TY - JOUR AB - In plant cells, environmental stressors promote changes in connectivity between the cortical ER and the PM. Although this process is tightly regulated in space and time, the molecular signals and structural components mediating these changes in inter-organelle communication are only starting to be characterized. In this report, we confirm the presence of a putative tethering complex containing the synaptotagmins 1 and 5 (SYT1 and SYT5) and the Ca2+ and lipid binding protein 1 (CLB1/SYT7). This complex is enriched at ER-PM contact sites (EPCS), have slow responses to changes in extracellular Ca2+, and display severe cytoskeleton-dependent rearrangements in response to the trivalent lanthanum (La3+) and gadolinium (Gd3+) rare earth elements (REEs). Although REEs are generally used as non-selective cation channel blockers at the PM, here we show that the slow internalization of REEs into the cytosol underlies the activation of the Ca2+/Calmodulin intracellular signaling, the accumulation of phosphatidylinositol-4-phosphate (PI4P) at the PM, and the cytoskeleton-dependent rearrangement of the SYT1/SYT5 EPCS complexes. We propose that the observed EPCS rearrangements act as a slow adaptive response to sustained stress conditions, and that this process involves the accumulation of stress-specific phosphoinositides species at the PM. AU - Lee, E AU - Vila Nova Santana, B AU - Samuels, E AU - Benitez-Fuente, F AU - Corsi, E AU - Botella, MA AU - Perez-Sancho, J AU - Vanneste, S AU - Friml, Jiří AU - Macho, A AU - Alves Azevedo, A AU - Rosado, A ID - 7646 IS - 14 JF - Journal of Experimental Botany SN - 0022-0957 TI - Rare earth elements induce cytoskeleton-dependent and PI4P-associated rearrangement of SYT1/SYT5 ER-PM contact site complexes in Arabidopsis VL - 71 ER - TY - JOUR AB - The agricultural green revolution spectacularly enhanced crop yield and lodging resistance with modified DELLA-mediated gibberellin signaling. However, this was achieved at the expense of reduced nitrogen-use efficiency (NUE). Recently, Wu et al. revealed novel gibberellin signaling that provides a blueprint for improving tillering and NUE in Green Revolution varieties (GRVs). AU - Xue, Huidan AU - Zhang, Yuzhou AU - Xiao, Guanghui ID - 7686 IS - 6 JF - Trends in Plant Science SN - 1360-1385 TI - Neo-gibberellin signaling: Guiding the next generation of the green revolution VL - 25 ER - TY - JOUR AB - Hormonal signalling in animals often involves direct transcription factor-hormone interactions that modulate gene expression. In contrast, plant hormone signalling is most commonly based on de-repression via the degradation of transcriptional repressors. Recently, we uncovered a non-canonical signalling mechanism for the plant hormone auxin whereby auxin directly affects the activity of the atypical auxin response factor (ARF), ETTIN towards target genes without the requirement for protein degradation. Here we show that ETTIN directly binds auxin, leading to dissociation from co-repressor proteins of the TOPLESS/TOPLESS-RELATED family followed by histone acetylation and induction of gene expression. This mechanism is reminiscent of animal hormone signalling as it affects the activity towards regulation of target genes and provides the first example of a DNA-bound hormone receptor in plants. Whilst auxin affects canonical ARFs indirectly by facilitating degradation of Aux/IAA repressors, direct ETTIN-auxin interactions allow switching between repressive and de-repressive chromatin states in an instantly-reversible manner. AU - Kuhn, André AU - Ramans Harborough, Sigurd AU - McLaughlin, Heather M AU - Natarajan, Bhavani AU - Verstraeten, Inge AU - Friml, Jiří AU - Kepinski, Stefan AU - Østergaard, Lars ID - 7793 JF - eLife SN - 2050-084X TI - Direct ETTIN-auxin interaction controls chromatin states in gynoecium development VL - 9 ER - TY - JOUR AB - Directional transport of the phytohormone auxin is a versatile, plant-specific mechanism regulating many aspects of plant development. The recently identified plant hormones, strigolactones (SLs), are implicated in many plant traits; among others, they modify the phenotypic output of PIN-FORMED (PIN) auxin transporters for fine-tuning of growth and developmental responses. Here, we show in pea and Arabidopsis that SLs target processes dependent on the canalization of auxin flow, which involves auxin feedback on PIN subcellular distribution. D14 receptor- and MAX2 F-box-mediated SL signaling inhibits the formation of auxin-conducting channels after wounding or from artificial auxin sources, during vasculature de novo formation and regeneration. At the cellular level, SLs interfere with auxin effects on PIN polar targeting, constitutive PIN trafficking as well as clathrin-mediated endocytosis. Our results identify a non-transcriptional mechanism of SL action, uncoupling auxin feedback on PIN polarity and trafficking, thereby regulating vascular tissue formation and regeneration. AU - Zhang, J AU - Mazur, E AU - Balla, J AU - Gallei, Michelle C AU - Kalousek, P AU - Medveďová, Z AU - Li, Y AU - Wang, Y AU - Prat, Tomas AU - Vasileva, Mina K AU - Reinöhl, V AU - Procházka, S AU - Halouzka, R AU - Tarkowski, P AU - Luschnig, C AU - Brewer, PB AU - Friml, Jiří ID - 8138 IS - 1 JF - Nature Communications SN - 2041-1723 TI - Strigolactones inhibit auxin feedback on PIN-dependent auxin transport canalization VL - 11 ER - TY - JOUR AU - He, Peng AU - Zhang, Yuzhou AU - Xiao, Guanghui ID - 8271 IS - 9 JF - Molecular Plant SN - 16742052 TI - Origin of a subgenome and genome evolution of allotetraploid cotton species VL - 13 ER - TY - JOUR AB - Cytokinins are mobile multifunctional plant hormones with roles in development and stress resilience. Although their Histidine Kinase receptors are substantially localised to the endoplasmic reticulum, cellular sites of cytokinin perception and importance of spatially heterogeneous cytokinin distribution continue to be debated. Here we show that cytokinin perception by plasma membrane receptors is an effective additional path for cytokinin response. Readout from a Two Component Signalling cytokinin-specific reporter (TCSn::GFP) closely matches intracellular cytokinin content in roots, yet we also find cytokinins in extracellular fluid, potentially enabling action at the cell surface. Cytokinins covalently linked to beads that could not pass the plasma membrane increased expression of both TCSn::GFP and Cytokinin Response Factors. Super-resolution microscopy of GFP-labelled receptors and diminished TCSn::GFP response to immobilised cytokinins in cytokinin receptor mutants, further indicate that receptors can function at the cell surface. We argue that dual intracellular and surface locations may augment flexibility of cytokinin responses. AU - Antoniadi, Ioanna AU - Novák, Ondřej AU - Gelová, Zuzana AU - Johnson, Alexander J AU - Plíhal, Ondřej AU - Simerský, Radim AU - Mik, Václav AU - Vain, Thomas AU - Mateo-Bonmatí, Eduardo AU - Karady, Michal AU - Pernisová, Markéta AU - Plačková, Lenka AU - Opassathian, Korawit AU - Hejátko, Jan AU - Robert, Stéphanie AU - Friml, Jiří AU - Doležal, Karel AU - Ljung, Karin AU - Turnbull, Colin ID - 8337 JF - Nature Communications TI - Cell-surface receptors enable perception of extracellular cytokinins VL - 11 ER - TY - JOUR AB - Spontaneously arising channels that transport the phytohormone auxin provide positional cues for self-organizing aspects of plant development such as flexible vasculature regeneration or its patterning during leaf venation. The auxin canalization hypothesis proposes a feedback between auxin signaling and transport as the underlying mechanism, but molecular players await discovery. We identified part of the machinery that routes auxin transport. The auxin-regulated receptor CAMEL (Canalization-related Auxin-regulated Malectin-type RLK) together with CANAR (Canalization-related Receptor-like kinase) interact with and phosphorylate PIN auxin transporters. camel and canar mutants are impaired in PIN1 subcellular trafficking and auxin-mediated PIN polarization, which macroscopically manifests as defects in leaf venation and vasculature regeneration after wounding. The CAMEL-CANAR receptor complex is part of the auxin feedback that coordinates polarization of individual cells during auxin canalization. AU - Hajny, Jakub AU - Prat, Tomas AU - Rydza, N AU - Rodriguez Solovey, Lesia AU - Tan, Shutang AU - Verstraeten, Inge AU - Domjan, David AU - Mazur, E AU - Smakowska-Luzan, E AU - Smet, W AU - Mor, E AU - Nolf, J AU - Yang, B AU - Grunewald, W AU - Molnar, Gergely AU - Belkhadir, Y AU - De Rybel, B AU - Friml, Jiří ID - 8721 IS - 6516 JF - Science SN - 0036-8075 TI - Receptor kinase module targets PIN-dependent auxin transport during canalization VL - 370 ER - TY - JOUR AB - Peptides derived from non-functional precursors play important roles in various developmental processes, but also in (a)biotic stress signaling. Our (phospho)proteome-wide analyses of C-terminally encoded peptide 5 (CEP5)-mediated changes revealed an impact on abiotic stress-related processes. Drought has a dramatic impact on plant growth, development and reproduction, and the plant hormone auxin plays a role in drought responses. Our genetic, physiological, biochemical and pharmacological results demonstrated that CEP5-mediated signaling is relevant for osmotic and drought stress tolerance in Arabidopsis, and that CEP5 specifically counteracts auxin effects. Specifically, we found that CEP5 signaling stabilizes AUX/IAA transcriptional repressors, suggesting the existence of a novel peptide-dependent control mechanism that tunes auxin signaling. These observations align with the recently described role of AUX/IAAs in stress tolerance and provide a novel role for CEP5 in osmotic and drought stress tolerance. AU - Smith, S AU - Zhu, S AU - Joos, L AU - Roberts, I AU - Nikonorova, N AU - Vu, LD AU - Stes, E AU - Cho, H AU - Larrieu, A AU - Xuan, W AU - Goodall, B AU - van de Cotte, B AU - Waite, JM AU - Rigal, A AU - R Harborough, SR AU - Persiau, G AU - Vanneste, S AU - Kirschner, GK AU - Vandermarliere, E AU - Martens, L AU - Stahl, Y AU - Audenaert, D AU - Friml, Jiří AU - Felix, G AU - Simon, R AU - Bennett, M AU - Bishopp, A AU - De Jaeger, G AU - Ljung, K AU - Kepinski, S AU - Robert, S AU - Nemhauser, J AU - Hwang, I AU - Gevaert, K AU - Beeckman, T AU - De Smet, I ID - 7949 IS - 8 JF - Molecular & Cellular Proteomics TI - The CEP5 peptide promotes abiotic stress tolerance, as revealed by quantitative proteomics, and attenuates the AUX/IAA equilibrium in Arabidopsis VL - 19 ER - TY - JOUR AB - Cell polarity is a fundamental feature of all multicellular organisms. In plants, prominent cell polarity markers are PIN auxin transporters crucial for plant development. To identify novel components involved in cell polarity establishment and maintenance, we carried out a forward genetic screening with PIN2:PIN1-HA;pin2 Arabidopsis plants, which ectopically express predominantly basally localized PIN1 in the root epidermal cells leading to agravitropic root growth. From the screen, we identified the regulator of PIN polarity 12 (repp12) mutation, which restored gravitropic root growth and caused PIN1-HA polarity switch from basal to apical side of root epidermal cells. Complementation experiments established the repp12 causative mutation as an amino acid substitution in Aminophospholipid ATPase3 (ALA3), a phospholipid flippase with predicted function in vesicle formation. ala3 T-DNA mutants show defects in many auxin-regulated processes, in asymmetric auxin distribution and in PIN trafficking. Analysis of quintuple and sextuple mutants confirmed a crucial role of ALA proteins in regulating plant development and in PIN trafficking and polarity. Genetic and physical interaction studies revealed that ALA3 functions together with GNOM and BIG3 ARF GEFs. Taken together, our results identified ALA3 flippase as an important interactor and regulator of ARF GEF functioning in PIN polarity, trafficking and auxin-mediated development. AU - Zhang, Xixi AU - Adamowski, Maciek AU - Marhavá, Petra AU - Tan, Shutang AU - Zhang, Yuzhou AU - Rodriguez Solovey, Lesia AU - Zwiewka, Marta AU - Pukyšová, Vendula AU - Sánchez, Adrià Sans AU - Raxwal, Vivek Kumar AU - Hardtke, Christian S. AU - Nodzynski, Tomasz AU - Friml, Jiří ID - 7619 IS - 5 JF - The Plant Cell SN - 1040-4651 TI - Arabidopsis flippases cooperate with ARF GTPase exchange factors to regulate the trafficking and polarity of PIN auxin transporters VL - 32 ER - TY - JOUR AB - Clathrin-mediated endocytosis (CME) and its core endocytic machinery are evolutionarily conserved across all eukaryotes. In mammals, the heterotetrameric adaptor protein complex-2 (AP-2) sorts plasma membrane (PM) cargoes into vesicles through the recognition of motifs based on tyrosine or di-leucine in their cytoplasmic tails. However, in plants, very little is known on how PM proteins are sorted for CME and whether similar motifs are required. In Arabidopsis thaliana, the brassinosteroid (BR) receptor, BR INSENSITIVE1 (BRI1), undergoes endocytosis that depends on clathrin and AP-2. Here we demonstrate that BRI1 binds directly to the medium AP-2 subunit, AP2M. The cytoplasmic domain of BRI1 contains five putative canonical surface-exposed tyrosine-based endocytic motifs. The tyrosine-to-phenylalanine substitution in Y898KAI reduced BRI1 internalization without affecting its kinase activity. Consistently, plants carrying the BRI1Y898F mutation were hypersensitive to BRs. Our study demonstrates that AP-2-dependent internalization of PM proteins via the recognition of functional tyrosine motifs also operates in plants. AU - Liu, D AU - Kumar, R AU - LAN, Claus AU - Johnson, Alexander J AU - Siao, W AU - Vanhoutte, I AU - Wang, P AU - Bender, KW AU - Yperman, K AU - Martins, S AU - Zhao, X AU - Vert, G AU - Van Damme, D AU - Friml, Jiří AU - Russinova, E ID - 8607 IS - 11 JF - Plant Cell SN - 1040-4651 TI - Endocytosis of BRASSINOSTEROID INSENSITIVE1 is partly driven by a canonical tyrosine-based Motif VL - 32 ER - TY - JOUR AB - The TPLATE complex (TPC) is a key endocytic adaptor protein complex in plants. TPC in Arabidopsis (Arabidopsis thaliana) contains six evolutionarily conserved subunits and two plant-specific subunits, AtEH1/Pan1 and AtEH2/Pan1, although cytoplasmic proteins are not associated with the hexameric subcomplex in the cytoplasm. To investigate the dynamic assembly of the octameric TPC at the plasma membrane (PM), we performed state-of-the-art dual-color live cell imaging at physiological and lowered temperatures. Lowering the temperature slowed down endocytosis, thereby enhancing the temporal resolution of the differential recruitment of endocytic components. Under both normal and lowered temperature conditions, the core TPC subunit TPLATE and the AtEH/Pan1 proteins exhibited simultaneous recruitment at the PM. These results, together with co-localization analysis of different TPC subunits, allow us to conclude that TPC in plant cells is not recruited to the PM sequentially but as an octameric complex. AU - Wang, J AU - Mylle, E AU - Johnson, Alexander J AU - Besbrugge, N AU - De Jaeger, G AU - Friml, Jiří AU - Pleskot, R AU - van Damme, D ID - 7695 IS - 3 JF - Plant Physiology SN - 0032-0889 TI - High temporal resolution reveals simultaneous plasma membrane recruitment of TPLATE complex subunits VL - 183 ER - TY - JOUR AB - * Morphogenesis and adaptive tropic growth in plants depend on gradients of the phytohormone auxin, mediated by the membrane‐based PIN‐FORMED (PIN) auxin transporters. PINs localize to a particular side of the plasma membrane (PM) or to the endoplasmic reticulum (ER) to directionally transport auxin and maintain intercellular and intracellular auxin homeostasis, respectively. However, the molecular cues that confer their diverse cellular localizations remain largely unknown. * In this study, we systematically swapped the domains between ER‐ and PM‐localized PIN proteins, as well as between apical and basal PM‐localized PINs from Arabidopsis thaliana , to shed light on why PIN family members with similar topological structures reside at different membrane compartments within cells. * Our results show that not only do the N‐ and C‐terminal transmembrane domains (TMDs) and central hydrophilic loop contribute to their differential subcellular localizations and cellular polarity, but that the pairwise‐matched N‐ and C‐terminal TMDs resulting from intramolecular domain–domain coevolution are also crucial for their divergent patterns of localization. * These findings illustrate the complexity of the evolutionary path of PIN proteins in acquiring their plethora of developmental functions and adaptive growth in plants. AU - Zhang, Yuzhou AU - Hartinger, Corinna AU - Wang, Xiaojuan AU - Friml, Jiří ID - 7697 IS - 5 JF - New Phytologist SN - 0028-646X TI - Directional auxin fluxes in plants by intramolecular domain‐domain co‐evolution of PIN auxin transporters VL - 227 ER - TY - JOUR AB - Previously, we reported that the allelic de-etiolated by zinc (dez) and trichome birefringence (tbr) mutants exhibit photomorphogenic development in the dark, which is enhanced by high Zn. TRICHOME BIREFRINGENCE-LIKE proteins had been implicated in transferring acetyl groups to various hemicelluloses. Pectin O-acetylation levels were lower in dark-grown dez seedlings than in the wild type. We observed Zn-enhanced photomorphogenesis in the dark also in the reduced wall acetylation 2 (rwa2-3) mutant, which exhibits lowered O-acetylation levels of cell wall macromolecules including pectins and xyloglucans, supporting a role for cell wall macromolecule O-acetylation in the photomorphogenic phenotypes of rwa2-3 and dez. Application of very short oligogalacturonides (vsOGs) restored skotomorphogenesis in dark-grown dez and rwa2-3. Here we demonstrate that in dez, O-acetylation of non-pectin cell wall components, notably of xyloglucan, is enhanced. Our results highlight the complexity of cell wall homeostasis and indicate against an influence of xyloglucan O-acetylation on light-dependent seedling development. AU - Sinclair, Scott A AU - Gille, S. AU - Pauly, M. AU - Krämer, U. ID - 7417 IS - 1 JF - Plant Signaling & Behavior SN - 1559-2324 TI - Regulation of acetylation of plant cell wall components is complex and responds to external stimuli VL - 15 ER - TY - THES AB - The plant hormone auxin plays indispensable roles in plant growth and development. An essential level of regulation in auxin action is the directional auxin transport within cells. The establishment of auxin gradient in plant tissue has been attributed to local auxin biosynthesis and directional intercellular auxin transport, which both are controlled by various environmental and developmental signals. It is well established that asymmetric auxin distribution in cells is achieved by polarly localized PIN-FORMED (PIN) auxin efflux transporters. Despite the initial insights into cellular mechanisms of PIN polarization obtained from the last decades, the molecular mechanism and specific regulators mediating PIN polarization remains elusive. In this thesis, we aim to find novel players in PIN subcellular polarity regulation during Arabidopsis development. We first characterize the physiological effect of piperonylic acid (PA) on Arabidopsis hypocotyl gravitropic bending and PIN polarization. Secondly, we reveal the importance of SCFTIR1/AFB auxin signaling pathway in shoot gravitropism bending termination. In addition, we also explore the role of myosin XI complex, and actin cytoskeleton in auxin feedback regulation on PIN polarity. In Chapter 1, we give an overview of the current knowledge about PIN-mediated auxin fluxes in various plant tropic responses. In Chapter 2, we study the physiological effect of PA on shoot gravitropic bending. Our results show that PA treatment inhibits auxin-mediated PIN3 repolarization by interfering with PINOID and PIN3 phosphorylation status, ultimately leading to hyperbending hypocotyls. In Chapter 3, we provide evidence to show that the SCFTIR1/AFB nuclear auxin signaling pathway is crucial and required for auxin-mediated PIN3 repolarization and shoot gravitropic bending termination. In Chapter 4, we perform a phosphoproteomics approach and identify the motor protein Myosin XI and its binding protein, the MadB2 family, as an essential regulator of PIN polarity for auxin-canalization related developmental processes. In Chapter 5, we demonstrate the vital role of actin cytoskeleton in auxin feedback on PIN polarity by regulating PIN subcellular trafficking. Overall, the data presented in this PhD thesis brings novel insights into the PIN polar localization regulation that resulted in the (re)establishment of the polar auxin flow and gradient in response to environmental stimuli during plant development. AU - Han, Huibin ID - 8589 SN - 2663-337X TI - Novel insights into PIN polarity regulation during Arabidopsis development ER - TY - JOUR AU - Han, Huibin AU - Rakusova, Hana AU - Verstraeten, Inge AU - Zhang, Yuzhou AU - Friml, Jiří ID - 7643 IS - 5 JF - Plant Physiology SN - 0032-0889 TI - SCF TIR1/AFB auxin signaling for bending termination during shoot gravitropism VL - 183 ER - TY - JOUR AB - Earlier, we demonstrated that transcript levels of METAL TOLERANCE PROTEIN2 (MTP2) and of HEAVY METAL ATPase2 (HMA2) increase strongly in roots of Arabidopsis upon prolonged zinc (Zn) deficiency and respond to shoot physiological Zn status, and not to the local Zn status in roots. This provided evidence for shoot-to-root communication in the acclimation of plants to Zn deficiency. Zn-deficient soils limit both the yield and quality of agricultural crops and can result in clinically relevant nutritional Zn deficiency in human populations. Implementing Zn deficiency during cultivation of the model plant Arabidopsis thaliana on agar-solidified media is difficult because trace element contaminations are present in almost all commercially available agars. Here, we demonstrate root morphological acclimations to Zn deficiency on agar-solidified medium following the effective removal of contaminants. These advancements allow reproducible phenotyping toward understanding fundamental plant responses to deficiencies of Zn and other essential trace elements. AU - Sinclair, Scott A AU - Krämer, U. ID - 7416 IS - 1 JF - Plant Signaling & Behavior SN - 1559-2324 TI - Generation of effective zinc-deficient agar-solidified media allows identification of root morphology changes in response to zinc limitation VL - 15 ER - TY - JOUR AB - The widely used non-steroidal anti-inflammatory drugs (NSAIDs) are derivatives of the phytohormone salicylic acid (SA). SA is well known to regulate plant immunity and development, whereas there have been few reports focusing on the effects of NSAIDs in plants. Our studies here reveal that NSAIDs exhibit largely overlapping physiological activities to SA in the model plant Arabidopsis. NSAID treatments lead to shorter and agravitropic primary roots and inhibited lateral root organogenesis. Notably, in addition to the SA-like action, which in roots involves binding to the protein phosphatase 2A (PP2A), NSAIDs also exhibit PP2A-independent effects. Cell biological and biochemical analyses reveal that many NSAIDs bind directly to and inhibit the chaperone activity of TWISTED DWARF1, thereby regulating actin cytoskeleton dynamics and subsequent endosomal trafficking. Our findings uncover an unexpected bioactivity of human pharmaceuticals in plants and provide insights into the molecular mechanism underlying the cellular action of this class of anti-inflammatory compounds. AU - Tan, Shutang AU - Di Donato, Martin AU - Glanc, Matous AU - Zhang, Xixi AU - Klíma, Petr AU - Liu, Jie AU - Bailly, Aurélien AU - Ferro, Noel AU - Petrášek, Jan AU - Geisler, Markus AU - Friml, Jiří ID - 8943 IS - 9 JF - Cell Reports TI - Non-steroidal anti-inflammatory drugs target TWISTED DWARF1-regulated actin dynamics and auxin transport-mediated plant development VL - 33 ER - TY - JOUR AB - Wound healing in plant tissues, consisting of rigid cell wall-encapsulated cells, represents a considerable challenge and occurs through largely unknown mechanisms distinct from those in animals. Owing to their inability to migrate, plant cells rely on targeted cell division and expansion to regenerate wounds. Strict coordination of these wound-induced responses is essential to ensure efficient, spatially restricted wound healing. Single-cell tracking by live imaging allowed us to gain mechanistic insight into the wound perception and coordination of wound responses after laser-based wounding in Arabidopsis root. We revealed a crucial contribution of the collapse of damaged cells in wound perception and detected an auxin increase specific to cells immediately adjacent to the wound. This localized auxin increase balances wound-induced cell expansion and restorative division rates in a dose-dependent manner, leading to tumorous overproliferation when the canonical TIR1 auxin signaling is disrupted. Auxin and wound-induced turgor pressure changes together also spatially define the activation of key components of regeneration, such as the transcription regulator ERF115. Our observations suggest that the wound signaling involves the sensing of collapse of damaged cells and a local auxin signaling activation to coordinate the downstream transcriptional responses in the immediate wound vicinity. AU - Hörmayer, Lukas AU - Montesinos López, Juan C AU - Marhavá, Petra AU - Benková, Eva AU - Yoshida, Saiko AU - Friml, Jiří ID - 8002 IS - 26 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - Wounding-induced changes in cellular pressure and localized auxin signalling spatially coordinate restorative divisions in roots VL - 117 ER - TY - JOUR AB - Plants, like other multicellular organisms, survive through a delicate balance between growth and defense against pathogens. Salicylic acid (SA) is a major defense signal in plants, and the perception mechanism as well as downstream signaling activating the immune response are known. Here, we identify a parallel SA signaling that mediates growth attenuation. SA directly binds to A subunits of protein phosphatase 2A (PP2A), inhibiting activity of this complex. Among PP2A targets, the PIN2 auxin transporter is hyperphosphorylated in response to SA, leading to changed activity of this important growth regulator. Accordingly, auxin transport and auxin-mediated root development, including growth, gravitropic response, and lateral root organogenesis, are inhibited. This study reveals how SA, besides activating immunity, concomitantly attenuates growth through crosstalk with the auxin distribution network. Further analysis of this dual role of SA and characterization of additional SA-regulated PP2A targets will provide further insights into mechanisms maintaining a balance between growth and defense. AU - Tan, Shutang AU - Abas, Melinda F AU - Verstraeten, Inge AU - Glanc, Matous AU - Molnar, Gergely AU - Hajny, Jakub AU - Lasák, Pavel AU - Petřík, Ivan AU - Russinova, Eugenia AU - Petrášek, Jan AU - Novák, Ondřej AU - Pospíšil, Jiří AU - Friml, Jiří ID - 7427 IS - 3 JF - Current Biology SN - 09609822 TI - Salicylic acid targets protein phosphatase 2A to attenuate growth in plants VL - 30 ER - TY - JOUR AB - Plant survival depends on vascular tissues, which originate in a self‐organizing manner as strands of cells co‐directionally transporting the plant hormone auxin. The latter phenomenon (also known as auxin canalization) is classically hypothesized to be regulated by auxin itself via the effect of this hormone on the polarity of its own intercellular transport. Correlative observations supported this concept, but molecular insights remain limited. In the current study, we established an experimental system based on the model Arabidopsis thaliana, which exhibits auxin transport channels and formation of vasculature strands in response to local auxin application. Our methodology permits the genetic analysis of auxin canalization under controllable experimental conditions. By utilizing this opportunity, we confirmed the dependence of auxin canalization on a PIN‐dependent auxin transport and nuclear, TIR1/AFB‐mediated auxin signaling. We also show that leaf venation and auxin‐mediated PIN repolarization in the root require TIR1/AFB signaling. Further studies based on this experimental system are likely to yield better understanding of the mechanisms underlying auxin transport polarization in other developmental contexts. AU - Mazur, E AU - Kulik, Ivan AU - Hajny, Jakub AU - Friml, Jiří ID - 7500 IS - 5 JF - New Phytologist SN - 0028-646x TI - Auxin canalization and vascular tissue formation by TIR1/AFB-mediated auxin signaling in arabidopsis VL - 226 ER - TY - THES AB - Self-organization is a hallmark of plant development manifested e.g. by intricate leaf vein patterns, flexible formation of vasculature during organogenesis or its regeneration following wounding. Spontaneously arising channels transporting the phytohormone auxin, created by coordinated polar localizations of PIN-FORMED 1 (PIN1) auxin exporter, provide positional cues for these as well as other plant patterning processes. To find regulators acting downstream of auxin and the TIR1/AFB auxin signaling pathway essential for PIN1 coordinated polarization during auxin canalization, we performed microarray experiments. Besides the known components of general PIN polarity maintenance, such as PID and PIP5K kinases, we identified and characterized a new regulator of auxin canalization, the transcription factor WRKY DNA-BINDING PROTEIN 23 (WRKY23). Next, we designed a subsequent microarray experiment to further uncover other molecular players, downstream of auxin-TIR1/AFB-WRKY23 involved in the regulation of auxin-mediated PIN repolarization. We identified a novel and crucial part of the molecular machinery underlying auxin canalization. The auxin-regulated malectin-type receptor-like kinase CAMEL and the associated leucine-rich repeat receptor-like kinase CANAR target and directly phosphorylate PIN auxin transporters. camel and canar mutants are impaired in PIN1 subcellular trafficking and auxin-mediated repolarization leading to defects in auxin transport, ultimately to leaf venation and vasculature regeneration defects. Our results describe the CAMEL-CANAR receptor complex, which is required for auxin feed-back on its own transport and thus for coordinated tissue polarization during auxin canalization. AU - Hajny, Jakub ID - 8822 SN - 2663-337X TI - Identification and characterization of the molecular machinery of auxin-dependent canalization during vasculature formation and regeneration ER - TY - JOUR AB - Flowering plants display the highest diversity among plant species and have notably shaped terrestrial landscapes. Nonetheless, the evolutionary origin of their unprecedented morphological complexity remains largely an enigma. Here, we show that the coevolution of cis-regulatory and coding regions of PIN-FORMED (PIN) auxin transporters confined their expression to certain cell types and directed their subcellular localization to particular cell sides, which together enabled dynamic auxin gradients across tissues critical to the complex architecture of flowering plants. Extensive intraspecies and interspecies genetic complementation experiments with PINs from green alga up to flowering plant lineages showed that PIN genes underwent three subsequent, critical evolutionary innovations and thus acquired a triple function to regulate the development of three essential components of the flowering plant Arabidopsis: shoot/root, inflorescence, and floral organ. Our work highlights the critical role of functional innovations within the PIN gene family as essential prerequisites for the origin of flowering plants. AU - Zhang, Yuzhou AU - Rodriguez Solovey, Lesia AU - Li, Lanxin AU - Zhang, Xixi AU - Friml, Jiří ID - 8986 IS - 50 JF - Science Advances TI - Functional innovations of PIN auxin transporters mark crucial evolutionary transitions during rise of flowering plants VL - 6 ER - TY - JOUR AB - Drought and salt stress are the main environmental cues affecting the survival, development, distribution, and yield of crops worldwide. MYB transcription factors play a crucial role in plants’ biological processes, but the function of pineapple MYB genes is still obscure. In this study, one of the pineapple MYB transcription factors, AcoMYB4, was isolated and characterized. The results showed that AcoMYB4 is localized in the cell nucleus, and its expression is induced by low temperature, drought, salt stress, and hormonal stimulation, especially by abscisic acid (ABA). Overexpression of AcoMYB4 in rice and Arabidopsis enhanced plant sensitivity to osmotic stress; it led to an increase in the number stomata on leaf surfaces and lower germination rate under salt and drought stress. Furthermore, in AcoMYB4 OE lines, the membrane oxidation index, free proline, and soluble sugar contents were decreased. In contrast, electrolyte leakage and malondialdehyde (MDA) content increased significantly due to membrane injury, indicating higher sensitivity to drought and salinity stresses. Besides the above, both the expression level and activities of several antioxidant enzymes were decreased, indicating lower antioxidant activity in AcoMYB4 transgenic plants. Moreover, under osmotic stress, overexpression of AcoMYB4 inhibited ABA biosynthesis through a decrease in the transcription of genes responsible for ABA synthesis (ABA1 and ABA2) and ABA signal transduction factor ABI5. These results suggest that AcoMYB4 negatively regulates osmotic stress by attenuating cellular ABA biosynthesis and signal transduction pathways. AU - Chen, Huihuang AU - Lai, Linyi AU - Li, Lanxin AU - Liu, Liping AU - Jakada, Bello Hassan AU - Huang, Youmei AU - He, Qing AU - Chai, Mengnan AU - Niu, Xiaoping AU - Qin, Yuan ID - 8283 IS - 16 JF - International Journal of Molecular Sciences SN - 16616596 TI - AcoMYB4, an Ananas comosus L. MYB transcription factor, functions in osmotic stress through negative regulation of ABA signaling VL - 21 ER - TY - JOUR AB - Clathrin-mediated endocytosis (CME) is a crucial cellular process implicated in many aspects of plant growth, development, intra- and inter-cellular signaling, nutrient uptake and pathogen defense. Despite these significant roles, little is known about the precise molecular details of how it functions in planta. In order to facilitate the direct quantitative study of plant CME, here we review current routinely used methods and present refined, standardized quantitative imaging protocols which allow the detailed characterization of CME at multiple scales in plant tissues. These include: (i) an efficient electron microscopy protocol for the imaging of Arabidopsis CME vesicles in situ, thus providing a method for the detailed characterization of the ultra-structure of clathrin-coated vesicles; (ii) a detailed protocol and analysis for quantitative live-cell fluorescence microscopy to precisely examine the temporal interplay of endocytosis components during single CME events; (iii) a semi-automated analysis to allow the quantitative characterization of global internalization of cargos in whole plant tissues; and (iv) an overview and validation of useful genetic and pharmacological tools to interrogate the molecular mechanisms and function of CME in intact plant samples. AU - Johnson, Alexander J AU - Gnyliukh, Nataliia AU - Kaufmann, Walter AU - Narasimhan, Madhumitha AU - Vert, G AU - Bednarek, SY AU - Friml, Jiří ID - 8139 IS - 15 JF - Journal of Cell Science SN - 0021-9533 TI - Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis VL - 133 ER - TY - JOUR AB - The interorganelle communication mediated by membrane contact sites (MCSs) is an evolutionary hallmark of eukaryotic cells. MCS connections enable the nonvesicular exchange of information between organelles and allow them to coordinate responses to changing cellular environments. In plants, the importance of MCS components in the responses to environmental stress has been widely established, but the molecular mechanisms regulating interorganelle connectivity during stress still remain opaque. In this report, we use the model plant Arabidopsis thaliana to show that ionic stress increases endoplasmic reticulum (ER)–plasma membrane (PM) connectivity by promoting the cortical expansion of synaptotagmin 1 (SYT1)-enriched ER–PM contact sites (S-EPCSs). We define differential roles for the cortical cytoskeleton in the regulation of S-EPCS dynamics and ER–PM connectivity, and we identify the accumulation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] at the PM as a molecular signal associated with the ER–PM connectivity changes. Our study highlights the functional conservation of EPCS components and PM phosphoinositides as modulators of ER–PM connectivity in eukaryotes, and uncovers unique aspects of the spatiotemporal regulation of ER–PM connectivity in plants. AU - Lee, Eunkyoung AU - Vanneste, Steffen AU - Pérez-Sancho, Jessica AU - Benitez-Fuente, Francisco AU - Strelau, Matthew AU - Macho, Alberto P. AU - Botella, Miguel A. AU - Friml, Jiří AU - Rosado, Abel ID - 5908 IS - 4 JF - Proceedings of the National Academy of Sciences of the United States of America TI - Ionic stress enhances ER–PM connectivity via phosphoinositide-associated SYT1 contact site expansion in Arabidopsis VL - 116 ER - TY - JOUR AB - Multicellular development requires coordinated cell polarization relative to body axes, and translation to oriented cell division 1–3 . In plants, it is unknown how cell polarities are connected to organismal axes and translated to division. Here, we identify Arabidopsis SOSEKI proteins that integrate apical–basal and radial organismal axes to localize to polar cell edges. Localization does not depend on tissue context, requires cell wall integrity and is defined by a transferrable, protein-specific motif. A Domain of Unknown Function in SOSEKI proteins resembles the DIX oligomerization domain in the animal Dishevelled polarity regulator. The DIX-like domain self-interacts and is required for edge localization and for influencing division orientation, together with a second domain that defines the polar membrane domain. Our work shows that SOSEKI proteins locally interpret global polarity cues and can influence cell division orientation. Furthermore, this work reveals that, despite fundamental differences, cell polarity mechanisms in plants and animals converge on a similar protein domain. AU - Yoshida, Saiko AU - Van Der Schuren, Alja AU - Van Dop, Maritza AU - Van Galen, Luc AU - Saiga, Shunsuke AU - Adibi, Milad AU - Möller, Barbara AU - Ten Hove, Colette A. AU - Marhavy, Peter AU - Smith, Richard AU - Friml, Jiří AU - Weijers, Dolf ID - 6023 IS - 2 JF - Nature Plants TI - A SOSEKI-based coordinate system interprets global polarity cues in arabidopsis VL - 5 ER - TY - JOUR AB - Abiotic stress poses constant challenges for plant survival and is a serious problem for global agricultural productivity. On a molecular level, stress conditions result in elevation of reactive oxygen species (ROS) production causing oxidative stress associated with oxidation of proteins and nucleic acids as well as impairment of membrane functions. Adaptation of root growth to ROS accumulation is facilitated through modification of auxin and cytokinin hormone homeostasis. Here, we report that in Arabidopsis root meristem, ROS-induced changes of auxin levels correspond to decreased abundance of PIN auxin efflux carriers at the plasma membrane (PM). Specifically, increase in H2O2 levels affects PIN2 endocytic recycling. We show that the PIN2 intracellular trafficking during adaptation to oxidative stress requires the function of the ADP-ribosylation factor (ARF)-guanine-nucleotide exchange factor (GEF) BEN1, an actin-associated regulator of the trafficking from the PM to early endosomes and, presumably, indirectly, trafficking to the vacuoles. We propose that H2O2 levels affect the actin dynamics thus modulating ARF-GEF-dependent trafficking of PIN2. This mechanism provides a way how root growth acclimates to stress and adapts to a changing environment. AU - Zwiewka, Marta AU - Bielach, Agnieszka AU - Tamizhselvan, Prashanth AU - Madhavan, Sharmila AU - Ryad, Eman Elrefaay AU - Tan, Shutang AU - Hrtyan, Mónika AU - Dobrev, Petre AU - Vanková, Radomira AU - Friml, Jiří AU - Tognetti, Vanesa B. ID - 6104 IS - 2 JF - Plant and Cell Physiology SN - 0032-0781 TI - Root adaptation to H2O2-induced oxidative stress by ARF-GEF BEN1- and cytoskeleton-mediated PIN2 trafficking VL - 60 ER - TY - JOUR AB - Gravitropism is an adaptive response that orients plant growth parallel to the gravity vector. Asymmetric distribution of the phytohormone auxin is a necessary prerequisite to the tropic bending both in roots and shoots. During hypocotyl gravitropic response, the PIN3 auxin transporter polarizes within gravity-sensing cells to redirect intercellular auxin fluxes. First gravity-induced PIN3 polarization to the bottom cell mem- branes leads to the auxin accumulation at the lower side of the organ, initiating bending and, later, auxin feedback-mediated repolarization restores symmetric auxin distribution to terminate bending. Here, we per- formed a forward genetic screen to identify regulators of both PIN3 polarization events during gravitropic response. We searched for mutants with defective PIN3 polarizations based on easy-to-score morphological outputs of decreased or increased gravity-induced hypocotyl bending. We identified the number of hypocotyl reduced bending (hrb) and hypocotyl hyperbending (hhb) mutants, revealing that reduced bending corre- lated typically with defective gravity-induced PIN3 relocation whereas all analyzed hhb mutants showed defects in the second, auxin-mediated PIN3 relocation. Next-generation sequencing-aided mutation map- ping identified several candidate genes, including SCARECROW and ACTIN2, revealing roles of endodermis specification and actin cytoskeleton in the respective gravity- and auxin-induced PIN polarization events. The hypocotyl gravitropism screen thus promises to provide novel insights into mechanisms underlying cell polarity and plant adaptive development. AU - Rakusová, Hana AU - Han, Huibin AU - Valošek, Petr AU - Friml, Jiří ID - 6262 IS - 6 JF - The Plant Journal SN - 0960-7412 TI - Genetic screen for factors mediating PIN polarization in gravistimulated Arabidopsis thaliana hypocotyls VL - 98 ER - TY - JOUR AB - Nitrate regulation of root stem cell activity is auxin-dependent. AU - Wang, Y AU - Gong, Z AU - Friml, Jiří AU - Zhang, J ID - 6261 IS - 1 JF - Plant Physiology SN - 0032-0889 TI - Nitrate modulates the differentiation of root distal stem cells VL - 180 ER - TY - JOUR AB - Root gravitropism is one of the most important processes allowing plant adaptation to the land environment. Auxin plays a central role in mediating root gravitropism, but how auxin contributes to gravitational perception and the subsequent response is still unclear. Here, we showed that the local auxin maximum/gradient within the root apex, which is generated by the PIN directional auxin transporters, regulates the expression of three key starch granule synthesis genes, SS4, PGM and ADG1, which in turn influence the accumulation of starch granules that serve as a statolith perceiving gravity. Moreover, using the cvxIAA‐ccvTIR1 system, we also showed that TIR1‐mediated auxin signaling is required for starch granule formation and gravitropic response within root tips. In addition, axr3 mutants showed reduced auxin‐mediated starch granule accumulation and disruption of gravitropism within the root apex. Our results indicate that auxin‐mediated statolith production relies on the TIR1/AFB‐AXR3‐mediated auxin signaling pathway. In summary, we propose a dual role for auxin in gravitropism: the regulation of both gravity perception and response. AU - Zhang, Yuzhou AU - He, P AU - Ma, X AU - Yang, Z AU - Pang, C AU - Yu, J AU - Wang, G AU - Friml, Jiří AU - Xiao, G ID - 6504 IS - 2 JF - New Phytologist SN - 0028-646x TI - Auxin-mediated statolith production for root gravitropism VL - 224 ER - TY - JOUR AB - Cell polarity is crucial for the coordinated development of all multicellular organisms. In plants, this is exemplified by the PIN-FORMED (PIN) efflux carriers of the phytohormone auxin: The polar subcellular localization of the PINs is instructive to the directional intercellular auxin transport, and thus to a plethora of auxin-regulated growth and developmental processes. Despite its importance, the regulation of PIN polar subcellular localization remains poorly understood. Here, we have employed advanced live-cell imaging techniques to study the roles of microtubules and actin microfilaments in the establishment of apical polar localization of PIN2 in the epidermis of the Arabidopsis root meristem. We report that apical PIN2 polarity requires neither intact actin microfilaments nor microtubules, suggesting that the primary spatial cue for polar PIN distribution is likely independent of cytoskeleton-guided endomembrane trafficking. AU - Glanc, Matous AU - Fendrych, Matyas AU - Friml, Jiří ID - 6611 IS - 6 JF - Biomolecules TI - PIN2 polarity establishment in arabidopsis in the absence of an intact cytoskeleton VL - 9 ER - TY - JOUR AB - An important adaptation during colonization of land by plants is gravitropic growth of roots, which enabled roots to reach water and nutrients, and firmly anchor plants in the ground. Here we provide insights into the evolution of an efficient root gravitropic mechanism in the seed plants. Architectural innovation, with gravity perception constrained in the root tips along with a shootward transport route for the phytohormone auxin, appeared only upon the emergence of seed plants. Interspecies complementation and protein domain swapping revealed functional innovations within the PIN family of auxin transporters leading to the evolution of gravitropism-specific PINs. The unique apical/shootward subcellular localization of PIN proteins is the major evolutionary innovation that connected the anatomically separated sites of gravity perception and growth response via the mobile auxin signal. We conclude that the crucial anatomical and functional components emerged hand-in-hand to facilitate the evolution of fast gravitropic response, which is one of the major adaptations of seed plants to dry land. AU - Zhang, Yuzhou AU - Xiao, G AU - Wang, X AU - Zhang, Xixi AU - Friml, Jiří ID - 6778 JF - Nature Communications SN - 2041-1723 TI - Evolution of fast root gravitropism in seed plants VL - 10 ER - TY - JOUR AB - Plants have a remarkable capacity to adjust their growth and development to elevated ambient temperatures. Increased elongation growth of roots, hypocotyls and petioles in warm temperatures are hallmarks of seedling thermomorphogenesis. In the last decade, significant progress has been made to identify the molecular signaling components regulating these growth responses. Increased ambient temperature utilizes diverse components of the light sensing and signal transduction network to trigger growth adjustments. However, it remains unknown whether temperature sensing and responses are universal processes that occur uniformly in all plant organs. Alternatively, temperature sensing may be confined to specific tissues or organs, which would require a systemic signal that mediates responses in distal parts of the plant. Here we show that Arabidopsis (Arabidopsis thaliana) seedlings show organ-specific transcriptome responses to elevated temperatures, and that thermomorphogenesis involves both autonomous and organ-interdependent temperature sensing and signaling. Seedling roots can sense and respond to temperature in a shoot-independent manner, whereas shoot temperature responses require both local and systemic processes. The induction of cell elongation in hypocotyls requires temperature sensing in cotyledons, followed by generation of a mobile auxin signal. Subsequently, auxin travels to the hypocotyl where it triggers local brassinosteroid-induced cell elongation in seedling stems, which depends upon a distinct, permissive temperature sensor in the hypocotyl. AU - Bellstaedt, Julia AU - Trenner, Jana AU - Lippmann, Rebecca AU - Poeschl, Yvonne AU - Zhang, Xixi AU - Friml, Jiří AU - Quint, Marcel AU - Delker, Carolin ID - 6366 IS - 2 JF - Plant Physiology SN - 0032-0889 TI - A mobile auxin signal connects temperature sensing in cotyledons with growth responses in hypocotyls VL - 180 ER - TY - JOUR AB - The plant hormone auxin has crucial roles in almost all aspects of plant growth and development. Concentrations of auxin vary across different tissues, mediating distinct developmental outcomes and contributing to the functional diversity of auxin. However, the mechanisms that underlie these activities are poorly understood. Here we identify an auxin signalling mechanism, which acts in parallel to the canonical auxin pathway based on the transport inhibitor response1 (TIR1) and other auxin receptor F-box (AFB) family proteins (TIR1/AFB receptors)1,2, that translates levels of cellular auxin to mediate differential growth during apical-hook development. This signalling mechanism operates at the concave side of the apical hook, and involves auxin-mediated C-terminal cleavage of transmembrane kinase 1 (TMK1). The cytosolic and nucleus-translocated C terminus of TMK1 specifically interacts with and phosphorylates two non-canonical transcriptional repressors of the auxin or indole-3-acetic acid (Aux/IAA) family (IAA32 and IAA34), thereby regulating ARF transcription factors. In contrast to the degradation of Aux/IAA transcriptional repressors in the canonical pathway, the newly identified mechanism stabilizes the non-canonical IAA32 and IAA34 transcriptional repressors to regulate gene expression and ultimately inhibit growth. The auxin–TMK1 signalling pathway originates at the cell surface, is triggered by high levels of auxin and shares a partially overlapping set of transcription factors with the TIR1/AFB signalling pathway. This allows distinct interpretations of different concentrations of cellular auxin, and thus enables this versatile signalling molecule to mediate complex developmental outcomes. AU - Cao, Min AU - Chen, Rong AU - Li, Pan AU - Yu, Yongqiang AU - Zheng, Rui AU - Ge, Danfeng AU - Zheng, Wei AU - Wang, Xuhui AU - Gu, Yangtao AU - Gelová, Zuzana AU - Friml, Jiří AU - Zhang, Heng AU - Liu, Renyi AU - He, Jun AU - Xu, Tongda ID - 6259 JF - Nature SN - 0028-0836 TI - TMK1-mediated auxin signalling regulates differential growth of the apical hook VL - 568 ER - TY - JOUR AB - PIN-FORMED (PIN) transporters mediate directional, intercellular movement of the phytohormone auxin in land plants. To elucidate the evolutionary origins of this developmentally crucial mechanism, we analysed the single PIN homologue of a simple green alga Klebsormidium flaccidum. KfPIN functions as a plasma membrane-localized auxin exporter in land plants and heterologous models. While its role in algae remains unclear, PIN-driven auxin export is probably an ancient and conserved trait within streptophytes. AU - Skokan, Roman AU - Medvecká, Eva AU - Viaene, Tom AU - Vosolsobě, Stanislav AU - Zwiewka, Marta AU - Müller, Karel AU - Skůpa, Petr AU - Karady, Michal AU - Zhang, Yuzhou AU - Janacek, Dorina P. AU - Hammes, Ulrich Z. AU - Ljung, Karin AU - Nodzyński, Tomasz AU - Petrášek, Jan AU - Friml, Jiří ID - 7106 IS - 11 JF - Nature Plants SN - 2055-0278 TI - PIN-driven auxin transport emerged early in streptophyte evolution VL - 5 ER - TY - JOUR AB - Roots grow downwards parallel to the gravity vector, to anchor a plant in soil and acquire water and nutrients, using a gravitropic mechanism dependent on the asymmetric distribution of the phytohormone auxin. Recently, Chang et al. demonstrate that asymmetric distribution of another phytohormone, cytokinin, directs root growth towards higher water content. AU - Sinclair, Scott A AU - Friml, Jiří ID - 7143 JF - Cell Research SN - 1001-0602 TI - Defying gravity: a plant's quest for moisture VL - 29 ER - TY - JOUR AB - During infection pathogens secrete small molecules, termed effectors, to manipulate and control the interaction with their specific hosts. Both the pathogen and the plant are under high selective pressure to rapidly adapt and co-evolve in what is usually referred to as molecular arms race. Components of the host’s immune system form a network that processes information about molecules with a foreign origin and damage-associated signals, integrating them with developmental and abiotic cues to adapt the plant’s responses. Both in the case of nucleotide-binding leucine-rich repeat receptors and leucine-rich repeat receptor kinases interaction networks have been extensively characterized. However, little is known on whether pathogenic effectors form complexes to overcome plant immunity and promote disease. Ustilago maydis, a biotrophic fungal pathogen that infects maize plants, produces effectors that target hubs in the immune network of the host cell. Here we assess the capability of U. maydis effector candidates to interact with each other, which may play a crucial role during the infection process. Using a systematic yeast-two-hybrid approach and based on a preliminary pooled screen, we selected 63 putative effectors for one-on-one matings with a library of nearly 300 effector candidates. We found that 126 of these effector candidates interacted either with themselves or other predicted effectors. Although the functional relevance of the observed interactions remains elusive, we propose that the observed abundance in complex formation between effectors adds an additional level of complexity to effector research and should be taken into consideration when studying effector evolution and function. Based on this fundamental finding, we suggest various scenarios which could evolutionarily drive the formation and stabilization of an effector interactome. AU - Alcântara, André AU - Bosch, Jason AU - Nazari, Fahimeh AU - Hoffmann, Gesa AU - Gallei, Michelle C AU - Uhse, Simon AU - Darino, Martin A. AU - Olukayode, Toluwase AU - Reumann, Daniel AU - Baggaley, Laura AU - Djamei, Armin ID - 7182 IS - 11 JF - Frontiers in Plant Science TI - Systematic Y2H screening reveals extensive effector-complex formation VL - 10 ER - TY - JOUR AB - Clathrin-mediated endocytosis (CME) is a highly conserved and essential cellular process in eukaryotic cells, but its dynamic and vital nature makes it challenging to study using classical genetics tools. In contrast, although small molecules can acutely and reversibly perturb CME, the few chemical CME inhibitors that have been applied to plants are either ineffective or show undesirable side effects. Here, we identify the previously described endosidin9 (ES9) as an inhibitor of clathrin heavy chain (CHC) function in both Arabidopsis and human cells through affinity-based target isolation, in vitro binding studies and X-ray crystallography. Moreover, we present a chemically improved ES9 analog, ES9-17, which lacks the undesirable side effects of ES9 while retaining the ability to target CHC. ES9 and ES9-17 have expanded the chemical toolbox used to probe CHC function, and present chemical scaffolds for further design of more specific and potent CHC inhibitors across different systems. AU - Dejonghe, Wim AU - Sharma, Isha AU - Denoo, Bram AU - De Munck, Steven AU - Lu, Qing AU - Mishev, Kiril AU - Bulut, Haydar AU - Mylle, Evelien AU - De Rycke, Riet AU - Vasileva, Mina K AU - Savatin, Daniel V. AU - Nerinckx, Wim AU - Staes, An AU - Drozdzecki, Andrzej AU - Audenaert, Dominique AU - Yperman, Klaas AU - Madder, Annemieke AU - Friml, Jiří AU - Van Damme, Daniël AU - Gevaert, Kris AU - Haucke, Volker AU - Savvides, Savvas N. AU - Winne, Johan AU - Russinova, Eugenia ID - 6377 IS - 6 JF - Nature Chemical Biology SN - 15524450 TI - Disruption of endocytosis through chemical inhibition of clathrin heavy chain function VL - 15 ER - TY - THES AB - The development and growth of Arabidopsis thaliana is regulated by a combination of genetic programing and also by the environmental influences. An important role in these processes play the phytohormones and among them, auxin is crucial as it controls many important functions. It is transported through the whole plant body by creating local and temporal concentration maxima and minima, which have an impact on the cell status, tissue and organ identity. Auxin has the property to undergo a directional and finely regulated cell-to-cell transport, which is enabled by the transport proteins, localized on the plasma membrane. An important role in this process have the PIN auxin efflux proteins, which have an asymmetric/polar subcellular localization and determine the directionality of the auxin transport. During the last years, there were significant advances in understanding how the trafficking molecular machineries function, including studies on molecular interactions, function, subcellular localization and intracellular distribution. However, there is still a lack of detailed characterization on the steps of endocytosis, exocytosis, endocytic recycling and degradation. Due to this fact, I focused on the identification of novel trafficking factors and better characterization of the intracellular trafficking pathways. My PhD thesis consists of an introductory chapter, three experimental chapters, a chapter containing general discussion, conclusions and perspectives and also an appendix chapter with published collaborative papers. The first chapter is separated in two different parts: I start by a general introduction to auxin biology and then I introduce the trafficking pathways in the model plant Arabidopsis thaliana. Then, I explain also the phosphorylation-signals for polar targeting and also the roles of the phytohormone strigolactone. The second chapter includes the characterization of bar1/sacsin mutant, which was identified in a forward genetic screen for novel trafficking components in Arabidopsis thaliana, where by the implementation of an EMS-treated pPIN1::PIN1-GFP marker line and by using the established inhibitor of ARF-GEFs, Brefeldin A (BFA) as a tool to study trafficking processes, we identified a novel factor, which is mediating the adaptation of the plant cell to ARF-GEF inhibition. The mutation is in a previously uncharacterized gene, encoding a very big protein that we, based on its homologies, called SACSIN with domains suggesting roles as a molecular chaperon or as a component of the ubiquitin-proteasome system. Our physiology and imaging studies revealed that SACSIN is a crucial plant cell component of the adaptation to the ARF-GEF inhibition. The third chapter includes six subchapters, where I focus on the role of the phytohormone strigolactone, which interferes with auxin feedback on PIN internalization. Strigolactone moderates the polar auxin transport by increasing the internalization of the PIN auxin efflux carriers, which reduces the canalization related growth responses. In addition, I also studied the role of phosphorylation in the strigolactone regulation of auxin feedback on PIN internalization. In this chapter I also present my results on the MAX2-dependence of strigolactone-mediated root growth inhibition and I also share my results on the auxin metabolomics profiling after application of GR24. In the fourth chapter I studied the effect of two small molecules ES-9 and ES9-17, which were identified from a collection of small molecules with the property to impair the clathrin-mediated endocytosis. In the fifth chapter, I discuss all my observations and experimental findings and suggest alternative hypothesis to interpret my results. In the appendix there are three collaborative published projects. In the first, I participated in the characterization of the role of ES9 as a small molecule, which is inhibitor of clathrin- mediated endocytosis in different model organisms. In the second paper, I contributed to the characterization of another small molecule ES9-17, which is a non-protonophoric analog of ES9 and also impairs the clathrin-mediated endocytosis not only in plant cells, but also in mammalian HeLa cells. Last but not least, I also attach another paper, where I tried to establish the grafting method as a technique in our lab to study canalization related processes. AU - Vasileva, Mina K ID - 7172 TI - Molecular mechanisms of endomembrane trafficking in Arabidopsis thaliana ER - TY - JOUR AB - Plasmodesmata (PD) are plant-specific membrane-lined channels that create cytoplasmic and membrane continuities between adjacent cells, thereby facilitating cell–cell communication and virus movement. Plant cells have evolved diverse mechanisms to regulate PD plasticity in response to numerous environmental stimuli. In particular, during defense against plant pathogens, the defense hormone, salicylic acid (SA), plays a crucial role in the regulation of PD permeability in a callose-dependent manner. Here, we uncover a mechanism by which plants restrict the spreading of virus and PD cargoes using SA signaling by increasing lipid order and closure of PD. We showed that exogenous SA application triggered the compartmentalization of lipid raft nanodomains through a modulation of the lipid raft-regulatory protein, Remorin (REM). Genetic studies, superresolution imaging, and transmission electron microscopy observation together demonstrated that Arabidopsis REM1.2 and REM1.3 are crucial for plasma membrane nanodomain assembly to control PD aperture and functionality. In addition, we also found that a 14-3-3 epsilon protein modulates REM clustering and membrane nanodomain compartmentalization through its direct interaction with REM proteins. This study unveils a molecular mechanism by which the key plant defense hormone, SA, triggers membrane lipid nanodomain reorganization, thereby regulating PD closure to impede virus spreading. AU - Huang, D AU - Sun, Y AU - Ma, Z AU - Ke, M AU - Cui, Y AU - Chen, Z AU - Chen, C AU - Ji, C AU - Tran, TM AU - Yang, L AU - Lam, SM AU - Han, Y AU - Shu, G AU - Friml, Jiří AU - Miao, Y AU - Jiang, L AU - Chen, X ID - 6999 IS - 42 JF - Proceedings of the National Academy of Sciences of the United States of America SN - 0027-8424 TI - Salicylic acid-mediated plasmodesmal closure via Remorin-dependent lipid organization VL - 116 ER - TY - THES AB - Clathrin-Mediated Endocytosis (CME) is an aspect of cellular trafficking that is constantly regulated for mediating developmental and physiological responses. The main aim of my thesis is to decipher the basic mechanisms of CME and post-endocytic trafficking in the whole multicellular organ systems of Arabidopsis. The first chapter of my thesis describes the search for new components involved in CME. Tandem affinity purification was conducted using CLC and its interacting partners were identified. Amongst the identified proteins were the Auxilin-likes1 and 2 (Axl1/2), putative uncoating factors, for which we made a full functional analysis. Over-expression of Axl1/2 causes extreme modifications in the dynamics of the machinery proteins and inhibition of endocytosis altogether. However the loss of function of the axl1/2 did not present any cellular or physiological phenotype, meaning Auxilin-likes do not form the major uncoating machinery. The second chapter of my thesis describes the establishment/utilisation of techniques to capture the dynamicity and the complexity of CME and post-endocytic trafficking. We have studied the development of endocytic pits at the PM – specifically, the mode of membrane remodeling during pit development and the role of actin in it, given plant cells possess high turgor pressure. Utilizing the improved z-resolution of TIRF and VAEM techniques, we captured the time-lapse of the endocytic events at the plasma membrane; and using particle detection software, we quantitatively analysed all the endocytic trajectories in an unbiased way to obtain the endocytic rate of the system. This together with the direct analysis of cargo internalisation from the PM provided an estimate on the endocytic potential of the cell. We also developed a methodology for ultrastructural analysis of different populations of Clathrin-Coated Structures (CCSs) in both PM and endomembranes in unroofed protoplasts. Structural analysis, together with the intensity profile of CCSs at the PM show that the mode of CCP development at the PM follows ‘Constant curvature model’; meaning that clathrin polymerisation energy is a major contributing factor of membrane remodeling. In addition, other analyses clearly show that actin is not required for membrane remodeling during invagination or any other step of CCP development, despite the prevalent high turgor pressure. However, actin is essential in orchestrating the post-endocytic trafficking of CCVs facilitating the EE formation. We also observed that the uncoating process post-endocytosis is not immediate; an alternative mechanism of uncoating – Sequential multi-step process – functions in the cell. Finally we also looked at one of the important physiological stimuli modulating the process – hormone, auxin. auxin has been known to influence CME before. We have made a detailed study on the concentration-time based effect of auxin on the machinery proteins, CCP development, and the specificity of cargoes endocytosed. To this end, we saw no general effect of auxin on CME at earlier time points. However, very low concentration of IAA, such as 50nM, accelerates endocytosis of specifically PIN2 through CME. Such a tight regulatory control with high specificity to PIN2 could be essential in modulating its polarity. AU - Narasimhan, Madhumitha ID - 6269 SN - 2663-337X TI - Clathrin-Mediated endocytosis, post-endocytic trafficking and their regulatory controls in plants ER - TY - JOUR AB - A process of restorative patterning in plant roots correctly replaces eliminated cells to heal local injuries despite the absence of cell migration, which underpins wound healing in animals. Patterning in plants relies on oriented cell divisions and acquisition of specific cell identities. Plants regularly endure wounds caused by abiotic or biotic environmental stimuli and have developed extraordinary abilities to restore their tissues after injuries. Here, we provide insight into a mechanism of restorative patterning that repairs tissues after wounding. Laser-assisted elimination of different cells in Arabidopsis root combined with live-imaging tracking during vertical growth allowed analysis of the regeneration processes in vivo. Specifically, the cells adjacent to the inner side of the injury re-activated their stem cell transcriptional programs. They accelerated their progression through cell cycle, coordinately changed the cell division orientation, and ultimately acquired de novo the correct cell fates to replace missing cells. These observations highlight existence of unknown intercellular positional signaling and demonstrate the capability of specified cells to re-acquire stem cell programs as a crucial part of the plant-specific mechanism of wound healing. AU - Marhavá, Petra AU - Hörmayer, Lukas AU - Yoshida, Saiko AU - Marhavy, Peter AU - Benková, Eva AU - Friml, Jiří ID - 6351 IS - 4 JF - Cell SN - 00928674 TI - Re-activation of stem cell pathways for pattern restoration in plant wound healing VL - 177 ER - TY - JOUR AB - Plants as sessile organisms are constantly under attack by herbivores, rough environmental situations, or mechanical pressure. These challenges often lead to the induction of wounds or destruction of already specified and developed tissues. Additionally, wounding makes plants vulnerable to invasion by pathogens, which is why wound signalling often triggers specific defence responses. To stay competitive or, eventually, survive under these circumstances, plants need to regenerate efficiently, which in rigid, tissue migration-incompatible plant tissues requires post-embryonic patterning and organogenesis. Now, several studies used laser-assisted single cell ablation in the Arabidopsis root tip as a minimal wounding proxy. Here, we discuss their findings and put them into context of a broader spectrum of wound signalling, pathogen responses and tissue as well as organ regeneration. AU - Hörmayer, Lukas AU - Friml, Jiří ID - 6943 JF - Current Opinion in Plant Biology SN - 1369-5266 TI - Targeted cell ablation-based insights into wound healing and restorative patterning VL - 52 ER - TY - JOUR AB - Polar auxin transport plays a pivotal role in plant growth and development. PIN auxin efflux carriers regulate directional auxin movement by establishing local auxin maxima, minima, and gradients that drive multiple developmental processes and responses to environmental signals. Auxin has been proposed to modulate its own transport by regulating subcellular PIN trafficking via processes such as clathrin-mediated PIN endocytosis and constitutive recycling. Here, we further investigated the mechanisms by which auxin affects PIN trafficking by screening auxin analogs and identified pinstatic acid (PISA) as a positive modulator of polar auxin transport in Arabidopsis thaliana. PISA had an auxin-like effect on hypocotyl elongation and adventitious root formation via positive regulation of auxin transport. PISA did not activate SCFTIR1/AFB signaling and yet induced PIN accumulation at the cell surface by inhibiting PIN internalization from the plasma membrane. This work demonstrates PISA to be a promising chemical tool to dissect the regulatory mechanisms behind subcellular PIN trafficking and auxin transport. AU - Oochi, A AU - Hajny, Jakub AU - Fukui, K AU - Nakao, Y AU - Gallei, Michelle C AU - Quareshy, M AU - Takahashi, K AU - Kinoshita, T AU - Harborough, SR AU - Kepinski, S AU - Kasahara, H AU - Napier, RM AU - Friml, Jiří AU - Hayashi, KI ID - 6260 IS - 2 JF - Plant Physiology SN - 0032-0889 TI - Pinstatic acid promotes auxin transport by inhibiting PIN internalization VL - 180 ER - TY - JOUR AB - Cortical microtubule arrays in elongating epidermal cells in both the root and stem of plants have the propensity of dynamic reorientations that are correlated with the activation or inhibition of growth. Factors regulating plant growth, among them the hormone auxin, have been recognized as regulators of microtubule array orientations. Some previous work in the field has aimed at elucidating the causal relationship between cell growth, the signaling of auxin or other growth-regulating factors, and microtubule array reorientations, with various conclusions. Here, we revisit this problem of causality with a comprehensive set of experiments in Arabidopsis thaliana, using the now available pharmacological and genetic tools. We use isolated, auxin-depleted hypocotyls, an experimental system allowing for full control of both growth and auxin signaling. We demonstrate that reorientation of microtubules is not directly triggered by an auxin signal during growth activation. Instead, reorientation is triggered by the activation of the growth process itself and is auxin-independent in its nature. We discuss these findings in the context of previous relevant work, including that on the mechanical regulation of microtubule array orientation. AU - Adamowski, Maciek AU - Li, Lanxin AU - Friml, Jiří ID - 6627 IS - 13 JF - International Journal of Molecular Sciences TI - Reorientation of cortical microtubule arrays in the hypocotyl of arabidopsis thaliana is induced by the cell growth process and independent of auxin signaling VL - 20 ER - TY - CHAP AB - Adventitious roots (AR) are de novo formed roots that emerge from any part of the plant or from callus in tissue culture, except root tissue. The plant tissue origin and the method by which they are induced determine the physiological properties of emerged ARs. Hence, a standard method encompassing all types of AR does not exist. Here we describe a method for the induction and analysis of AR that emerge from the etiolated hypocotyl of dicot plants. The hypocotyl is formed during embryogenesis and shows a determined developmental pattern which usually does not involve AR formation. However, the hypocotyl shows propensity to form de novo roots under specific circumstances such as removal of the root system, high humidity or flooding, or during de-etiolation. The hypocotyl AR emerge from a pericycle-like cell layer surrounding the vascular tissue of the central cylinder, which is reminiscent to the developmental program of lateral roots. Here we propose an easy protocol for in vitro hypocotyl AR induction from etiolated Arabidopsis seedlings. AU - Trinh, Hoang AU - Verstraeten, Inge AU - Geelen, Danny ID - 408 SN - 1064-3745 T2 - Root Development TI - In vitro assay for induction of adventitious rooting on intact arabidopsis hypocotyls VL - 1761 ER - TY - CHAP AB - Immunolocalization is a valuable tool for cell biology research that allows to rapidly determine the localization and expression levels of endogenous proteins. In plants, whole-mount in situ immunolocalization remains a challenging method, especially in tissues protected by waxy layers and complex cell wall carbohydrates. Here, we present a robust method for whole-mount in situ immunolocalization in primary root meristems and lateral root primordia in Arabidopsis thaliana. For good epitope preservation, fixation is done in an alkaline paraformaldehyde/glutaraldehyde mixture. This fixative is suitable for detecting a wide range of proteins, including integral transmembrane proteins and proteins peripherally attached to the plasma membrane. From initiation until emergence from the primary root, lateral root primordia are surrounded by several layers of differentiated tissues with a complex cell wall composition that interferes with the efficient penetration of all buffers. Therefore, immunolocalization in early lateral root primordia requires a modified method, including a strong solvent treatment for removal of hydrophobic barriers and a specific cocktail of cell wall-degrading enzymes. The presented method allows for easy, reliable, and high-quality in situ detection of the subcellular localization of endogenous proteins in primary and lateral root meristems without the need of time-consuming crosses or making translational fusions to fluorescent proteins. AU - Karampelias, Michael AU - Tejos, Ricardo AU - Friml, Jirí AU - Vanneste, Steffen ED - Ristova, Daniela ED - Barbez, Elke ID - 411 T2 - Root Development. Methods and Protocols TI - Optimized whole mount in situ immunolocalization for Arabidopsis thaliana root meristems and lateral root primordia VL - 1761 ER - TY - JOUR AB - Asymmetric auxin distribution is instrumental for the differential growth that causes organ bending on tropic stimuli and curvatures during plant development. Local differences in auxin concentrations are achieved mainly by polarized cellular distribution of PIN auxin transporters, but whether other mechanisms involving auxin homeostasis are also relevant for the formation of auxin gradients is not clear. Here we show that auxin methylation is required for asymmetric auxin distribution across the hypocotyl, particularly during its response to gravity. We found that loss-of-function mutants in Arabidopsis IAA CARBOXYL METHYLTRANSFERASE1 (IAMT1) prematurely unfold the apical hook, and that their hypocotyls are impaired in gravitropic reorientation. This defect is linked to an auxin-dependent increase in PIN gene expression, leading to an increased polar auxin transport and lack of asymmetric distribution of PIN3 in the iamt1 mutant. Gravitropic reorientation in the iamt1 mutant could be restored with either endodermis-specific expression of IAMT1 or partial inhibition of polar auxin transport, which also results in normal PIN gene expression levels. We propose that IAA methylation is necessary in gravity-sensing cells to restrict polar auxin transport within the range of auxin levels that allow for differential responses. AU - Abbas, Mohamad AU - Hernández, García J AU - Pollmann, Stephan AU - Samodelov, Sophia L AU - Kolb, Martina AU - Friml, Jirí AU - Hammes, Ulrich Z AU - Zurbriggen, Matias D AU - Blázquez, Miguel AU - Alabadí, David ID - 203 IS - 26 JF - PNAS TI - Auxin methylation is required for differential growth in Arabidopsis VL - 115 ER - TY - JOUR AB - CLE peptides have been implicated in various developmental processes of plants and mediate their responses to environmental stimuli. However, the biological relevance of most CLE genes remains to be functionally characterized. Here, we report that CLE9, which is expressed in stomata, acts as an essential regulator in the induction of stomatal closure. Exogenous application of CLE9 peptides or overexpression of CLE9 effectively led to stomatal closure and enhanced drought tolerance, whereas CLE9 loss-of-function mutants were sensitivity to drought stress. CLE9-induced stomatal closure was impaired in abscisic acid (ABA)-deficient mutants, indicating that ABA is required for CLE9-medaited guard cell signalling. We further deciphered that two guard cell ABA-signalling components, OST1 and SLAC1, were responsible for CLE9-induced stomatal closure. MPK3 and MPK6 were activated by the CLE9 peptide, and CLE9 peptides failed to close stomata in mpk3 and mpk6 mutants. In addition, CLE9 peptides stimulated the induction of hydrogen peroxide (H2O2) and nitric oxide (NO) synthesis associated with stomatal closure, which was abolished in the NADPH oxidase-deficient mutants or nitric reductase mutants, respectively. Collectively, our results reveal a novel ABA-dependent function of CLE9 in the regulation of stomatal apertures, thereby suggesting a potential role of CLE9 in the stress acclimatization of plants. AU - Zhang, Luosha AU - Shi, Xiong AU - Zhang, Yutao AU - Wang, Jiajing AU - Yang, Jingwei AU - Ishida, Takashi AU - Jiang, Wenqian AU - Han, Xiangyu AU - Kang, Jingke AU - Wang, Xuening AU - Pan, Lixia AU - Lv, Shuo AU - Cao, Bing AU - Zhang, Yonghong AU - Wu, Jinbin AU - Han, Huibin AU - Hu, Zhubing AU - Cui, Langjun AU - Sawa, Shinichiro AU - He, Junmin AU - Wang, Guodong ID - 5830 JF - Plant Cell and Environment SN - 01407791 TI - CLE9 peptide-induced stomatal closure is mediated by abscisic acid, hydrogen peroxide, and nitric oxide in arabidopsis thaliana ER - TY - JOUR AB - The plant hormone gibberellic acid (GA) is a crucial regulator of growth and development. The main paradigm of GA signaling puts forward transcriptional regulation via the degradation of DELLA transcriptional repressors. GA has also been shown to regulate tropic responses by modulation of the plasma membrane incidence of PIN auxin transporters by an unclear mechanism. Here we uncovered the cellular and molecular mechanisms by which GA redirects protein trafficking and thus regulates cell surface functionality. Photoconvertible reporters revealed that GA balances the protein traffic between the vacuole degradation route and recycling back to the cell surface. Low GA levels promote vacuolar delivery and degradation of multiple cargos, including PIN proteins, whereas high GA levels promote their recycling to the plasma membrane. This GA effect requires components of the retromer complex, such as Sorting Nexin 1 (SNX1) and its interacting, microtubule (MT)-associated protein, the Cytoplasmic Linker-Associated Protein (CLASP1). Accordingly, GA regulates the subcellular distribution of SNX1 and CLASP1, and the intact MT cytoskeleton is essential for the GA effect on trafficking. This GA cellular action occurs through DELLA proteins that regulate the MT and retromer presumably via their interaction partners Prefoldins (PFDs). Our study identified a branching of the GA signaling pathway at the level of DELLA proteins, which, in parallel to regulating transcription, also target by a nontranscriptional mechanism the retromer complex acting at the intersection of the degradation and recycling trafficking routes. By this mechanism, GA can redirect receptors and transporters to the cell surface, thus coregulating multiple processes, including PIN-dependent auxin fluxes during tropic responses. AU - Salanenka, Yuliya AU - Verstraeten, Inge AU - Löfke, Christian AU - Tabata, Kaori AU - Naramoto, Satoshi AU - Glanc, Matous AU - Friml, Jirí ID - 428 IS - 14 JF - PNAS TI - Gibberellin DELLA signaling targets the retromer complex to redirect protein trafficking to the plasma membrane VL - 115 ER -