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.
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.
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.
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.
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.
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.
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.