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Publikationen - Molekulare Signalverarbeitung

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Publikation

Kopycki, J., Wieduwild, E., Kohlschmidt, J., Brandt, W., Stepanova, A.N., Alonso, J.M., 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

Navarro-Quezada, A. & Schumann, N. & Quint, M. Plant F-Box protein evolution is determined by lineage-specific timing of major gene family expansion waves PLoS One 8, e68672, (2013) DOI: 10.1371/journal.pone.0068672

F-box proteins (FBPs) represent one of the largest and fastest evolving gene/protein families in the plant kingdom. The FBP superfamily can be divided in several subfamilies characterized by different C-terminal protein-protein interaction domains that recruit targets for proteasomal degradation. Hence, a clear picture of their phylogeny and molecular evolution is of special interest for the general understanding of evolutionary histories of multi-domain and/or large protein families in plants. In an effort to further understand the molecular evolution of F-box family proteins, we asked whether the largest subfamily in Arabidopsis thaliana, which carries a C-terminal F-box associated domain (FBA proteins) shares evolutionary patterns and signatures of selection with other FBPs. To address this question, we applied phylogenetic and molecular evolution analyses in combination with the evaluation of transcriptional profiles. Based on the 2219 FBA proteins we de novo identified in 34 completely sequenced plant genomes, we compared their evolutionary patterns to a previously analyzed large subfamily carrying C-terminal kelch repeats. We found that these two large FBP subfamilies generally tend to evolve by massive waves of duplication, followed by sequence conservation of the F-box domain and sequence diversification of the target recruiting domain. We conclude that the earlier in evolutionary time a major wave of expansion occurred, the more pronounced these selection signatures are. As a consequence, when performing cross species comparisons among FBP subfamilies, significant differences will be observed in the selective signatures of protein-protein interaction domains. Depending on the species, the investigated subfamilies comprise up to 45% of the complete superfamily, indicating that other subfamilies possibly follow similar modes of evolution.

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

Bürstenbinder, K., Savchenko, T., Müller, J., Adamson, A.W., Stamm, G., Kwong, R., Zipp, B.J. & Dhurvas Chandrasekaran, D. & 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

Acosta, I.F., Gasperini, D., Chételat, A., Stolz, S., Santuari, L. & Farmer, E.E. Role of NINJA in root jasmonate signaling. In: PNAS 110 (38), 15473-15478, (2013) DOI: 10.1073/pnas.1307910110

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Publikation

Wasternack, C. & Hause, B. Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in Annals of Botany Annals of Botany 111, 1021-1058, (2013) DOI: 10.1093/aob/mct067

Background: Jasmonates are important regulators in plant responses to biotic and abiotic stresses as well as in

development. Synthesized from lipid-constituents, the initially formed jasmonic acid is converted to different

metabolites including the conjugate with isoleucine. Important new components of jasmonate signalling including

its receptor were identified, providing deeper insight into the role of jasmonate signalling pathways in stress

responses and development.

Scope: The present review is an update of the review on jasmonates published in this journal in 2007. New data

of the last five years are described with emphasis on metabolites of jasmonates, on jasmonate perception and

signalling, on cross-talk to other plant hormones and on jasmonate signalling in response to herbivores and pathogens,

in symbiotic interactions, in flower development, in root growth and in light perception.

Conclusions: The last few years have seen breakthroughs in the identification of JASMONATE ZIM DOMAIN

(JAZ) proteins and their interactors such as transcription factors and co-repressors, and the crystallization of the

jasmonate receptor as well as of the enzyme conjugating jasmonate to amino acids. Now, the complex nature of

networks of jasmonate signalling in stress responses and development including hormone cross-talk can beaddressed.

Bücher und Buchkapitel

Wasternack, C. & Hause, B. Benno Parthier und die Jasmonatforschung in Halle. In: Benno Parthier und die Jasmonatforschung in Halle Nova Acta Leopoldina, NF Supplementum 28, 29-38, (2013) ISBN: ISBN 978-3-8047-3209-4

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Publikation

Dekkers, B.J.W., Pearce, S., van Bolderen-Veldkamp, R.P., Marshall, A., Widera, P., Gilbert, J., Drost, H.-G., Basseli, G.W., Müller, K., King, J.R., Wood, A.T.A., Grosse, I., Quint, M., Krasnogor, N., Leubner-Metzger, G. & Holdsworth, M.J. & Bentsink, L. Transcriptional Dynamics of Two Seed Compartments with Opposing Roles in Arabidopsis Seed Germination Plant Physiol 163, 205-215, (2013)

Seed germination is a critical stage in the plant life cycle and the first step toward successful plant establishment. Therefore, understanding

germination is of important ecological and agronomical relevance. Previous research revealed that different seed compartments (testa,

endosperm, and embryo) control germination, but little is known about the underlying spatial and temporal transcriptome changes that

lead to seed germination. We analyzed genome-wide expression in germinating Arabidopsis (Arabidopsis thaliana) seedswith both temporal

and spatial detail and provide Web-accessible visualizations of the data reported (vseed.nottingham.ac.uk). We show the potential of this highresolution

data set for the construction ofmeaningful coexpression networks, which provide insight into the genetic control of germination.

The data set reveals two transcriptional phases during germination that are separated by testa rupture. The first phase is marked by large

transcriptome changes as the seed switches from a dry, quiescent state to a hydrated and active state. At the end of this first transcriptional

phase, the number of differentially expressed genes between consecutive time points drops. This increases again at testa rupture, the start of

the second transcriptional phase. Transcriptome data indicate a role for mechano-induced signaling at this stage and subsequently highlight

the fates of the endosperm and radicle: senescence and growth, respectively. Finally, using a phylotranscriptomic approach, we show that

expression levels of evolutionarily young genes drop during the first transcriptional phase and increase during the second phase.Evolutionarily old genes show an opposite pattern, suggesting a more conserved transcriptome prior to the completion of germination.

Publikation

Huang, H. & Quint, M. & Gray, W.M. The eta7/csn3-3 auxin response mutant of Arabidopsis defines a novel function for the CSN3 subunit of the COP9 signalosome  PLoS One 8, e66578, (2013)

TIR1/AFBTIR1

The COP9 signalosome (CSN) is an eight subunit protein complex conserved in all higher eukaryotes. In Arabidopsis thaliana, the CSN regulates auxin response by removing the ubiquitin-like protein NEDD8/RUB1 from the CUL1 subunit of the SCF

ubiquitin-ligase (deneddylation). Previously described null mutations in any CSN subunit result in the pleiotropic cop/det/fus phenotype and cause seedling lethality, hampering the study of CSN functions in plant development. In a genetic screen to identify enhancers of the auxin response defects conferred by the tir1-1 mutation, we identified a viable csn mutant of subunit 3 (CSN3), designated eta7/csn3-3. In addition to enhancing tir1-1 mutant phenotypes, the csn3-3 mutation alone confers several phenotypes indicative of impaired auxin signaling including auxin resistant root growth and diminished auxin responsive gene expression. Unexpectedly however, csn3-3 plants are not defective in either the CSN-mediated deneddylation of CUL1 or in SCF

-mediated degradation of Aux/IAA proteins. These findings suggest that csn3-3 is an atypical csn mutant that defines a novel CSN or CSN3-specific function. Consistent with this possibility, we observe dramatic differences in double mutant interactions between csn3-3 and other auxin signaling mutants compared to another weak csn mutant, csn1-10. Lastly, unlike other csn mutants, assembly of the CSN holocomplex is unaffected in csn3-3 plants. However, we detected a small CSN3-containing protein complex that is altered in csn3-3 plants. We hypothesize that in addition to its role in the CSN as a cullin deneddylase, CSN3 functions in a distinct protein complex that is required for proper auxin signaling.

Publikation

Poeschl, Y., Delker, C., Trenner, J., Ullrich, K. & Quint, M. & Grosse, I. Optimized probe masking for comparative transcriptomics of closely related species. PLOS ONE 8, e78497, (2013)

Microarrays are commonly applied to study the transcriptome of specific species. However, many available microarrays are

restricted to model organisms, and the design of custom microarrays for other species is often not feasible. Hence,

transcriptomics approaches of non-model organisms as well as comparative transcriptomics studies among two or more

species often make use of cost-intensive RNAseq studies or, alternatively, by hybridizing transcripts of a query species to a

microarray of a closely related species. When analyzing these cross-species microarray expression data, differences in the

transcriptome of the query species can cause problems, such as the following: (i) lower hybridization accuracy of probes due

to mismatches or deletions, (ii) probes binding multiple transcripts of different genes, and (iii) probes binding transcripts of

non-orthologous genes. So far, methods for (i) exist, but these neglect (ii) and (iii). Here, we propose an approach for

comparative transcriptomics addressing problems (i) to (iii), which retains only transcript-specific probes binding transcripts

of orthologous genes. We apply this approach to an Arabidopsis lyrata expression data set measured on a microarray

designed for Arabidopsis thaliana, and compare it to two alternative approaches, a sequence-based approach and a genomic

DNA hybridization-based approach. We investigate the number of retained probe sets, and we validate the resulting

expression responses by qRT-PCR. We find that the proposed approach combines the benefit of sequence-based stringency

and accuracy while allowing the expression analysis of much more genes than the alternative sequence-based approach. As

an added benefit, the proposed approach requires probes to detect transcripts of orthologous genes only, which provides asuperior base for biological interpretation of the measured expression responses.

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