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Bassal, M.; Majovsky, P.; Thieme, D.; Herr, T.; Abukhalaf, M.; Ayash, M.; Al Shweiki, M. R.; Proksch, C.; Hmedat, A.; Ziegler, J.; Neumann, S.; Hoehenwarter, W. Reshaping of the Arabidopsis thaliana proteome landscape and co-regulation of proteins in development and immunity bioRxiv (2020) DOI: 10.1101/2020.03.09.978627

Proteome remodeling is a fundamental adaptive response and proteins in complex and functionally related proteins are often co-expressed. Using a deep sampling strategy we define Arabidopsis thaliana tissue core proteomes at around 10,000 proteins per tissue and absolutely quantify (copy numbers per cell) nearly 16,000 proteins throughout the plant lifecycle. A proteome wide survey of global post translational modification revealed amino acid exchanges pointing to potential conservation of translational infidelity in eukaryotes. Correlation analysis of protein abundance uncovered potentially new tissue and age specific roles of entire signaling modules regulating transcription in photosynthesis, seed development and senescence and abscission. Among others, the data suggest a potential function of RD26 and other NAC transcription factors in seed development related to desiccation tolerance as well as a possible function of Cysteine-rich Receptor-like Kinases (CRKs) as ROS sensors in senescence. All of the components of ribosome biogenesis factor (RBF) complexes were co-expressed tissue and age specifically indicating functional promiscuity in the assembly of these little described protein complexes in Arabidopsis. Treatment of seedlings with flg22 for 16 hours allowed us to characterize proteome architecture in basal immunity in detail. The results were complemented with parallel reaction monitoring (PRM) targeted proteomics, phytohormone, amino acid and transcript measurements. We obtained strong evidence of suppression of jasmonate (JA) and JA-Ile levels by deconjugation and hydroxylation via IAA-ALA RESISTANT3 (IAR3) and JASMONATE-INDUCED OXYGENASE 2 (JOX2) under the control of JASMONATE INSENSITIVE 1 (MYC2). This previously unknown regulatory switch is another part of the puzzle of the as yet understudied role of JA in pattern triggered immunity. The extensive coverage of the Arabidopsis proteome in various biological scenarios presents a rich resource to plant biologists that we make available to the community.

Niemeyer, M.; Moreno Castillo, E.; Ihling, C. H.; Iacobucci, C.; Wilde, V.; Hellmuth, A.; Hoehenwarter, W.; Samodelov, S. L.; Zurbriggen, M. D.; Kastritis, P. L.; Sinz, A.; Calderón Villalobos, L. I. A. Flexibility of intrinsically disordered degrons in AUX/IAA proteins reinforces auxin co-receptor assemblies Nat Commun 11, 2277, (2020) DOI: 10.1038/s41467-020-16147-2

Cullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5 and their AUX/IAA targets perceive the phytohormone auxin. The F-box protein TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, by adopting biochemical, structural proteomics and in vivo approaches we unveil how flexibility in AUX/IAAs and regions in TIR1 affect their conformational ensemble allowing surface accessibility of degrons. We resolve TIR1·auxin·IAA7 and TIR1·auxin·IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) in the degron’s vicinity, cooperatively position AUX/IAAs on TIR1. We identify essential residues at the TIR1 N- and C-termini, which provide non-native interaction interfaces with IDRs and the folded PB1 domain of AUX/IAAs. We thereby establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation, and might provide conformational flexibility for a multiplicity of functional states.

Niemeyer, M.; Moreno Castillo, E.; Ihling, C. H.; Iacobucci, C.; Wilde, V.; Hellmuth, A.; Hoehenwarter, W.; Samodelov, S. L.; Zurbriggen, M. D.; Kastritis, P. L.; Sinz, A.; Calderón Villalobos, L. I. A. Flexibility of intrinsically disordered degrons in AUX/IAA proteins reinforces auxin receptor assemblies bioRxiv (2019) DOI: 10.1101/787770

Cullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5 and their ubiquitylation targets, AUX/IAAs, sense auxin concentrations in the nucleus. TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, we resolved TIR1·auxin·IAA7 and TIR1·auxin·IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) in the degron′s vicinity, cooperatively position AUX/IAAs on TIR1. The AUX/IAA PB1 interaction domain also assists in non-native contacts, affecting AUX/IAA dynamic interaction states. Our results establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation and might provide conformational flexibility for adopting a multiplicity of functional states. We postulate IDRs in distinct members of the AUX/IAA family to be an adaptive signature for protein interaction and initiation region for proteasome recruitment.

Winkler, M.; Niemeyer, M.; Hellmuth, A.; Janitza, P.; Christ, G.; Samodelov, S. L.; Wilde, V.; Majovsky, P.; Trujillo, M.; Zurbriggen, M. D.; Hoehenwarter, W.; Quint, M.; Calderón Villalobos, L. I. A. Variation in auxin sensing guides AUX/IAA transcriptional repressor ubiquitylation and destruction. Nature Commun. 8, 15706, (2017) DOI: 10.1038/ncomms15706

Auxin is a small molecule morphogen that bridges SCFTIR1/AFB-AUX/IAA co-receptor interactions leading to ubiquitylation and proteasome-dependent degradation of AUX/IAA transcriptional repressors. Here, we systematically dissect auxin sensing by SCFTIR1-IAA6 and SCFTIR1-IAA19 co-receptor complexes, and assess IAA6/IAA19 ubiquitylation in vitro and IAA6/IAA19 degradation in vivo. We show that TIR1-IAA19 and TIR1-IAA6 have distinct auxin affinities that correlate with ubiquitylation and turnover dynamics of the AUX/IAA. We establish a system to track AUX/IAA ubiquitylation in IAA6 and IAA19 in vitro and show that it occurs in flexible hotspots in degron-flanking regions adorned with specific Lys residues. We propose that this signature is exploited during auxin-mediated SCFTIR1-AUX/IAA interactions. We present evidence for an evolving AUX/IAA repertoire, typified by the IAA6/IAA19 ohnologues, that discriminates the range of auxin concentrations found in plants. We postulate that the intrinsic flexibility of AUX/IAAs might bias their ubiquitylation and destruction kinetics enabling specific auxin responses.

Hoehenwarter, W.; Mönchgesang, S.; Neumann, S.; Majovsky, P.; Abel, S.; Müller, J. Comparative expression profiling reveals a role of the root apoplast in local phosphate response BMC Plant Biol 16 , 106, (2016) DOI: 10.1186/s12870-016-0790-8

BackgroundPlant adaptation to limited phosphate availability comprises a wide range of responses to conserve and remobilize internal phosphate sources and to enhance phosphate acquisition. Vigorous restructuring of root system architecture provides a developmental strategy for topsoil exploration and phosphate scavenging. Changes in external phosphate availability are locally sensed at root tips and adjust root growth by modulating cell expansion and cell division. The functionally interacting Arabidopsis genes, LOW PHOSPHATE RESPONSE 1 and 2 (LPR1/LPR2) and PHOSPHATE DEFICIENCY RESPONSE 2 (PDR2), are key components of root phosphate sensing. We recently demonstrated that the LOW PHOSPHATE RESPONSE 1 - PHOSPHATE DEFICIENCY RESPONSE 2 (LPR1-PDR2) module mediates apoplastic deposition of ferric iron (Fe3+) in the growing root tip during phosphate limitation. Iron deposition coincides with sites of reactive oxygen species generation and triggers cell wall thickening and callose accumulation, which interfere with cell-to-cell communication and inhibit root growth.ResultsWe took advantage of the opposite phosphate-conditional root phenotype of the phosphate deficiency response 2 mutant (hypersensitive) and low phosphate response 1 and 2 double mutant (insensitive) to investigate the phosphate dependent regulation of gene and protein expression in roots using genome-wide transcriptome and proteome analysis. We observed an overrepresentation of genes and proteins that are involved in the regulation of iron homeostasis, cell wall remodeling and reactive oxygen species formation, and we highlight a number of candidate genes with a potential function in root adaptation to limited phosphate availability. Our experiments reveal that FERRIC REDUCTASE DEFECTIVE 3 mediated, apoplastic iron redistribution, but not intracellular iron uptake and iron storage, triggers phosphate-dependent root growth modulation. We further highlight expressional changes of several cell wall-modifying enzymes and provide evidence for adjustment of the pectin network at sites of iron accumulation in the root.ConclusionOur study reveals new aspects of the elaborate interplay between phosphate starvation responses and changes in iron homeostasis. The results emphasize the importance of apoplastic iron redistribution to mediate phosphate-dependent root growth adjustment and suggest an important role for citrate in phosphate-dependent apoplastic iron transport. We further demonstrate that root growth modulation correlates with an altered expression of cell wall modifying enzymes and changes in the pectin network of the phosphate-deprived root tip, supporting the hypothesis that pectins are involved in iron binding and/or phosphate mobilization.

Parry, G.; Calderón Villalobos, L.I.; Prigge, M.; Peret, B.; Dharmasiri, S.; Itoh, H.; Lechner, E.; Gray, W.M.; Bennett, M.; Estelle, M. Complex regulation of the TIR/AFB family of auxin receptors Proc Natl Acad Sci USA 106(52), 22540-22545, (2009) DOI: 10.1073/pnas.0911967106

Auxin regulates most aspects of plant growth and development. The hormone is perceived by the TIR1/AFB family of F-box proteins acting in concert with the Aux/IAA transcriptional repressors. Arabidopsis plants that lack members of the TIR1/AFB family are auxin resistant and display a variety of growth defects. However, little is known about the functional differences between individual members of the family. Phylogenetic studies reveal that the TIR1/AFB proteins are conserved across land plant lineages and fall into four clades. Three of these subgroups emerged before separation of angiosperms and gymnosperms whereas the last emerged before the monocot-eudicot split. This evolutionary history suggests that the members of each clade have distinct functions. To explore this possibility in Arabidopsis, we have analyzed a range of mutant genotypes, generated promoter swap transgenic lines, and performed in vitro binding assays between individual TIR1/AFB and Aux/IAA proteins. Our results indicate that the TIR1/AFB proteins have distinct biochemical activities and that TIR1 and AFB2 are the dominant auxin receptors in the seedling root. Further, we demonstrate that TIR1, AFB2, and AFB3, but not AFB1 exhibit significant posttranscriptional regulation. The microRNA miR393 is expressed in a pattern complementary to that of the auxin receptors and appears to regulate TIR1/AFB expression. However our data suggest that this regulation is complex. Our results suggest that differences between members of the auxin receptor family may contribute to the complexity of auxin response.

Quint, M.; Barkawi, L.S.; Fan, K.T.; Cohen, J.D.; Gray, W.M. Arabidopsis IAR4 modulates auxin response by regulating auxin homeostasis Plant Physiol 150, 748-758, (2009) DOI: 10.1104/pp.109.136671

In a screen for enhancers of tir1-1 auxin resistance, we identified two novel alleles of the putative mitochondrial pyruvate dehydrogenase E1α-subunit, IAA-Alanine Resistant4 (IAR4). In addition to enhancing the auxin response defects of tir1-1, iar4 single mutants exhibit numerous auxin-related phenotypes including auxin-resistant root growth and reduced lateral root development, as well as defects in primary root growth, root hair initiation, and root hair elongation. Remarkably, all of these iar4 mutant phenotypes were rescued when endogenous indole-3-acetic acid (IAA) levels were increased by growth at high temperature or overexpression of the YUCCA1 IAA biosynthetic enzyme, suggesting that iar4 mutations may alter IAA homeostasis rather than auxin response. Consistent with this possibility, iar4 mutants exhibit increased Aux/IAA stability compared to wild type under basal conditions, but not in response to an auxin treatment. Measurements of free IAA levels detected no significant difference between iar4-3 and wild-type controls. However, we consistently observed significantly higher levels of IAA-amino acid conjugates in the iar4-3 mutant. Furthermore, using stable isotope-labeled IAA precursors, we observed a significant increase in the relative utilization of the Trp-independent IAA biosynthetic pathway in iar4-3. We therefore suggest that the auxin phenotypes of iar4 mutants are the result of altered IAA homeostasis.

Zhang, W.; Ito, H.; Quint, M.; Huang, H.; Noël, L.D.; Gray, W.M. Genetic analysis of CAND1-CUL1 interactions in Arabidopsis supports a role for CAND1-mediated cycling of the SCFTIR1 complex Proc Natl Acad Sci 105, 8470-8475, (2008) DOI: 10.1073/pnas.0804144105

SKP1-Cullin1-F-box protein (SCF) ubiquitin-ligases regulate numerous aspects of eukaryotic growth and development. Cullin-Associated and Neddylation-Dissociated (CAND1) modulates SCF function through its interactions with the CUL1 subunit. Although biochemical studies with human CAND1 suggested that CAND1 plays a negative regulatory role by sequestering CUL1 and preventing SCF complex assembly, genetic studies in Arabidopsis have shown that cand1 mutants exhibit reduced SCF activity, demonstrating that CAND1 is required for optimal SCF function in vivo. Together, these genetic and biochemical studies have suggested a model of CAND1-mediated cycles of SCF complex assembly and disassembly. Here, using the SCFTIR1 complex of the Arabidopsis auxin response pathway, we test the SCF cycling model with Arabidopsis mutant derivatives of CAND1 and CUL1 that have opposing effects on the CAND1CUL1 interaction. We find that the disruption of the CAND1CUL1 interaction results in an increased abundance of assembled SCFTIR1 complex. In contrast, stabilization of the CAND1CUL1 interaction diminishes SCFTIR1 complex abundance. The fact that both decreased and increased CAND1CUL1 interactions result in reduced SCFTIR1 activity in vivo strongly supports the hypothesis that CAND1-mediated cycling is required for optimal SCF function.

Quint, M.; Gray, W.M. Auxin signaling Curr Opin Plant Biol 9, 448-453, (2006) DOI: 10.1016/j.pbi.2006.07.006

Auxin regulates a host of plant developmental and physiological processes, including embryogenesis, vascular differentiation, organogenesis, tropic growth, and root and shoot architecture. Genetic and biochemical studies carried out over the past decade have revealed that much of this regulation involves the SCFTIR1/AFB-mediated proteolysis of the Aux/IAA family of transcriptional regulators. With the recent finding that the TRANSPORT INHIBITOR RESPONSE1 (TIR1)/AUXIN SIGNALING F-BOX (AFB) proteins also function as auxin receptors, a potentially complete, and surprisingly simple, signaling pathway from perception to transcriptional response is now before us. However, understanding how this seemingly simple pathway controls the myriad of specific auxin responses remains a daunting challenge, and compelling evidence exists for SCFTIR1/AFB-independent auxin signaling pathways.

Quint, M.; Ito, H.; Zhang, W.; Gray, W.M. Characterization of a novel temperature-sensitive allele of the CUL1/AXR6 subunit of SCF ubiquitin-ligases Plant J 43, 371-383, (2005)

Selective protein degradation by the ubiquitin-proteasome pathway has emerged as a key regulatory mechanism in a wide variety of cellular processes. The selective components of this pathway are the E3 ubiquitin-ligases which act downstream of the ubiquitin-activating and -conjugating enzymes to identify specific substrates for ubiquitinylation. SCF-type ubiquitin-ligases are the most abundant class of E3 enzymes in Arabidopsis. In a genetic screen for enhancers of the tir1-1 auxin response defect, we identified eta1/axr6-3, a recessive and temperature-sensitive mutation in the CUL1 core component of the SCFTIR1 complex. The axr6-3 mutation interferes with Skp1 binding, thus preventing SCF complex assembly. axr6-3 displays a pleiotropic phenotype with defects in numerous SCF-regulated pathways including auxin signaling, jasmonate signaling, flower development, and photomorphogenesis. We used axr6-3 as a tool for identifying pathways likely to be regulated by SCF-mediated proteolysis and propose new roles for SCF regulation of the far-red light/phyA and sugar signaling pathways. The recessive inheritance and the temperature-sensitive nature of the pleiotropically acting axr6-3 mutation opens promising possibilities for the identification and investigation of SCF-regulated pathways in Arabidopsis.
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