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Publikationen - Molekulare Signalverarbeitung

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Bücher und Buchkapitel

Klemm, S.; Buhl, J.; Möller, B.; Bürstenbinder, K.; Quantitative analysis of microtubule organization in leaf epidermis pavement cells (Hussey, P.J., Wang, P.). The Plant Cytoskeleton 2604, 43-61, (2023) ISBN: 978-1-0716-2866-9 DOI: 10.1007/978-1-0716-2867-6_4

Leaf epidermis pavement cells form highly complex shapes with interlocking lobes and necks at their anticlinal walls. The microtubule cytoskeleton plays essential roles in pavement cell morphogenesis, in particular at necks. Vice versa, shape generates stress patterns that regulate microtubule organization. Genetic or pharmacological perturbations that affect pavement cell shape often affect microtubule organization. Pavement cell shape and microtubule organization are therefore closely interconnected. Here, we present commonly used approaches for the quantitative analysis of pavement cell shape characteristics and of microtubule organization. In combination with ablation experiments, these methods can be applied to investigate how different genotypes (or treatments) affect the organization and stress responsiveness of the microtubule cytoskeleton.
Publikation

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 ER-PM contact sites Curr. Biol. 31, 1251-1260, (2021) DOI: 10.1016/j.cub.2020.12.009

In plants, the cortical endoplasmic reticulum (ER) network is connected to the plasma membrane (PM) through the ER-PM contact sites (EPCSs), whose structures are maintained by EPCS resident proteins and the cytoskeleton.1-7 Strong co-alignment between EPCSs and the cytoskeleton is observed in plants,1,8 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 fluorescence resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (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 and EPCS structure. Their loss-of-function mutants, net3a/NET3C RNAi, klcr1, 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 cytoskeletal structure and ER morphology at the EPCS and for normal plant cell morphogenesis.
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.
Bücher und Buchkapitel

Möller, B.; Poeschl, Y.; Klemm, S.; Bürstenbinder, K.; Morphological Analysis of Leaf Epidermis Pavement Cells with PaCeQuant (Cvrčková, F. & Žárský, V., eds.). Methods Mol. Biol. 1992, 329-349, (2019) ISBN: 978-1-4939-9469-4 DOI: 10.1007/978-1-4939-9469-4_22

Morphological analysis of cell shapes requires segmentation of cell contours from input images and subsequent extraction of meaningful shape descriptors that provide the basis for qualitative and quantitative assessment of shape characteristics. Here, we describe the publicly available ImageJ plugin PaCeQuant and its associated R package PaCeQuantAna, which provides a pipeline for fully automatic segmentation, feature extraction, statistical analysis, and graphical visualization of cell shape properties. PaCeQuant is specifically well suited for analysis of jigsaw puzzle-like leaf epidermis pavement cells from 2D input images and supports the quantification of global, contour-based, skeleton-based, and pavement cell-specific shape descriptors.
Preprints

Mitra, D.; Kumari, P.; Quegwer, J.; Klemm, S.; Möller, 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

Fonseca, S.; Chini, A.; Hamberg, M.; Adie, B.; Porzel, A.; Kramell, R.; Miersch, O.; Wasternack, C.; Solano, R.; (+)-7-iso-Jasmonoyl-L-isoleucine is the endogenous bioactive jasmonate Nat. Chem. Biol. 5, 344-350, (2009) DOI: 10.1038/nchembio.161

Hormone-triggered activation of the jasmonate signaling pathway in Arabidopsis thaliana requires SCFCOI1-mediated proteasome degradation of JAZ repressors. (−)-JA-L-Ile is the proposed bioactive hormone, and SCFCOI1 is its likely receptor. We found that the biological activity of (−)-JA-L-Ile is unexpectedly low compared to coronatine and the synthetic isomer (+)-JA-L-Ile, which suggests that the stereochemical orientation of the cyclopentanone-ring side chains greatly affects receptor binding. Detailed GC-MS and HPLC analyses showed that the (−)-JA-L-Ile preparations currently used in ligand binding studies contain small amounts of the C7 epimer (+)-7-iso-JA-L-Ile. Purification of each of these molecules demonstrated that pure (−)-JA-L-Ile is inactive and that the active hormone is (+)-7-iso-JA-L-Ile, which is also structurally more similar to coronatine. In addition, we show that pH changes promote conversion of (+)-7-iso-JA-L-Ile to the inactive (−)-JA-L-Ile form, thus providing a simple mechanism that can regulate hormone activity through epimerization.
Publikation

BERGER, S.; Weichert, H.; Porzel, A.; Wasternack, C.; Kühn, H.; Feussner, I.; Enzymatic and non-enzymatic lipid peroxidation in leaf development BBA-Mol. Cell Biol. Lipids 1533, 266-276, (2001) DOI: 10.1016/S1388-1981(01)00161-5

Enzymatic and non-enzymatic lipid peroxidation has been implicated in programmed cell death, which is a major process of leaf senescence. To test this hypothesis we developed a high-performance liquid chromatography (HPLC) method for a simultaneous analysis of the major hydro(pero)xy polyenoic fatty acids. Quantities of lipid peroxidation products in leaves of different stages of development including natural senescence indicated a strong increase in the level of oxygenated polyenoic fatty acids (PUFAs) during the late stages of leaf senescence. Comprehensive structural elucidation of the oxygenation products by means of HPLC, gas chromatography/mass spectrometry and 1H nuclear magnetic resonance suggested a non-enzymatic origin. However, in some cases a small share of specifically oxidized PUFAs was identified suggesting involvement of lipid peroxidizing enzymes. To inspect the possible role of enzymatic lipid peroxidation in leaf senescence, we analyzed the abundance of lipoxygenases (LOXs) in rosette leaves of Arabidopsis. LOXs and their product (9Z,11E,13S,15Z)-13-hydroperoxy-9,11,15-octadecatrienoic acid were exclusively detected in young green leaves. In contrast, in senescing leaves the specific LOX products were overlaid by large amounts of stereo-random lipid peroxidation products originating from non-enzymatic oxidation. These data indicate a limited contribution of LOXs to total lipid peroxidation, and a dominant role of non-enzymatic lipid peroxidation in late stages of leaf development.
Publikation

Miersch, O.; Porzel, A.; Wasternack, C.; Microbial conversion of jasmonates - hydroxylations by Aspergillus niger Phytochemistry 50, 1147-1152, (1999) DOI: 10.1016/S0031-9422(98)00698-0

Aspergillus niger is able to hydroxylate the pentenyl side chain of (−)-jasmonic acid (JA) leading to (11S)- (−)-hydroxy-JA/ (11R)- (−)-hydroxy-JA (2:1) and (−)-11,12-didehydro-JA. Methyl (−)-jasmonate (JA-Me) is converted upon hydrolysis. During prolonged cultivation or at non-optimized isolation procedures, the 11-hydroxy- (9Z)-pentenyl side chain may isomerize to (10E)-9-hydroxy- and (9E)-11-hydroxy-compounds by allylic rearrangement. The fungus hydroxylates (±)-9,10-dihydro-JA at position C-11 into 11j-hydroxy-9,10-dihydro-JA. As JA-Me, the methyl dihydro-JA is hydroxylated only upon hydrolysis into the free acid.
Publikation

Kramell, R.; Porzel, A.; Miersch, O.; Schneider, G.; Wasternack, C.; Chromatographic resolution of peptide-like conjugates of jasmonic acid and of cucurbic acid isomers J. Chromatogr. A 847, 103-107, (1999) DOI: 10.1016/S0021-9673(99)00335-0

The chiral separation of peptide-like conjugates of jasmonic acid and of cucurbic acid isomers was investigated by liquid chromatography on Chiralpak AS and Nucleodex β-PM. The retention sequences reflect distinct chromatographic properties with respect to the chirality of the jasmonic acid part or of the cucurbic acid isomers. The chromatographic behaviour of the amide conjugates on a reversed-phase C18 column provides evidence for the resolution of diastereomeric conjugates depending on the chirality of both constituents of the conjugate molecule. The chromatographic procedures are suitable for the analytical and preparative separation of such conjugates.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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