosyltransferase UGT74B1 (UDP-glycosyltransferase 74B1) catalyses the penultimate step in the core biosynthetic pathway of glucosinolates, a group of natural products with important functions in plant defence against pests and pathogens. We found that mutation of the highly conserved Ser284 to leucine [wei9-1 (weak ethylene insensitive)] caused only very mild morphological and metabolic phenotypes, in dramatic contrast with knockout mutants, indicating that steady state glucosinolate levels are actively regulated even in unchallenged plants. Analysis of the effects of the mutation via a structural modelling approach indicated that the affected serine interacts directly with UDP-glucose, but also predicted alterations in acceptor substrate affinity and the kcat value, sparking an interest in the kinetic behaviour of the wild-type enzyme. Initial velocity and inhibition studies revealed that UGT74B1 is not inhibited by its glycoside product. Together with the effects of the missense mutation, these findings are most consistent with a partial rapid equilibrium ordered mechanism. This model explains the lack of product inhibition observed both in vitro and in vivo, illustrating a general mechanism whereby enzymes can continue to function even at very high product/precursor ratios.
Publikation

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.
Publikation

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.
Publikation

Abel, S.; Auxin Is Surfacing ACS Chem. Biol. 2, 380-384, (2007) DOI: 10.1021/cb7001158

Indole-3-acetic acid (IAA or auxin) is essential throughout the life cycle of a plant. It controls diverse cellular processes, including gene expression. The hormone is perceived by a ubiquitin protein ligase (E3) and triggers the rapid destruction of repressors, called Aux/IAA proteins. The first structural model of a plant hormone receptor illustrates how auxin promotes Aux/IAA substrate recruitment by extending the hydrophobic protein-interaction surface. This work establishes a novel mechanism of E3 regulation by small molecules and promises a novel strategy for the treatment of human disorders associated with defective ubiquitin-dependent proteolysis.
Publikation

Vigliocco, A.; Alemano, S.; Miersch, O.; Alvarez, D.; Abdala, G.; Endogenous jasmonates in dry and imbibed sunflower seeds from plants grown at different soil moisture contents Seed Sci. Res. 17, 91-98, (2007) DOI: 10.1017/S0960258507708371

In this study, we characterized two sunflower (Helianthus annuus L.) lines with differential sensitivity to drought, the sensitive line B59 and the tolerant line B71. Using both lines, we compared the content of endogenous jasmonates (JAs) in dry and imbibed seeds from plants grown under irrigation and drought. Jasmonic acid (JA), 12-oxo-phytodienoic acid (OPDA), 11-hydroxyjasmonate (11-OH-JA) and 12-hydroxyjasmonate (12-OH-JA) were detected in dry and imbibed sunflower seeds. Seeds from plants grown under drought had a lower content of total JAs and exhibited higher germination percentages than seeds from irrigated plants, demonstrating that environmental conditions have a strong influence on the progeny. OPDA and 12-OH-JA were the main compounds found in dry seeds of both lines. Imbibed seeds showed an enhanced amount of total JAs with respect to dry seeds produced by plants grown in both soil moisture conditions. Imbibition triggered a dramatic OPDA increase in the embryo, suggesting a role of this compound in germination. We conclude that JAs patterns vary during sunflower germination and that the environmental conditions experienced by the mother plant modify the hormonal content of the seed progeny.
Publikation

Wang, Q.; Grubb, C. D.; Abel, S.; Direct analysis of single leaf disks for chemopreventive glucosinolates Phytochem. Anal. 13, 152-157, (2002) DOI: 10.1002/pca.636

Natural isothiocyanates, produced during plant tissue damage from methionine‐derived glucosinolates, are potent inducers of mammalian phase 2 detoxification enzymes such as quinone reductase (QR). A greatly simplified bioassay for glucosinolates based on induction and colorimetric detection of QR activity in murine hepatoma cells is described. It is demonstrated that excised leaf disks of Arabidopsis thaliana (ecotype Columbia) can directly and reproducibly substitute for cell‐free leaf extracts as inducers of murine QR, which reduces sample preparation to a minimum and maximizes throughput. A comparison of 1 and 3 mm diameter leaf disks indicated that QR inducer potency was proportional to disk circumference (extent of tissue damage) rather than to area. When compared to the QR inducer potency of the corresponding amount of extract, 1 mm leaf disks were equally effective, whereas 3 mm disks were 70% as potent. The QR inducer potency of leaf disks correlated positively with the content of methionine‐derived glucosinolates, as shown by the analysis of wild‐type plants and mutant lines with lower or higher glucosinolate content. Thus, the microtitre plate‐based assay of single leaf disks provides a robust and inexpensive visual method for rapidly screening large numbers of plants in mapping populations or mutant collections and may be applicable to other glucosinolate‐producing species.
Publikation

Vigliocco, A.; Bonamico, B.; Alemano, S.; Miersch, O.; Abdala, G.; Stimulation of jasmonic acid production in Zea Mays L. infected by the maize rough dwarf virus - Río Cuarto. Reversion of symptoms by salicylic acid Biocell 26, 369-374, (2002)

In the present paper we study the possible biological relevance of endogenous jasmonic acid (JA) and exogenous salicylic acid (SA) in a plant-microbial system maize-virus. The virus disease "Mal de Río Cuarto" is caused by the maize rough dwarf virus - Río Cuarto. The characteristic symptoms are the appearance of galls or "enations" in leaves, shortening of the stem internodes, poor radical system and general stunting. Changes in JA and protein pattern in maize control and infected plants of a virus-tolerant cultivar were investigated. Healthy and infected-leaf discs were collected for JA measurement at different post-infection times (20, 40, 60 and 68 days). JA was also measured in roots on day 60 after infection. For SDS-PAGE protein analysis, leaf discs were also harvested on day 60 after infection. Infected leaves showed higher levels of JA than healthy leaves, and the rise in endogenous JA coincided with the enation formation. The soluble protein amount did not show differences between infected and healthy leaves; moreover, no difference in the expression of soluble protein was revealed by SDS-PAGE. Our results show that the octadecanoid pathway was stimulated in leaves and roots of the tolerant maize cultivar when infected by this virus. This finding, together with fewer plants with the disease symptoms, suggest that higher foliar and roots JA content may be related to disease tolerance. SA exogenous treatment caused the reversion of the dwarfism symptom.
Publikation

Laskowski, M. J.; Dreher, K. A.; Gehring, M. A.; Abel, S.; Gensler, A. L.; Sussex, I. M.; FQR1, a Novel Primary Auxin-Response Gene, Encodes a Flavin Mononucleotide-Binding Quinone Reductase Plant Physiol. 128, 578-590, (2002) DOI: 10.1104/pp.010581

FQR1 is a novel primary auxin-response gene that codes for a flavin mononucleotide-binding flavodoxin-like quinone reductase. Accumulation of FQR1 mRNA begins within 10 min of indole-3-acetic acid application and reaches a maximum of approximately 10-fold induction 30 min after treatment. This increase in FQR1 mRNA abundance is not diminished by the protein synthesis inhibitor cycloheximide, demonstrating thatFQR1 is a primary auxin-response gene. Sequence analysis reveals that FQR1 belongs to a family of flavin mononucleotide-binding quinone reductases. Partially purified His-tagged FQR1 isolated fromEscherichia coli catalyzes the transfer of electrons from NADH and NADPH to several substrates and exhibits in vitro quinone reductase activity. Overexpression of FQR1 in plants leads to increased levels of FQR1 protein and quinone reductase activity, indicating that FQR1 functions as a quinone reductase in vivo. In mammalian systems, glutathione S-transferases and quinone reductases are classified as phase II detoxification enzymes. We hypothesize that the auxin-inducible glutathioneS-transferases and quinone reductases found in plants also act as detoxification enzymes, possibly to protect against auxin-induced oxidative stress.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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