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Publikationen - Molekulare Signalverarbeitung

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Preprints

Zang, J.; Klemm, S.; Pain, C.; Duckney, P.; Bao, Z.; Stamm, G.; Kriechbaumer, V.; Bürstenbinder, K.; Hussey, P. J.; Wang, P. A Novel Plant Actin-Microtubule Bridging Complex Regulates Cytoskeletal and ER Structure at Endoplasmic Reticulum-Plasma Membrane Contact Sites (EPCS) SSRN Electronic Journal (2020) DOI: 10.2139/ssrn.3581370

In plants, the cortical ER network is connected to the plasma membrane through the ER-PM contact sites (EPCS), whose structures are maintained by EPCS resident proteins and the cytoskeleton. Strong co-alignment between EPCS and the cytoskeleton is observed in plants, but little is known of how the cytoskeleton is maintained and regulated at the EPCS. Here we have used a yeast-two-hybrid screen and subsequent in vivo interaction studies in plants by FRET-FLIM analysis, to identify two microtubule binding proteins, KLCR1 (Kinesin Light Chain Related protein 1) and IQD2 (IQ67-Domain 2) that interact with the actin binding protein NET3C and form a component of plant EPCS, that mediates the link between the actin and microtubule networks. The NET3C-KLCR1-IQD2 module, acting as an actin-microtubule bridging complex, has a direct influence on ER morphology. Their loss of function mutants, net3a/NET3C RNAi, 0klcr1 or iqd2, exhibit defects in pavement cell morphology which we suggest is linked to the disorganization of both actin filaments and microtubules. In conclusion, our results reveal a novel cytoskeletal associated complex, which is essential for the maintenance and organization of both cytoskeletal structure and ER morphology at the EPCS, and for normal plant cell morphogenesis.
Publikation

Mitra, D.; Klemm, S.; Kumari, P.; Quegwer, J.; Möller, B.; Poeschl, Y.; Pflug, P.; Stamm, G.; Abel, S.; Bürstenbinder, K. Microtubule-associated protein IQ67 DOMAIN5 regulates morphogenesis of leaf pavement cells in Arabidopsis thaliana J Exp Bot 70, 529-543, (2019) DOI: 10.1093/jxb/ery395

Plant microtubules form a highly dynamic intracellular network with important roles for regulating cell division, cell proliferation and cell morphology. Its organization and dynamics are coordinated by various microtubule-associated proteins (MAPs) that integrate environmental and developmental stimuli to fine-tune and adjust cytoskeletal arrays. IQ67 DOMAIN (IQD) proteins recently emerged as a class of plant-specific MAPs with largely unknown functions. Here, using a reverse genetics approach, we characterize Arabidopsis IQD5 in terms of its expression domains, subcellular localization and biological functions. We show that IQD5 is expressed mostly in vegetative tissues, where it localizes to cortical microtubule arrays. Our phenotypic analysis of iqd5 loss-of-function lines reveals functions of IQD5 in pavement cell (PC) shape morphogenesis. Histochemical analysis of cell wall composition further suggests reduced rates of cellulose deposition in anticlinal cell walls, which correlate with reduced anisotropic expansion. Lastly, we demonstrate IQD5-dependent recruitment of calmodulin calcium sensors to cortical microtubule arrays and provide first evidence for important roles of calcium in regulation of PC morphogenesis. Our work thus identifies IQD5 as a novel player in PC shape regulation, and, for the first time, links calcium signaling to developmental processes that regulate anisotropic growth in PCs.
Publikation

García, M. L.; Bó, E. D.; da Graça, J. V.; Gago-Zachert, S.; Hammond, J.; Moreno, P.; Natsuaki, T.; Pallás, V.; Navarro, J. A.; Reyes, C. A.; Luna, G. R.; Sasaya, T.; Tzanetakis, I. E.; Vaira, A. M.; Verbeek, M.; ICTV Report Consortium Corrigendum: ICTV Virus Taxonomy Profile: Ophioviridae J Gen Virol 99, 949-949, (2018) DOI: 10.1099/jgv.0.001093

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Preprints

Mitra, D.; Kumari, P.; Quegwer, J.; Klemm, S.; Moeller, B.; Poeschl, Y.; Pflug, P.; Stamm, G.; Abel, S.; Bürstenbinder, K. Microtubule-associated protein IQ67 DOMAIN5 regulates interdigitation of leaf pavement cells in Arabidopsis thaliana bioRxiv (2018) DOI: 10.1101/268466

Plant microtubules form a highly dynamic intracellular network with important roles for regulating cell division, cell proliferation and cell morphology. Its organization and dynamics are coordinated by various microtubule-associated proteins (MAPs) that integrate environmental and developmental stimuli to fine-tune and adjust cytoskeletal arrays. IQ67 DOMAIN (IQD) proteins recently emerged as a class of plant-specific MAPs with largely unknown functions. Here, using a reverse genetics approach, we characterize Arabidopsis IQD5 in terms of its expression domains, subcellular localization and biological functions. We show that IQD5 is expressed mostly in vegetative tissues, where it localizes to cortical microtubule arrays. Our phenotypic analysis of iqd5 loss-of-function lines reveals functions of IQD5 in pavement cell (PC) shape morphogenesis, as indicated by reduced interdigitation of neighboring cells in the leaf epidermis of iqd5 mutants. Histochemical analysis of cell wall composition further suggests reduced rates of cellulose deposition in anticlinal cell walls, which correlate with reduced asymmetric expansion. Lastly, we provide evidence for IQD5-dependent recruitment of calmodulin calcium sensors to cortical microtubule arrays. Our work thus identifies IQD5 as a novel player in PC shape regulation, and, for the first time, links calcium signaling to developmental processes that regulate multi-polar growth in PCs.
Publikation

García, M. L.; Bó, E. D.; da Graça, J. V.; Gago-Zachert, S.; Hammond, J.; Moreno, P.; Natsuaki, T.; Pallás, V.; Navarro, J. A.; Reyes, C. A.; Luna, G. R.; Sasaya, T.; Tzanetakis, I. E.; Vaira, A. M.; Verbeek, M.; ICTV Report Consortium ICTV Virus Taxonomy Profile: Ophioviridae J Gen Virol 98 , 1161-1162, (2017) DOI: 10.1099/jgv.0.000836

Ophioviridae,The Ophioviridae is a family of filamentous plant viruses, with single-stranded negative, and possibly ambisense, RNA genomes of 11.3–12.5 kb divided into 3–4 segments, each encapsidated separately. Virions are naked filamentous nucleocapsids, forming kinked circles of at least two different contour lengths. The sole genus, Ophiovirus, includes seven species. Four ophioviruses are soil-transmitted and their natural hosts include trees, shrubs, vegetables and bulbous or corm-forming ornamentals, both monocots and dicots. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the which is available at http://www.ictv.global/report/ophioviridae.
Publikation

Bürstenbinder, K.; Möller, B.; Plötner; R.; Stamm, G.; Hause, G.; Mitra, D.; Abel, S. The IQD family of calmodulin-binding proteins links calcium signaling to microtubules, membrane subdomains, and the nucleus. Plant Physiol 173, 1692-1708, (2017) DOI: 10.1104/pp.16.01743

Calcium (Ca2+) signaling and dynamic reorganization of the cytoskeleton are essential processes for the coordination and control of plant cell shape and cell growth. Calmodulin (CaM) and closely related CaM-like polypeptides (CML) are principal sensors of Ca2+ signals. CaM/CMLs decode and relay information encrypted by the second messenger via differential interactions with a wide spectrum of targets to modulate their diverse biochemical activities. The plant-specific IQ67-DOMAIN (IQD) family emerged as the possibly largest class of CaM interacting proteins with undefined molecular functions and biological roles. Here, we show that the 33 members of the IQD family in Arabidopsis thaliana differentially localize, using GFP-tagged proteins, to multiple and distinct subcellular sites, including microtubule (MT) arrays, plasma membrane microdomains, and nuclear compartments. Intriguingly, the various IQD-specific localization patterns coincide with the subcellular patterns of IQD-dependent recruitment of CaM, suggesting that the diverse IQD members sequester Ca2+-CaM signaling modules to specific subcellular sites for precise regulation of Ca2+-dependent processes. Because MT localization is a hallmark of most IQD family members, we quantitatively analyzed GFP-labeled MT arrays in tobacco cells transiently expressing GFP-IQD fusions and observed IQD-specific MT patterns, which point to a role of IQDs in MT organization and dynamics. Indeed, stable overexpression of select IQD proteins in Arabidopsis altered cellular MT orientation, cell shape, and organ morphology. Because IQDs share biochemical properties with scaffold proteins, we propose that IQD families provide an assortment of platform proteins for integrating CaM-dependent Ca2+ signaling at multiple cellular sites to regulate cell function, shape, and growth. 
Publikation

Gasperini, D.; Acosta, I. F.; Farmer, E. E. Cotyledon Wounding of Arabidopsis Seedlings Bio Protoc 6, e1712, (2016) DOI: 10.21769/BioProtoc.1712

Damage to plant organs through both biotic and abiotic injury is very common in nature. Arabidopsis thaliana 5-day-old (5-do) seedlings represent an excellent system in which to study plant responses to mechanical wounding, both at the site of the damage and in distal unharmed tissues. Seedlings of wild type, transgenic or mutant lines subjected to single or repetitive cotyledon wounding can be used to quantify morphological alterations (e.g., root length, Gasperini et al., 2015), analyze the dynamics of reporter genes in vivo (Larrieu et al., 2015; Gasperini et al., 2015), follow transcriptional changes by quantitative RT-PCR (Acosta et al., 2013; Gasperini et al., 2015) or examine additional aspects of the wound response with a plethora of downstream procedures. Here we illustrate how to rapidly and reliably wound cotyledons of young seedlings, and show the behavior of two promoters driving the expression of β-glucuronidase (GUS) in entire seedlings and in the primary root meristem, following single or repetitive cotyledon wounding respectively. We describe two procedures that can be easily adapted to specific experimental needs.
Publikation

Gasperini, D.; Chauvin, A.; Acosta, I. F.; Kurenda, A.; Stolz, S.; Chételat, A.; Wolfender, J.-L.; Farmer, E. E. Axial and Radial Oxylipin Transport Plant Physiol 169, 2244-2254, (2015) DOI: 10.1104/pp.15.01104

Jasmonates are oxygenated lipids (oxylipins) that control defense gene expression in response to cell damage in plants. How mobile are these potent mediators within tissues? Exploiting a series of 13-lipoxygenase (13-lox) mutants in Arabidopsis (Arabidopsis thaliana) that displays impaired jasmonic acid (JA) synthesis in specific cell types and using JA-inducible reporters, we mapped the extent of the transport of endogenous jasmonates across the plant vegetative growth phase. In seedlings, we found that jasmonate (or JA precursors) could translocate axially from wounded shoots to unwounded roots in a LOX2-dependent manner. Grafting experiments with the wild type and JA-deficient mutants confirmed shoot-to-root oxylipin transport. Next, we used rosettes to investigate radial cell-to-cell transport of jasmonates. After finding that the LOX6 protein localized to xylem contact cells was not wound inducible, we used the lox234 triple mutant to genetically isolate LOX6 as the only JA precursor-producing LOX in the plant. When a leaf of this mutant was wounded, the JA reporter gene was expressed in distal leaves. Leaf sectioning showed that JA reporter expression extended from contact cells throughout the vascular bundle and into extravascular cells, revealing a radial movement of jasmonates. Our results add a crucial element to a growing picture of how the distal wound response is regulated in rosettes, showing that both axial (shoot-to-root) and radial (cell-to-cell) transport of oxylipins plays a major role in the wound response. The strategies developed herein provide unique tools with which to identify intercellular jasmonate transport routes.
Publikation

Gasperini, D.; Chételat, A.; Acosta, I. F.; Goossens, J.; Pauwels, L.; Goossens, A.; Dreos, R.; Alfonso, E.; Farmer, E. E. Multilayered Organization of Jasmonate Signalling in the Regulation of Root Growth PLOS Genet 11, e1005300, (2015) DOI: 10.1371/journal.pgen.1005300

Physical damage can strongly affect plant growth, reducing the biomass of developing organs situated at a distance from wounds. These effects, previously studied in leaves, require the activation of jasmonate (JA) signalling. Using a novel assay involving repetitive cotyledon wounding in Arabidopsis seedlings, we uncovered a function of JA in suppressing cell division and elongation in roots. Regulatory JA signalling components were then manipulated to delineate their relative impacts on root growth. The new transcription factor mutant myc2-322B was isolated. In vitro transcription assays and whole-plant approaches revealed that myc2-322B is a dosage-dependent gain-of-function mutant that can amplify JA growth responses. Moreover, myc2-322B displayed extreme hypersensitivity to JA that totally suppressed root elongation. The mutation weakly reduced root growth in undamaged plants but, when the upstream negative regulator NINJA was genetically removed, myc2-322B powerfully repressed root growth through its effects on cell division and cell elongation. Furthermore, in a JA-deficient mutant background, ninja1 myc2-322B still repressed root elongation, indicating that it is possible to generate JA-responses in the absence of JA. We show that NINJA forms a broadly expressed regulatory layer that is required to inhibit JA signalling in the apex of roots grown under basal conditions. By contrast, MYC2, MYC3 and MYC4 displayed cell layer-specific localisations and MYC3 and MYC4 were expressed in mutually exclusive regions. In nature, growing roots are likely subjected to constant mechanical stress during soil penetration that could lead to JA production and subsequent detrimental effects on growth. Our data reveal how distinct negative regulatory layers, including both NINJA-dependent and -independent mechanisms, restrain JA responses to allow normal root growth. Mechanistic insights from this work underline the importance of mapping JA signalling components to specific cell types in order to understand and potentially engineer the growth reduction that follows physical damage.
Publikation

Farmer, E. E.; Gasperini, D.; Acosta, I. F. The squeeze cell hypothesis for the activation of jasmonate synthesis in response to wounding New Phytol 204, 282-288, (2014) DOI: 10.1111/nph.12897

Jasmonates are lipid mediators that control defence gene expression in response to wounding and other environmental stresses. These small molecules can accumulate at distances up to several cm from sites of damage and this is likely to involve cell‐to‐cell jasmonate transport. Also, and independently of jasmonate synthesis, transport and perception, different long‐distance wound signals that stimulate distal jasmonate synthesis are propagated at apparent speeds of several cm min–1 to tissues distal to wounds in a mechanism that involves clade 3 GLUTAMATE RECEPTOR‐LIKE (GLR) genes. A search for jasmonate synthesis enzymes that might decode these signals revealed LOX6, a lipoxygenase that is necessary for much of the rapid accumulation of jasmonic acid at sites distal to wounds. Intriguingly, the LOX6 promoter is expressed in a distinct niche of cells that are adjacent to mature xylem vessels, a location that would make these contact cells sensitive to the release of xylem water column tension upon wounding. We propose a model in which rapid axial changes in xylem hydrostatic pressure caused by wounding travel through the vasculature and lead to slower, radially dispersed pressure changes that act in a clade 3 GLR‐dependent mechanism to promote distal jasmonate synthesis.
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