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 - TY - JOUR AB - Flowers have a species-specific functional life span that determines the time window in which pollination, fertilization and seed set can occur. The stigma tissue plays a key role in flower receptivity by intercepting pollen and initiating pollen tube growth toward the ovary. In this article, we show that a developmentally controlled cell death programme terminates the functional life span of stigma cells in Arabidopsis. We identified the leaf senescence regulator ORESARA1 (also known as ANAC092) and the previously uncharacterized KIRA1 (also known as ANAC074) as partially redundant transcription factors that modulate stigma longevity by controlling the expression of programmed cell death-associated genes. KIRA1 expression is sufficient to induce cell death and terminate floral receptivity, whereas lack of both KIRA1 and ORESARA1 substantially increases stigma life span. Surprisingly, the extension of stigma longevity is accompanied by only a moderate extension of flower receptivity, suggesting that additional processes participate in the control of the flower's receptive life span. AU - Gao, Zhen AU - Daneva, Anna AU - Salanenka, Yuliya AU - Van Durme, Matthias AU - Huysmans, Marlies AU - Lin, Zongcheng AU - De Winter, Freya AU - Vanneste, Steffen AU - Karimi, Mansour AU - Van De Velde, Jan AU - Vandepoele, Klaas AU - Van De Walle, Davy AU - Dewettinck, Koen AU - Lambrecht, Bart AU - Nowack, Moritz ID - 280 IS - 6 JF - Nature Plants TI - KIRA1 and ORESARA1 terminate flower receptivity by promoting cell death in the stigma of Arabidopsis VL - 4 ER - TY - JOUR AB - The angiosperm seed is composed of three genetically distinct tissues: the diploid embryo that originates from the fertilized egg cell, the triploid endosperm that is produced from the fertilized central cell, and the maternal sporophytic integuments that develop into the seed coat1. At the onset of embryo development in Arabidopsis thaliana, the zygote divides asymmetrically, producing a small apical embryonic cell and a larger basal cell that connects the embryo to the maternal tissue2. The coordinated and synchronous development of the embryo and the surrounding integuments, and the alignment of their growth axes, suggest communication between maternal tissues and the embryo. In contrast to animals, however, where a network of maternal factors that direct embryo patterning have been identified3,4, only a few maternal mutations have been described to affect embryo development in plants5–7. Early embryo patterning in Arabidopsis requires accumulation of the phytohormone auxin in the apical cell by directed transport from the suspensor8–10. However, the origin of this auxin has remained obscure. Here we investigate the source of auxin for early embryogenesis and provide evidence that the mother plant coordinates seed development by supplying auxin to the early embryo from the integuments of the ovule. We show that auxin response increases in ovules after fertilization, due to upregulated auxin biosynthesis in the integuments, and this maternally produced auxin is required for correct embryo development. AU - Robert, Hélène AU - Park, Chulmin AU - Gutièrrez, Carla AU - Wójcikowska, Barbara AU - Pěnčík, Aleš AU - Novák, Ondřej AU - Chen, Junyi AU - Grunewald, Wim AU - Dresselhaus, Thomas AU - Friml, Jirí AU - Laux, Thomas ID - 158 IS - 8 JF - Nature Plants TI - Maternal auxin supply contributes to early embryo patterning in Arabidopsis VL - 4 ER - TY - JOUR AB - AtNHX5 and AtNHX6 are endosomal Na+,K+/H+ antiporters that are critical for growth and development in Arabidopsis, but the mechanism behind their action remains unknown. Here, we report that AtNHX5 and AtNHX6, functioning as H+ leak, control auxin homeostasis and auxin-mediated development. We found that nhx5 nhx6 exhibited growth variations of auxin-related defects. We further showed that nhx5 nhx6 was affected in auxin homeostasis. Genetic analysis showed that AtNHX5 and AtNHX6 were required for the function of the ER-localized auxin transporter PIN5. Although AtNHX5 and AtNHX6 were co-localized with PIN5 at ER, they did not interact directly. Instead, the conserved acidic residues in AtNHX5 and AtNHX6, which are essential for exchange activity, were required for PIN5 function. AtNHX5 and AtNHX6 regulated the pH in ER. Overall, AtNHX5 and AtNHX6 may regulate auxin transport across the ER via the pH gradient created by their transport activity. H+-leak pathway provides a fine-tuning mechanism that controls cellular auxin fluxes. AU - Fan, Ligang AU - Zhao, Lei AU - Hu, Wei AU - Li, Weina AU - Novák, Ondřej AU - Strnad, Miroslav AU - Simon, Sibu AU - Friml, Jirí AU - Shen, Jinbo AU - Jiang, Liwen AU - Qiu, Quan ID - 462 JF - Plant, Cell and Environment TI - NHX antiporters regulate the pH of endoplasmic reticulum and auxin-mediated development VL - 41 ER - TY - JOUR AB - The phytohormone auxin is the information carrier in a plethora of developmental and physiological processes in plants(1). It has been firmly established that canonical, nuclear auxin signalling acts through regulation of gene transcription(2). Here, we combined microfluidics, live imaging, genetic engineering and computational modelling to reanalyse the classical case of root growth inhibition(3) by auxin. We show that Arabidopsis roots react to addition and removal of auxin by extremely rapid adaptation of growth rate. This process requires intracellular auxin perception but not transcriptional reprogramming. The formation of the canonical TIR1/AFB-Aux/IAA co-receptor complex is required for the growth regulation, hinting to a novel, non-transcriptional branch of this signalling pathway. Our results challenge the current understanding of root growth regulation by auxin and suggest another, presumably non-transcriptional, signalling output of the canonical auxin pathway. AU - Fendrych, Matyas AU - Akhmanova, Maria AU - Merrin, Jack AU - Glanc, Matous AU - Hagihara, Shinya AU - Takahashi, Koji AU - Uchida, Naoyuki AU - Torii, Keiko U AU - Friml, Jirí ID - 192 IS - 7 JF - Nature Plants TI - Rapid and reversible root growth inhibition by TIR1 auxin signalling VL - 4 ER - TY - JOUR AB - The intercellular transport of auxin is driven by PIN-formed (PIN) auxin efflux carriers. PINs are localized at the plasma membrane (PM) and on constitutively recycling endomembrane vesicles. Therefore, PINs can mediate auxin transport either by direct translocation across the PM or by pumping auxin into secretory vesicles (SVs), leading to its secretory release upon fusion with the PM. Which of these two mechanisms dominates is a matter of debate. Here, we addressed the issue with a mathematical modeling approach. We demonstrate that the efficiency of secretory transport depends on SV size, half-life of PINs on the PM, pH, exocytosis frequency and PIN density. 3D structured illumination microscopy (SIM) was used to determine PIN density on the PM. Combining this data with published values of the other parameters, we show that the transport activity of PINs in SVs would have to be at least 1000× greater than on the PM in order to produce a comparable macroscopic auxin transport. If both transport mechanisms operated simultaneously and PINs were equally active on SVs and PM, the contribution of secretion to the total auxin flux would be negligible. In conclusion, while secretory vesicle-mediated transport of auxin is an intriguing and theoretically possible model, it is unlikely to be a major mechanism of auxin transport inplanta. AU - Hille, Sander AU - Akhmanova, Maria AU - Glanc, Matous AU - Johnson, Alexander J AU - Friml, Jirí ID - 14 IS - 11 JF - International Journal of Molecular Sciences TI - Relative contribution of PIN-containing secretory vesicles and plasma membrane PINs to the directed auxin transport: Theoretical estimation VL - 19 ER - TY - JOUR AB - Wheat (Triticum ssp.) is one of the most important human food sources. However, this crop is very sensitive to temperature changes. Specifically, processes during wheat leaf, flower, and seed development and photosynthesis, which all contribute to the yield of this crop, are affected by high temperature. While this has to some extent been investigated on physiological, developmental, and molecular levels, very little is known about early signalling events associated with an increase in temperature. Phosphorylation-mediated signalling mechanisms, which are quick and dynamic, are associated with plant growth and development, also under abiotic stress conditions. Therefore, we probed the impact of a short-term and mild increase in temperature on the wheat leaf and spikelet phosphoproteome. In total, 3822 (containing 5178 phosphosites) and 5581 phosphopeptides (containing 7023 phosphosites) were identified in leaf and spikelet samples, respectively. Following statistical analysis, the resulting data set provides the scientific community with a first large-scale plant phosphoproteome under the control of higher ambient temperature. This community resource on the high temperature-mediated wheat phosphoproteome will be valuable for future studies. Our analyses also revealed a core set of common proteins between leaf and spikelet, suggesting some level of conserved regulatory mechanisms. Furthermore, we observed temperature-regulated interconversion of phosphoforms, which probably impacts protein activity. AU - Vu, Lam AU - Zhu, Tingting AU - Verstraeten, Inge AU - Van De Cotte, Brigitte AU - Gevaert, Kris AU - De Smet, Ive ID - 36 IS - 19 JF - Journal of Experimental Botany TI - Temperature-induced changes in the wheat phosphoproteome reveal temperature-regulated interconversion of phosphoforms VL - 69 ER - TY - JOUR AB - Land plants evolved from charophytic algae, among which Charophyceae possess the most complex body plans. We present the genome of Chara braunii; comparison of the genome to those of land plants identified evolutionary novelties for plant terrestrialization and land plant heritage genes. C. braunii employs unique xylan synthases for cell wall biosynthesis, a phragmoplast (cell separation) mechanism similar to that of land plants, and many phytohormones. C. braunii plastids are controlled via land-plant-like retrograde signaling, and transcriptional regulation is more elaborate than in other algae. The morphological complexity of this organism may result from expanded gene families, with three cases of particular note: genes effecting tolerance to reactive oxygen species (ROS), LysM receptor-like kinases, and transcription factors (TFs). Transcriptomic analysis of sexual reproductive structures reveals intricate control by TFs, activity of the ROS gene network, and the ancestral use of plant-like storage and stress protection proteins in the zygote. AU - Nishiyama, Tomoaki AU - Sakayama, Hidetoshi AU - De Vries, Jan AU - Buschmann, Henrik AU - Saint Marcoux, Denis AU - Ullrich, Kristian AU - Haas, Fabian AU - Vanderstraeten, Lisa AU - Becker, Dirk AU - Lang, Daniel AU - Vosolsobě, Stanislav AU - Rombauts, Stephane AU - Wilhelmsson, Per AU - Janitza, Philipp AU - Kern, Ramona AU - Heyl, Alexander AU - Rümpler, Florian AU - Calderón Villalobos, Luz AU - Clay, John AU - Skokan, Roman AU - Toyoda, Atsushi AU - Suzuki, Yutaka AU - Kagoshima, Hiroshi AU - Schijlen, Elio AU - Tajeshwar, Navindra AU - Catarino, Bruno AU - Hetherington, Alexander AU - Saltykova, Assia AU - Bonnot, Clemence AU - Breuninger, Holger AU - Symeonidi, Aikaterini AU - Radhakrishnan, Guru AU - Van Nieuwerburgh, Filip AU - Deforce, Dieter AU - Chang, Caren AU - Karol, Kenneth AU - Hedrich, Rainer AU - Ulvskov, Peter AU - Glöckner, Gernot AU - Delwiche, Charles AU - Petrášek, Jan AU - Van De Peer, Yves AU - Friml, Jirí AU - Beilby, Mary AU - Dolan, Liam AU - Kohara, Yuji AU - Sugano, Sumio AU - Fujiyama, Asao AU - Delaux, Pierre Marc AU - Quint, Marcel AU - Theissen, Gunter AU - Hagemann, Martin AU - Harholt, Jesper AU - Dunand, Christophe AU - Zachgo, Sabine AU - Langdale, Jane AU - Maumus, Florian AU - Van Der Straeten, Dominique AU - Gould, Sven B AU - Rensing, Stefan ID - 148 IS - 2 JF - Cell TI - The Chara genome: Secondary complexity and implications for plant terrestrialization VL - 174 ER - TY - JOUR AB - The trafficking of subcellular cargos in eukaryotic cells crucially depends on vesicle budding, a process mediated by ARF-GEFs (ADP-ribosylation factor guanine nucleotide exchange factors). In plants, ARF-GEFs play essential roles in endocytosis, vacuolar trafficking, recycling, secretion, and polar trafficking. Moreover, they are important for plant development, mainly through controlling the polar subcellular localization of PIN-FORMED (PIN) transporters of the plant hormone auxin. Here, using a chemical genetics screen in Arabidopsis thaliana, we identified Endosidin 4 (ES4), an inhibitor of eukaryotic ARF-GEFs. ES4 acts similarly to and synergistically with the established ARF-GEF inhibitor Brefeldin A and has broad effects on intracellular trafficking, including endocytosis, exocytosis, and vacuolar targeting. Additionally, Arabidopsis and yeast (Sacharomyces cerevisiae) mutants defective in ARF-GEF show altered sensitivity to ES4. ES4 interferes with the activation-based membrane association of the ARF1 GTPases, but not of their mutant variants that are activated independently of ARF-GEF activity. Biochemical approaches and docking simulations confirmed that ES4 specifically targets the SEC7 domain-containing ARF-GEFs. These observations collectively identify ES4 as a chemical tool enabling the study of ARF-GEF-mediated processes, including ARF-GEF-mediated plant development. AU - Kania, Urszula AU - Nodzyński, Tomasz AU - Lu, Qing AU - Hicks, Glenn R AU - Nerinckx, Wim AU - Mishev, Kiril AU - Peurois, Francois AU - Cherfils, Jacqueline AU - De, Rycke Riet Maria AU - Grones, Peter AU - Robert, Stéphanie AU - Russinova, Eugenia AU - Friml, Jirí ID - 147 IS - 10 JF - The Plant Cell SN - 1040-4651 TI - The inhibitor Endosidin 4 targets SEC7 domain-type ARF GTPase exchange factors and interferes with sub cellular trafficking in eukaryotes VL - 30 ER - TY - JOUR AB - The root cap protects the stem cell niche of angiosperm roots from damage. In Arabidopsis, lateral root cap (LRC) cells covering the meristematic zone are regularly lost through programmed cell death, while the outermost layer of the root cap covering the tip is repeatedly sloughed. Efficient coordination with stem cells producing new layers is needed to maintain a constant size of the cap. We present a signalling pair, the peptide IDA-LIKE1 (IDL1) and its receptor HAESA-LIKE2 (HSL2), mediating such communication. Live imaging over several days characterized this process from initial fractures in LRC cell files to full separation of a layer. Enhanced expression of IDL1 in the separating root cap layers resulted in increased frequency of sloughing, balanced with generation of new layers in a HSL2-dependent manner. Transcriptome analyses linked IDL1-HSL2 signalling to the transcription factors BEARSKIN1/2 and genes associated with programmed cell death. Mutations in either IDL1 or HSL2 slowed down cell division, maturation and separation. Thus, IDL1-HSL2 signalling potentiates dynamic regulation of the homeostatic balance between stem cell division and sloughing activity. AU - Shi, Chun Lin AU - Von Wangenheim, Daniel AU - Herrmann, Ullrich AU - Wildhagen, Mari AU - Kulik, Ivan AU - Kopf, Andreas AU - Ishida, Takashi AU - Olsson, Vilde AU - Anker, Mari Kristine AU - Albert, Markus AU - Butenko, Melinka A AU - Felix, Georg AU - Sawa, Shinichiro AU - Claassen, Manfred AU - Friml, Jirí AU - Aalen, Reidunn B ID - 146 IS - 8 JF - Nature Plants TI - The dynamics of root cap sloughing in Arabidopsis is regulated by peptide signalling VL - 4 ER - TY - JOUR AB - Strigolactones (SLs) are a relatively recent addition to the list of plant hormones that control different aspects of plant development. SL signalling is perceived by an α/β hydrolase, DWARF 14 (D14). A close homolog of D14, KARRIKIN INSENSTIVE2 (KAI2), is involved in perception of an uncharacterized molecule called karrikin (KAR). Recent studies in Arabidopsis identified the SUPPRESSOR OF MAX2 1 (SMAX1) and SMAX1-LIKE 7 (SMXL7) to be potential SCF–MAX2 complex-mediated proteasome targets of KAI2 and D14, respectively. Genetic studies on SMXL7 and SMAX1 demonstrated distinct developmental roles for each, but very little is known about these repressors in terms of their sequence features. In this study, we performed an extensive comparative analysis of SMXLs and determined their phylogenetic and evolutionary history in the plant lineage. Our results show that SMXL family members can be sub-divided into four distinct phylogenetic clades/classes, with an ancient SMAX1. Further, we identified the clade-specific motifs that have evolved and that might act as determinants of SL-KAR signalling specificity. These specificities resulted from functional diversities among the clades. Our results suggest that a gradual co-evolution of SMXL members with their upstream receptors D14/KAI2 provided an increased specificity to both the SL perception and response in land plants. AU - Moturu, Taraka Ramji AU - Thula, Sravankumar AU - Singh, Ravi Kumar AU - Nodzyński, Tomasz AU - Vařeková, Radka Svobodová AU - Friml, Jiří AU - Simon, Sibu ID - 10881 IS - 9 JF - Journal of Experimental Botany KW - Plant Science KW - Physiology SN - 0022-0957 TI - Molecular evolution and diversification of the SMXL gene family VL - 69 ER - TY - JOUR AB - Coordinated cell polarization in developing tissues is a recurrent theme in multicellular organisms. In plants, a directional distribution of the plant hormone auxin is at the core of many developmental programs. A feedback regulation of auxin on the polarized localization of PIN auxin transporters in individual cells has been proposed as a self-organizing mechanism for coordinated tissue polarization, but the molecular mechanisms linking auxin signalling to PIN-dependent auxin transport remain unknown. We performed a microarray-based approach to find regulators of the auxin-induced PIN relocation in the Arabidopsis thaliana root. We identified a subset of a family of phosphatidylinositol transfer proteins (PITP), the PATELLINs (PATL). Here, we show that PATLs are expressed in partially overlapping cells types in different tissues going through mitosis or initiating differentiation programs. PATLs are plasma membrane-associated proteins accumulated in Arabidopsis embryos, primary roots, lateral root primordia, and developing stomata. Higher order patl mutants display reduced PIN1 repolarization in response to auxin, shorter root apical meristem, and drastic defects in embryo and seedling development. This suggests PATLs redundantly play a crucial role in polarity and patterning in Arabidopsis. AU - Tejos, Ricardo AU - Rodríguez Furlán, Cecilia AU - Adamowski, Maciek AU - Sauer, Michael AU - Norambuena, Lorena AU - Friml, Jirí ID - 913 IS - 2 JF - Journal of Cell Science SN - 00219533 TI - PATELLINS are regulators of auxin mediated PIN1 relocation and plant development in Arabidopsis thaliana VL - 131 ER - TY - JOUR AB - Cell polarity, manifested by the localization of proteins to distinct polar plasma membrane domains, is a key prerequisite of multicellular life. In plants, PIN auxin transporters are prominent polarity markers crucial for a plethora of developmental processes. Cell polarity mechanisms in plants are distinct from other eukaryotes and still largely elusive. In particular, how the cell polarities are propagated and maintained following cell division remains unknown. Plant cytokinesis is orchestrated by the cell plate—a transient centrifugally growing endomembrane compartment ultimately forming the cross wall1. Trafficking of polar membrane proteins is typically redirected to the cell plate, and these will consequently have opposite polarity in at least one of the daughter cells2–5. Here, we provide mechanistic insights into post-cytokinetic re-establishment of cell polarity as manifested by the apical, polar localization of PIN2. We show that the apical domain is defined in a cell-intrinsic manner and that re-establishment of PIN2 localization to this domain requires de novo protein secretion and endocytosis, but not basal-to-apical transcytosis. Furthermore, we identify a PINOID-related kinase WAG1, which phosphorylates PIN2 in vitro6 and is transcriptionally upregulated specifically in dividing cells, as a crucial regulator of post-cytokinetic PIN2 polarity re-establishment. AU - Glanc, Matous AU - Fendrych, Matyas AU - Friml, Jirí ID - 5673 IS - 12 JF - Nature Plants SN - 2055-0278 TI - Mechanistic framework for cell-intrinsic re-establishment of PIN2 polarity after cell division VL - 4 ER - TY - JOUR AB - Clathrin-mediated endocytosis (CME) is a cellular trafficking process in which cargoes and lipids are internalized from the plasma membrane into vesicles coated with clathrin and adaptor proteins. CME is essential for many developmental and physiological processes in plants, but its underlying mechanism is not well characterised compared to that in yeast and animal systems. Here, we searched for new factors involved in CME in Arabidopsis thaliana by performing Tandem Affinity Purification of proteins that interact with clathrin light chain, a principal component of the clathrin coat. Among the confirmed interactors, we found two putative homologues of the clathrin-coat uncoating factor auxilin previously described in non-plant systems. Overexpression of AUXILIN-LIKE1 and AUXILIN-LIKE2 in A. thaliana caused an arrest of seedling growth and development. This was concomitant with inhibited endocytosis due to blocking of clathrin recruitment after the initial step of adaptor protein binding to the plasma membrane. By contrast, auxilin-like(1/2) loss-of-function lines did not present endocytosis-related developmental or cellular phenotypes under normal growth conditions. This work contributes to the on-going characterization of the endocytotic machinery in plants and provides a robust tool for conditionally and specifically interfering with CME in A. thaliana. AU - Adamowski, Maciek AU - Narasimhan, Madhumitha AU - Kania, Urszula AU - Glanc, Matous AU - De Jaeger, Geert AU - Friml, Jirí ID - 412 IS - 3 JF - The Plant Cell SN - 1040-4651 TI - A functional study of AUXILIN LIKE1 and 2 two putative clathrin uncoating factors in Arabidopsis VL - 30 ER - TY - JOUR AB - Auxin is unique among plant hormones due to its directional transport that is mediated by the polarly distributed PIN auxin transporters at the plasma membrane. The canalization hypothesis proposes that the auxin feedback on its polar flow is a crucial, plant-specific mechanism mediating multiple self-organizing developmental processes. Here, we used the auxin effect on the PIN polar localization in Arabidopsis thaliana roots as a proxy for the auxin feedback on the PIN polarity during canalization. We performed microarray experiments to find regulators of this process that act downstream of auxin. We identified genes that were transcriptionally regulated by auxin in an AXR3/IAA17- and ARF7/ARF19-dependent manner. Besides the known components of the PIN polarity, such as PID and PIP5K kinases, a number of potential new regulators were detected, among which the WRKY23 transcription factor, which was characterized in more detail. Gain- and loss-of-function mutants confirmed a role for WRKY23 in mediating the auxin effect on the PIN polarity. Accordingly, processes requiring auxin-mediated PIN polarity rearrangements, such as vascular tissue development during leaf venation, showed a higher WRKY23 expression and required the WRKY23 activity. Our results provide initial insights into the auxin transcriptional network acting upstream of PIN polarization and, potentially, canalization-mediated plant development. AU - Prat, Tomas AU - Hajny, Jakub AU - Grunewald, Wim AU - Vasileva, Mina K AU - Molnar, Gergely AU - Tejos, Ricardo AU - Schmid, Markus AU - Sauer, Michael AU - Friml, Jirí ID - 449 IS - 1 JF - PLoS Genetics TI - WRKY23 is a component of the transcriptional network mediating auxin feedback on PIN polarity VL - 14 ER - TY - JOUR AB - Intercellular distribution of the plant hormone auxin largely depends on the polar subcellular distribution of the plasma membrane PIN-FORMED (PIN) auxin transporters. PIN polarity switches in response to different developmental and environmental signals have been shown to redirect auxin fluxes mediating certain developmental responses. PIN phosphorylation at different sites and by different kinases is crucial for PIN function. Here we investigate the role of PIN phosphorylation during gravitropic response. Loss- and gain-of-function mutants in PINOID and related kinases but not in D6PK kinase as well as mutations mimicking constitutive dephosphorylated or phosphorylated status of two clusters of predicted phosphorylation sites partially disrupted PIN3 phosphorylation and caused defects in gravitropic bending in roots and hypocotyls. In particular, they impacted PIN3 polarity rearrangements in response to gravity and during feed-back regulation by auxin itself. Thus PIN phosphorylation, besides regulating transport activity and apical-basal targeting, is also important for the rapid polarity switches in response to environmental and endogenous signals. AU - Grones, Peter AU - Abas, Melinda F AU - Hajny, Jakub AU - Jones, Angharad AU - Waidmann, Sascha AU - Kleine Vehn, Jürgen AU - Friml, Jirí ID - 191 IS - 1 JF - Scientific Reports TI - PID/WAG-mediated phosphorylation of the Arabidopsis PIN3 auxin transporter mediates polarity switches during gravitropism VL - 8 ER - TY - JOUR AB - The rapid auxin-triggered growth of the Arabidopsis hypocotyls involves the nuclear TIR1/AFB-Aux/IAA signaling and is accompanied by acidification of the apoplast and cell walls (Fendrych et al., 2016). Here, we describe in detail the method for analysis of the elongation and the TIR1/AFB-Aux/IAA-dependent auxin response in hypocotyl segments as well as the determination of relative values of the cell wall pH. AU - Li, Lanxin AU - Krens, Gabriel AU - Fendrych, Matyas AU - Friml, Jirí ID - 442 IS - 1 JF - Bio-protocol TI - Real-time analysis of auxin response, cell wall pH and elongation in Arabidopsis thaliana Hypocotyls VL - 8 ER - TY - JOUR AB - In this review, we summarize the different biosynthesis-related pathways that contribute to the regulation of endogenous auxin in plants. We demonstrate that all known genes involved in auxin biosynthesis also have a role in root formation, from the initiation of a root meristem during embryogenesis to the generation of a functional root system with a primary root, secondary lateral root branches and adventitious roots. Furthermore, the versatile adaptation of root development in response to environmental challenges is mediated by both local and distant control of auxin biosynthesis. In conclusion, auxin homeostasis mediated by spatial and temporal regulation of auxin biosynthesis plays a central role in determining root architecture. AU - Olatunji, Damilola AU - Geelen, Danny AU - Verstraeten, Inge ID - 572 IS - 12 JF - International Journal of Molecular Sciences TI - Control of endogenous auxin levels in plant root development VL - 18 ER - TY - JOUR AB - Plant organs are typically organized into three main tissue layers. The middle ground tissue layer comprises the majority of the plant body and serves a wide range of functions, including photosynthesis, selective nutrient uptake and storage, and gravity sensing. Ground tissue patterning and maintenance in Arabidopsis are controlled by a well-established gene network revolving around the key regulator SHORT-ROOT (SHR). In contrast, it is completely unknown how ground tissue identity is first specified from totipotent precursor cells in the embryo. The plant signaling molecule auxin, acting through AUXIN RESPONSE FACTOR (ARF) transcription factors, is critical for embryo patterning. The auxin effector ARF5/MONOPTEROS (MP) acts both cell-autonomously and noncell-autonomously to control embryonic vascular tissue formation and root initiation, respectively. Here we show that auxin response and ARF activity cell-autonomously control the asymmetric division of the first ground tissue cells. By identifying embryonic target genes, we show that MP transcriptionally initiates the ground tissue lineage and acts upstream of the regulatory network that controls ground tissue patterning and maintenance. Strikingly, whereas the SHR network depends on MP, this MP function is, at least in part, SHR independent. Our study therefore identifies auxin response as a regulator of ground tissue specification in the embryonic root, and reveals that ground tissue initiation and maintenance use different regulators and mechanisms. Moreover, our data provide a framework for the simultaneous formation of multiple cell types by the same transcriptional regulator. AU - Möller, Barbara AU - Ten Hove, Colette AU - Xiang, Daoquan AU - Williams, Nerys AU - López, Lorena AU - Yoshida, Saiko AU - Smit, Margot AU - Datla, Raju AU - Weijers, Dolf ID - 657 IS - 12 JF - PNAS SN - 00278424 TI - Auxin response cell autonomously controls ground tissue initiation in the early arabidopsis embryo VL - 114 ER - TY - JOUR AB - The exocyst, a eukaryotic tethering complex, coregulates targeted exocytosis as an effector of small GTPases in polarized cell growth. In land plants, several exocyst subunits are encoded by double or triple paralogs, culminating in tens of EXO70 paralogs. Out of 23 Arabidopsis thaliana EXO70 isoforms, we analyzed seven isoforms expressed in pollen. Genetic and microscopic analyses of single mutants in EXO70A2, EXO70C1, EXO70C2, EXO70F1, EXO70H3, EXO70H5, and EXO70H6 genes revealed that only a loss-of-function EXO70C2 allele resulted in a significant male-specific transmission defect (segregation 40%:51%:9%) due to aberrant pollen tube growth. Mutant pollen tubes grown in vitro exhibited an enhanced growth rate and a decreased thickness of the tip cell wall, causing tip bursts. However, exo70C2 pollen tubes could frequently recover and restart their speedy elongation, resulting in a repetitive stop-and-go growth dynamics. A pollenspecific depletion of the closest paralog, EXO70C1, using artificial microRNA in the exo70C2 mutant background, resulted in a complete pollen-specific transmission defect, suggesting redundant functions of EXO70C1 and EXO70C2. Both EXO70C1 and EXO70C2, GFP tagged and expressed under the control of their native promoters, localized in the cytoplasm of pollen grains, pollen tubes, and also root trichoblast cells. The expression of EXO70C2-GFP complemented the aberrant growth of exo70C2 pollen tubes. The absent EXO70C2 interactions with core exocyst subunits in the yeast two-hybrid assay, cytoplasmic localization, and genetic effect suggest an unconventional EXO70 function possibly as a regulator of exocytosis outside the exocyst complex. In conclusion, EXO70C2 is a novel factor contributing to the regulation of optimal tip growth of Arabidopsis pollen tubes. AU - Synek, Lukáš AU - Vukašinović, Nemanja AU - Kulich, Ivan AU - Hála, Michal AU - Aldorfová, Klára AU - Fendrych, Matyas AU - Žárský, Viktor ID - 669 IS - 1 JF - Plant Physiology SN - 00320889 TI - EXO70C2 is a key regulatory factor for optimal tip growth of pollen VL - 174 ER - TY - JOUR AB - Plants are sessile organisms rooted in one place. The soil resources that plants require are often distributed in a highly heterogeneous pattern. To aid foraging, plants have evolved roots whose growth and development are highly responsive to soil signals. As a result, 3D root architecture is shaped by myriad environmental signals to ensure resource capture is optimised and unfavourable environments are avoided. The first signals sensed by newly germinating seeds — gravity and light — direct root growth into the soil to aid seedling establishment. Heterogeneous soil resources, such as water, nitrogen and phosphate, also act as signals that shape 3D root growth to optimise uptake. Root architecture is also modified through biotic interactions that include soil fungi and neighbouring plants. This developmental plasticity results in a ‘custom-made’ 3D root system that is best adapted to forage for resources in each soil environment that a plant colonises. AU - Morris, Emily AU - Griffiths, Marcus AU - Golebiowska, Agata AU - Mairhofer, Stefan AU - Burr Hersey, Jasmine AU - Goh, Tatsuaki AU - Von Wangenheim, Daniel AU - Atkinson, Brian AU - Sturrock, Craig AU - Lynch, Jonathan AU - Vissenberg, Kris AU - Ritz, Karl AU - Wells, Darren AU - Mooney, Sacha AU - Bennett, Malcolm ID - 722 IS - 17 JF - Current Biology SN - 09609822 TI - Shaping 3D root system architecture VL - 27 ER -