@article{13201, abstract = {As a crucial nitrogen source, nitrate (NO3−) is a key nutrient for plants. Accordingly, root systems adapt to maximize NO3− availability, a developmental regulation also involving the phytohormone auxin. Nonetheless, the molecular mechanisms underlying this regulation remain poorly understood. Here, we identify low-nitrate-resistant mutant (lonr) in Arabidopsis (Arabidopsis thaliana), whose root growth fails to adapt to low-NO3− conditions. lonr2 is defective in the high-affinity NO3− transporter NRT2.1. lonr2 (nrt2.1) mutants exhibit defects in polar auxin transport, and their low-NO3−-induced root phenotype depends on the PIN7 auxin exporter activity. NRT2.1 directly associates with PIN7 and antagonizes PIN7-mediated auxin efflux depending on NO3− levels. These results reveal a mechanism by which NRT2.1 in response to NO3− limitation directly regulates auxin transport activity and, thus, root growth. This adaptive mechanism contributes to the root developmental plasticity to help plants cope with changes in NO3− availability.}, author = {Wang, Yalu and Yuan, Zhi and Wang, Jinyi and Xiao, Huixin and Wan, Lu and Li, Lanxin and Guo, Yan and Gong, Zhizhong and Friml, Jiří and Zhang, Jing}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, number = {25}, publisher = {National Academy of Sciences}, title = {{The nitrate transporter NRT2.1 directly antagonizes PIN7-mediated auxin transport for root growth adaptation}}, doi = {10.1073/pnas.2221313120}, volume = {120}, year = {2023}, } @phdthesis{14510, author = {Gnyliukh, Nataliia}, isbn = {978-3-99078-037-4}, issn = {2663-337X}, keywords = {Clathrin-Mediated Endocytosis, vesicle scission, Dynamin-Related Protein 2, SH3P2, TPLATE complex, Total internal reflection fluorescence microscopy, Arabidopsis thaliana}, pages = {180}, publisher = {Institute of Science and Technology Austria}, title = {{Mechanism of clathrin-coated vesicle formation during endocytosis in plants}}, doi = {10.15479/at:ista:14510}, year = {2023}, } @article{10016, abstract = {Auxin has always been at the forefront of research in plant physiology and development. Since the earliest contemplations by Julius von Sachs and Charles Darwin, more than a century-long struggle has been waged to understand its function. This largely reflects the failures, successes, and inevitable progress in the entire field of plant signaling and development. Here I present 14 stations on our long and sometimes mystical journey to understand auxin. These highlights were selected to give a flavor of the field and to show the scope and limits of our current knowledge. A special focus is put on features that make auxin unique among phytohormones, such as its dynamic, directional transport network, which integrates external and internal signals, including self-organizing feedback. Accented are persistent mysteries and controversies. The unexpected discoveries related to rapid auxin responses and growth regulation recently disturbed our contentment regarding understanding of the auxin signaling mechanism. These new revelations, along with advances in technology, usher us into a new, exciting era in auxin research. }, author = {Friml, Jiří}, issn = {1943-0264}, journal = {Cold Spring Harbor Perspectives in Biology}, number = {5}, publisher = {Cold Spring Harbor Laboratory}, title = {{Fourteen stations of auxin}}, doi = {10.1101/cshperspect.a039859 }, volume = {14}, year = {2022}, } @article{10583, abstract = {The synthetic strigolactone (SL) analog, rac-GR24, has been instrumental in studying the role of SLs as well as karrikins because it activates the receptors DWARF14 (D14) and KARRIKIN INSENSITIVE 2 (KAI2) of their signaling pathways, respectively. Treatment with rac-GR24 modifies the root architecture at different levels, such as decreasing the lateral root density (LRD), while promoting root hair elongation or flavonol accumulation. Previously, we have shown that the flavonol biosynthesis is transcriptionally activated in the root by rac-GR24 treatment, but, thus far, the molecular players involved in that response have remained unknown. To get an in-depth insight into the changes that occur after the compound is perceived by the roots, we compared the root transcriptomes of the wild type and the more axillary growth2 (max2) mutant, affected in both SL and karrikin signaling pathways, with and without rac-GR24 treatment. Quantitative reverse transcription (qRT)-PCR, reporter line analysis and mutant phenotyping indicated that the flavonol response and the root hair elongation are controlled by the ELONGATED HYPOCOTYL 5 (HY5) and MYB12 transcription factors, but HY5, in contrast to MYB12, affects the LRD as well. Furthermore, we identified the transcription factors TARGET OF MONOPTEROS 5 (TMO5) and TMO5 LIKE1 as negative and the Mediator complex as positive regulators of the rac-GR24 effect on LRD. Altogether, hereby, we get closer toward understanding the molecular mechanisms that underlay the rac-GR24 responses in the root.}, author = {Struk, Sylwia and Braem, Lukas and Matthys, Cedrick and Walton, Alan and Vangheluwe, Nick and Van Praet, Stan and Jiang, Lingxiang and Baster, Pawel and De Cuyper, Carolien and Boyer, Francois-Didier and Stes, Elisabeth and Beeckman, Tom and Friml, Jiří and Gevaert, Kris and Goormachtig, Sofie}, issn = {1471-9053}, journal = {Plant & Cell Physiology}, keywords = {flavonols, MAX2, rac-Gr24, RNA-seq, root development, transcriptional regulation}, number = {1}, pages = {104--119}, publisher = {Oxford University Press}, title = {{Transcriptional analysis in the Arabidopsis roots reveals new regulators that link rac-GR24 treatment with changes in flavonol accumulation, root hair elongation and lateral root density}}, doi = {10.1093/pcp/pcab149}, volume = {63}, year = {2022}, } @article{10717, abstract = {Much of what we know about the role of auxin in plant development derives from exogenous manipulations of auxin distribution and signaling, using inhibitors, auxins and auxin analogs. In this context, synthetic auxin analogs, such as 1-Naphtalene Acetic Acid (1-NAA), are often favored over the endogenous auxin indole-3-acetic acid (IAA), in part due to their higher stability. While such auxin analogs have proven to be instrumental to reveal the various faces of auxin, they display in some cases distinct bioactivities compared to IAA. Here, we focused on the effect of auxin analogs on the accumulation of PIN proteins in Brefeldin A-sensitive endosomal aggregations (BFA bodies), and the correlation with the ability to elicit Ca 2+ responses. For a set of commonly used auxin analogs, we evaluated if auxin-analog induced Ca 2+ signaling inhibits PIN accumulation. Not all auxin analogs elicited a Ca 2+ response, and their differential ability to elicit Ca 2+ responses correlated partially with their ability to inhibit BFA-body formation. However, in tir1/afb and cngc14, 1-NAA-induced Ca 2+ signaling was strongly impaired, yet 1-NAA still could inhibit PIN accumulation in BFA bodies. This demonstrates that TIR1/AFB-CNGC14-dependent Ca 2+ signaling does not inhibit BFA body formation in Arabidopsis roots.}, author = {Wang, R and Himschoot, E and Grenzi, M and Chen, J and Safi, A and Krebs, M and Schumacher, K and Nowack, MK and Moeder, W and Yoshioka, K and Van Damme, D and De Smet, I and Geelen, D and Beeckman, T and Friml, Jiří and Costa, A and Vanneste, S}, issn = {1460-2431}, journal = {Journal of Experimental Botany}, number = {8}, publisher = {Oxford Academic}, title = {{Auxin analog-induced Ca2+ signaling is independent of inhibition of endosomal aggregation in Arabidopsis roots}}, doi = {10.1093/jxb/erac019}, volume = {73}, year = {2022}, } @article{10719, abstract = {Auxin, one of the first identified and most widely studied phytohormones, has been and will remain a hot topic in plant biology. After more than a century of passionate exploration, the mysteries of its synthesis, transport, signaling, and metabolism have largely been unlocked. Due to the rapid development of new technologies, new methods, and new genetic materials, the study of auxin has entered the fast lane over the past 30 years. Here, we highlight advances in understanding auxin signaling, including auxin perception, rapid auxin responses, TRANSPORT INHIBITOR RESPONSE 1 and AUXIN SIGNALING F-boxes (TIR1/AFBs)-mediated transcriptional and non-transcriptional branches, and the epigenetic regulation of auxin signaling. We also focus on feedback inhibition mechanisms that prevent the over-amplification of auxin signals. In addition, we cover the TRANSMEMBRANE KINASEs (TMKs)-mediated non-canonical signaling, which converges with TIR1/AFBs-mediated transcriptional regulation to coordinate plant growth and development. The identification of additional auxin signaling components and their regulation will continue to open new avenues of research in this field, leading to an increasingly deeper, more comprehensive understanding of how auxin signals are interpreted at the cellular level to regulate plant growth and development.}, author = {Yu, Z and Zhang, F and Friml, Jiří and Ding, Z}, issn = {1744-7909}, journal = {Journal of Integrative Plant Biology}, number = {2}, pages = {371--392}, publisher = {Wiley}, title = {{Auxin signaling: Research advances over the past 30 years}}, doi = {10.1111/jipb.13225}, volume = {64}, year = {2022}, } @article{10768, abstract = {Among the most fascinated properties of the plant hormone auxin is its ability to promote formation of its own directional transport routes. These gradually narrowing auxin channels form from the auxin source toward the sink and involve coordinated, collective polarization of individual cells. Once established, the channels provide positional information, along which new vascular strands form, for example, during organogenesis, regeneration, or leave venation. The main prerequisite of this still mysterious auxin canalization mechanism is a feedback between auxin signaling and its directional transport. This is manifested by auxin-induced re-arrangements of polar, subcellular localization of PIN-FORMED (PIN) auxin exporters. Immanent open questions relate to how position of auxin source and sink as well as tissue context are sensed and translated into tissue polarization and how cells communicate to polarize coordinately. Recently, identification of the first molecular players opens new avenues into molecular studies of this intriguing example of self-organizing plant development.}, author = {Hajny, Jakub and Tan, Shutang and Friml, Jiří}, issn = {1369-5266}, journal = {Current Opinion in Plant Biology}, number = {2}, publisher = {Elsevier}, title = {{Auxin canalization: From speculative models toward molecular players}}, doi = {10.1016/j.pbi.2022.102174}, volume = {65}, year = {2022}, } @article{10841, abstract = {In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein assembly polypeptide-1 (AP-1) complex operates as part of the secretory pathway at the trans-Golgi network (TGN), while the AP-2 complex and the TPLATE complex jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched TGN/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis (Arabidopsis thaliana) cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.}, author = {Dahhan, DA and Reynolds, GD and Cárdenas, JJ and Eeckhout, D and Johnson, Alexander J and Yperman, K and Kaufmann, Walter and Vang, N and Yan, X and Hwang, I and Heese, A and De Jaeger, G and Friml, Jiří and Van Damme, D and Pan, J and Bednarek, SY}, issn = {1532-298x}, journal = {Plant Cell}, number = {6}, pages = {2150--2173}, publisher = {Oxford Academic}, title = {{Proteomic characterization of isolated Arabidopsis clathrin-coated vesicles reveals evolutionarily conserved and plant-specific components}}, doi = {10.1093/plcell/koac071}, volume = {34}, year = {2022}, } @article{10888, abstract = {Despite the growing interest in using chemical genetics in plant research, small molecule target identification remains a major challenge. The cellular thermal shift assay coupled with high-resolution mass spectrometry (CETSA MS) that monitors changes in the thermal stability of proteins caused by their interactions with small molecules, other proteins, or posttranslational modifications, allows the discovery of drug targets or the study of protein–metabolite and protein–protein interactions mainly in mammalian cells. To showcase the applicability of this method in plants, we applied CETSA MS to intact Arabidopsis thaliana cells and identified the thermal proteome of the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, bikinin. A comparison between the thermal and the phosphoproteomes of bikinin revealed the auxin efflux carrier PIN-FORMED1 (PIN1) as a substrate of the Arabidopsis GSK3s that negatively regulate the brassinosteroid signaling. We established that PIN1 phosphorylation by the GSK3s is essential for maintaining its intracellular polarity that is required for auxin-mediated regulation of vascular patterning in the leaf, thus revealing cross-talk between brassinosteroid and auxin signaling.}, author = {Lu, Qing and Zhang, Yonghong and Hellner, Joakim and Giannini, Caterina and Xu, Xiangyu and Pauwels, Jarne and Ma, Qian and Dejonghe, Wim and Han, Huibin and Van De Cotte, Brigitte and Impens, Francis and Gevaert, Kris and De Smet, Ive and Friml, Jiří and Molina, Daniel Martinez and Russinova, Eugenia}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, number = {11}, publisher = {Proceedings of the National Academy of Sciences}, title = {{Proteome-wide cellular thermal shift assay reveals unexpected cross-talk between brassinosteroid and auxin signaling}}, doi = {10.1073/pnas.2118220119}, volume = {119}, year = {2022}, } @article{11589, abstract = {Calcium-dependent protein kinases (CPK) are key components of a wide array of signaling pathways, translating stress and nutrient signaling into the modulation of cellular processes such as ion transport and transcription. However, not much is known about CPKs in endomembrane trafficking. Here, we screened for CPKs that impact on root growth and gravitropism, by overexpressing constitutively active forms of CPKs under the control of an inducible promoter in Arabidopsis thaliana. We found that inducible overexpression of an constitutive active CPK30 (CA-CPK30) resulted in a loss of root gravitropism and ectopic auxin accumulation in the root tip. Immunolocalization revealed that CA-CPK30 roots have reduced PIN protein levels, PIN1 polarity defects and impaired Brefeldin A (BFA)-sensitive trafficking. Moreover, FM4-64 uptake was reduced, indicative of a defect in endocytosis. The effects on BFA-sensitive trafficking were not specific to PINs, as BFA could not induce aggregation of ARF1- and CHC-labeled endosomes in CA-CPK30. Interestingly, the interference with BFA-body formation, could be reverted by increasing the extracellular pH, indicating a pH-dependence of this CA-CPK30 effect. Altogether, our data reveal an important role for CPK30 in root growth regulation and endomembrane trafficking in Arabidopsis thaliana.}, author = {Wang, Ren and Himschoot, Ellie and Chen, Jian and Boudsocq, Marie and Geelen, Danny and Friml, Jiří and Beeckman, Tom and Vanneste, Steffen}, issn = {1664-462X}, journal = {Frontiers in Plant Science}, publisher = {Frontiers}, title = {{Constitutive active CPK30 interferes with root growth and endomembrane trafficking in Arabidopsis thaliana}}, doi = {10.3389/fpls.2022.862398}, volume = {13}, year = {2022}, } @article{11723, abstract = {Plant cell growth responds rapidly to various stimuli, adapting architecture to environmental changes. Two major endogenous signals regulating growth are the phytohormone auxin and the secreted peptides rapid alkalinization factors (RALFs). Both trigger very rapid cellular responses and also exert long-term effects [Du et al., Annu. Rev. Plant Biol. 71, 379–402 (2020); Blackburn et al., Plant Physiol. 182, 1657–1666 (2020)]. However, the way, in which these distinct signaling pathways converge to regulate growth, remains unknown. Here, using vertical confocal microscopy combined with a microfluidic chip, we addressed the mechanism of RALF action on growth. We observed correlation between RALF1-induced rapid Arabidopsis thaliana root growth inhibition and apoplast alkalinization during the initial phase of the response, and revealed that RALF1 reversibly inhibits primary root growth through apoplast alkalinization faster than within 1 min. This rapid apoplast alkalinization was the result of RALF1-induced net H+ influx and was mediated by the receptor FERONIA (FER). Furthermore, we investigated the cross-talk between RALF1 and the auxin signaling pathways during root growth regulation. The results showed that RALF-FER signaling triggered auxin signaling with a delay of approximately 1 h by up-regulating auxin biosynthesis, thus contributing to sustained RALF1-induced growth inhibition. This biphasic RALF1 action on growth allows plants to respond rapidly to environmental stimuli and also reprogram growth and development in the long term.}, author = {Li, Lanxin and Chen, Huihuang and Alotaibi, Saqer S. and Pěnčík, Aleš and Adamowski, Maciek and Novák, Ondřej and Friml, Jiří}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences}, keywords = {Multidisciplinary}, number = {31}, publisher = {Proceedings of the National Academy of Sciences}, title = {{RALF1 peptide triggers biphasic root growth inhibition upstream of auxin biosynthesis}}, doi = {10.1073/pnas.2121058119}, volume = {119}, year = {2022}, } @article{12053, abstract = {Strigolactones (SLs) are a class of phytohormones that regulate plant shoot branching and adventitious root development. However, little is known regarding the role of SLs in controlling the behavior of the smallest unit of the organism, the single cell. Here, taking advantage of a classic single-cell model offered by the cotton (Gossypium hirsutum) fiber cell, we show that SLs, whose biosynthesis is fine-tuned by gibberellins (GAs), positively regulate cell elongation and cell wall thickness by promoting the biosynthesis of very-long-chain fatty acids (VLCFAs) and cellulose, respectively. Furthermore, we identified two layers of transcription factors (TFs) involved in the hierarchical regulation of this GA-SL crosstalk. The top-layer TF GROWTH-REGULATING FACTOR 4 (GhGRF4) directly activates expression of the SL biosynthetic gene DWARF27 (D27) to increase SL accumulation in fiber cells and GAs induce GhGRF4 expression. SLs induce the expression of four second-layer TF genes (GhNAC100-2, GhBLH51, GhGT2, and GhB9SHZ1), which transmit SL signals downstream to two ketoacyl-CoA synthase genes (KCS) and three cellulose synthase (CesA) genes by directly activating their transcription. Finally, the KCS and CesA enzymes catalyze the biosynthesis of very long chain fatty acids and cellulose, respectively, to regulate development of high-grade cotton fibers. In addition to providing a theoretical basis for cotton fiber improvement, our results shed light on SL signaling in plant development at the single-cell level.}, author = {Tian, Z and Zhang, Yuzhou and Zhu, L and Jiang, B and Wang, H and Gao, R and Friml, Jiří and Xiao, G}, issn = {1532-298X}, journal = {The Plant Cell}, number = {12}, pages = {4816--4839}, publisher = {Oxford University Press}, title = {{Strigolactones act downstream of gibberellins to regulate fiber cell elongation and cell wall thickness in cotton (Gossypium hirsutum)}}, doi = {10.1093/plcell/koac270}, volume = {34}, year = {2022}, } @article{12052, abstract = {Directionality in the intercellular transport of the plant hormone auxin is determined by polar plasma membrane localization of PIN-FORMED (PIN) auxin transport proteins. However, apart from PIN phosphorylation at conserved motifs, no further determinants explicitly controlling polar PIN sorting decisions have been identified. Here we present Arabidopsis WAVY GROWTH 3 (WAV3) and closely related RING-finger E3 ubiquitin ligases, whose loss-of-function mutants show a striking apical-to-basal polarity switch in PIN2 localization in root meristem cells. WAV3 E3 ligases function as essential determinants for PIN polarity, acting independently from PINOID/WAG-dependent PIN phosphorylation. They antagonize ectopic deposition of de novo synthesized PIN proteins already immediately following completion of cell division, presumably via preventing PIN sorting into basal, ARF GEF-mediated trafficking. Our findings reveal an involvement of E3 ligases in the selective targeting of apically localized PINs in higher plants.}, author = {Konstantinova, N and Hörmayer, Lukas and Glanc, Matous and Keshkeih, R and Tan, Shutang and Di Donato, M and Retzer, K and Moulinier-Anzola, J and Schwihla, M and Korbei, B and Geisler, M and Friml, Jiří and Luschnig, C}, issn = {2041-1723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{WAVY GROWTH Arabidopsis E3 ubiquitin ligases affect apical PIN sorting decisions}}, doi = {10.1038/s41467-022-32888-8}, volume = {13}, year = {2022}, } @article{12054, abstract = {Polar auxin transport is unique to plants and coordinates their growth and development1,2. The PIN-FORMED (PIN) auxin transporters exhibit highly asymmetrical localizations at the plasma membrane and drive polar auxin transport3,4; however, their structures and transport mechanisms remain largely unknown. Here, we report three inward-facing conformation structures of Arabidopsis thaliana PIN1: the apo state, bound to the natural auxin indole-3-acetic acid (IAA), and in complex with the polar auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). The transmembrane domain of PIN1 shares a conserved NhaA fold5. In the substrate-bound structure, IAA is coordinated by both hydrophobic stacking and hydrogen bonding. NPA competes with IAA for the same site at the intracellular pocket, but with a much higher affinity. These findings inform our understanding of the substrate recognition and transport mechanisms of PINs and set up a framework for future research on directional auxin transport, one of the most crucial processes underlying plant development.}, author = {Yang, Z and Xia, J and Hong, J and Zhang, C and Wei, H and Ying, W and Sun, C and Sun, L and Mao, Y and Gao, Y and Tan, S and Friml, Jiří and Li, D and Liu, X and Sun, L}, issn = {1476-4687}, journal = {Nature}, number = {7927}, pages = {611--615}, publisher = {Springer Nature}, title = {{Structural insights into auxin recognition and efflux by Arabidopsis PIN1}}, doi = {10.1038/s41586-022-05143-9}, volume = {609}, year = {2022}, } @article{12121, abstract = {Autophagosomes are double-membraned vesicles that traffic harmful or unwanted cellular macromolecules to the vacuole for recycling. Although autophagosome biogenesis has been extensively studied, autophagosome maturation, i.e., delivery and fusion with the vacuole, remains largely unknown in plants. Here, we have identified an autophagy adaptor, CFS1, that directly interacts with the autophagosome marker ATG8 and localizes on both membranes of the autophagosome. Autophagosomes form normally in Arabidopsis thaliana cfs1 mutants, but their delivery to the vacuole is disrupted. CFS1’s function is evolutionarily conserved in plants, as it also localizes to the autophagosomes and plays a role in autophagic flux in the liverwort Marchantia polymorpha. CFS1 regulates autophagic flux by bridging autophagosomes with the multivesicular body-localized ESCRT-I component VPS23A, leading to the formation of amphisomes. Similar to CFS1-ATG8 interaction, disrupting the CFS1-VPS23A interaction blocks autophagic flux and renders plants sensitive to nitrogen starvation. Altogether, our results reveal a conserved vacuolar sorting hub that regulates autophagic flux in plants.}, author = {Zhao, Jierui and Bui, Mai Thu and Ma, Juncai and Künzl, Fabian and Picchianti, Lorenzo and De La Concepcion, Juan Carlos and Chen, Yixuan and Petsangouraki, Sofia and Mohseni, Azadeh and García-Leon, Marta and Gomez, Marta Salas and Giannini, Caterina and Gwennogan, Dubois and Kobylinska, Roksolana and Clavel, Marion and Schellmann, Swen and Jaillais, Yvon and Friml, Jiří and Kang, Byung-Ho and Dagdas, Yasin}, issn = {1540-8140}, journal = {Journal of Cell Biology}, keywords = {Cell Biology}, number = {12}, publisher = {Rockefeller University Press}, title = {{Plant autophagosomes mature into amphisomes prior to their delivery to the central vacuole}}, doi = {10.1083/jcb.202203139}, volume = {221}, year = {2022}, } @article{12130, abstract = {Germline determination is essential for species survival and evolution in multicellular organisms. In most flowering plants, formation of the female germline is initiated with specification of one megaspore mother cell (MMC) in each ovule; however, the molecular mechanism underlying this key event remains unclear. Here we report that spatially restricted auxin signaling promotes MMC fate in Arabidopsis. Our results show that the microRNA160 (miR160) targeted gene ARF17 (AUXIN RESPONSE FACTOR17) is required for promoting MMC specification by genetically interacting with the SPL/NZZ (SPOROCYTELESS/NOZZLE) gene. Alterations of auxin signaling cause formation of supernumerary MMCs in an ARF17- and SPL/NZZ-dependent manner. Furthermore, miR160 and ARF17 are indispensable for attaining a normal auxin maximum at the ovule apex via modulating the expression domain of PIN1 (PIN-FORMED1) auxin transporter. Our findings elucidate the mechanism by which auxin signaling promotes the acquisition of female germline cell fate in plants.}, author = {Huang, Jian and Zhao, Lei and Malik, Shikha and Gentile, Benjamin R. and Xiong, Va and Arazi, Tzahi and Owen, Heather A. and Friml, Jiří and Zhao, Dazhong}, issn = {2041-1723}, journal = {Nature Communications}, keywords = {General Physics and Astronomy, General Biochemistry, Genetics and Molecular Biology, General Chemistry, Multidisciplinary}, publisher = {Springer Nature}, title = {{Specification of female germline by microRNA orchestrated auxin signaling in Arabidopsis}}, doi = {10.1038/s41467-022-34723-6}, volume = {13}, year = {2022}, } @article{12239, abstract = {Biological systems are the sum of their dynamic three-dimensional (3D) parts. Therefore, it is critical to study biological structures in 3D and at high resolution to gain insights into their physiological functions. Electron microscopy of metal replicas of unroofed cells and isolated organelles has been a key technique to visualize intracellular structures at nanometer resolution. However, many of these methods require specialized equipment and personnel to complete them. Here, we present novel accessible methods to analyze biological structures in unroofed cells and biochemically isolated organelles in 3D and at nanometer resolution, focusing on Arabidopsis clathrin-coated vesicles (CCVs). While CCVs are essential trafficking organelles, their detailed structural information is lacking due to their poor preservation when observed via classical electron microscopy protocols experiments. First, we establish a method to visualize CCVs in unroofed cells using scanning transmission electron microscopy tomography, providing sufficient resolution to define the clathrin coat arrangements. Critically, the samples are prepared directly on electron microscopy grids, removing the requirement to use extremely corrosive acids, thereby enabling the use of this method in any electron microscopy lab. Secondly, we demonstrate that this standardized sample preparation allows the direct comparison of isolated CCV samples with those visualized in cells. Finally, to facilitate the high-throughput and robust screening of metal replicated samples, we provide a deep learning analysis method to screen the “pseudo 3D” morphologies of CCVs imaged with 2D modalities. Collectively, our work establishes accessible ways to examine the 3D structure of biological samples and provide novel insights into the structure of plant CCVs.}, author = {Johnson, Alexander J and Kaufmann, Walter and Sommer, Christoph M and Costanzo, Tommaso and Dahhan, Dana A. and Bednarek, Sebastian Y. and Friml, Jiří}, issn = {1674-2052}, journal = {Molecular Plant}, keywords = {Plant Science, Molecular Biology}, number = {10}, pages = {1533--1542}, publisher = {Elsevier}, title = {{Three-dimensional visualization of planta clathrin-coated vesicles at ultrastructural resolution}}, doi = {10.1016/j.molp.2022.09.003}, volume = {15}, year = {2022}, } @article{11489, abstract = {Much of plant development depends on cell-to-cell redistribution of the plant hormone auxin, which is facilitated by the plasma membrane (PM) localized PIN FORMED (PIN) proteins. Auxin export activity, developmental roles, subcellular trafficking, and polarity of PINs have been well studied, but their structure remains elusive besides a rough outline that they contain two groups of 5 alpha-helices connected by a large hydrophilic loop (HL). Here, we focus on the PIN1 HL as we could produce it in sufficient quantities for biochemical investigations to provide insights into its secondary structure. Circular dichroism (CD) studies revealed its nature as an intrinsically disordered protein (IDP), manifested by the increase of structure content upon thermal melting. Consistent with IDPs serving as interaction platforms, PIN1 loops homodimerize. PIN1 HL cytoplasmic overexpression in Arabidopsis disrupts early endocytic trafficking of PIN1 and PIN2 and causes defects in the cotyledon vasculature formation. In summary, we demonstrate that PIN1 HL has an intrinsically disordered nature, which must be considered to gain further structural insights. Some secondary structures may form transiently during pairing with known and yet-to-be-discovered interactors.}, author = {Bilanovičová, V and Rýdza, N and Koczka, L and Hess, M and Feraru, E and Friml, Jiří and Nodzyński, T}, issn = {1422-0067}, journal = {International Journal of Molecular Sciences}, number = {11}, pages = {6352}, publisher = {MDPI}, title = {{The hydrophilic loop of Arabidopsis PIN1 auxin efflux carrier harbors hallmarks of an intrinsically disordered protein}}, doi = {10.3390/ijms23116352}, volume = {23}, year = {2022}, } @article{12144, abstract = {The phytohormone auxin is the major coordinative signal in plant development1, mediating transcriptional reprogramming by a well-established canonical signalling pathway. TRANSPORT INHIBITOR RESPONSE 1 (TIR1)/AUXIN-SIGNALING F-BOX (AFB) auxin receptors are F-box subunits of ubiquitin ligase complexes. In response to auxin, they associate with Aux/IAA transcriptional repressors and target them for degradation via ubiquitination2,3. Here we identify adenylate cyclase (AC) activity as an additional function of TIR1/AFB receptors across land plants. Auxin, together with Aux/IAAs, stimulates cAMP production. Three separate mutations in the AC motif of the TIR1 C-terminal region, all of which abolish the AC activity, each render TIR1 ineffective in mediating gravitropism and sustained auxin-induced root growth inhibition, and also affect auxin-induced transcriptional regulation. These results highlight the importance of TIR1/AFB AC activity in canonical auxin signalling. They also identify a unique phytohormone receptor cassette combining F-box and AC motifs, and the role of cAMP as a second messenger in plants.}, author = {Qi, Linlin and Kwiatkowski, Mateusz and Chen, Huihuang and Hörmayer, Lukas and Sinclair, Scott A and Zou, Minxia and del Genio, Charo I. and Kubeš, Martin F. and Napier, Richard and Jaworski, Krzysztof and Friml, Jiří}, issn = {1476-4687}, journal = {Nature}, number = {7934}, pages = {133--138}, publisher = {Springer Nature}, title = {{Adenylate cyclase activity of TIR1/AFB auxin receptors in plants}}, doi = {10.1038/s41586-022-05369-7}, volume = {611}, year = {2022}, } @article{12120, abstract = {Plant root architecture flexibly adapts to changing nitrate (NO3−) availability in the soil; however, the underlying molecular mechanism of this adaptive development remains under-studied. To explore the regulation of NO3−-mediated root growth, we screened for low-nitrate-resistant mutant (lonr) and identified mutants that were defective in the NAC transcription factor NAC075 (lonr1) as being less sensitive to low NO3− in terms of primary root growth. We show that NAC075 is a mobile transcription factor relocating from the root stele tissues to the endodermis based on NO3− availability. Under low-NO3− availability, the kinase CBL-interacting protein kinase 1 (CIPK1) is activated, and it phosphorylates NAC075, restricting its movement from the stele, which leads to the transcriptional regulation of downstream target WRKY53, consequently leading to adapted root architecture. Our work thus identifies an adaptive mechanism involving translocation of transcription factor based on nutrient availability and leading to cell-specific reprogramming of plant root growth.}, author = {Xiao, Huixin and Hu, Yumei and Wang, Yaping and Cheng, Jinkui and Wang, Jinyi and Chen, Guojingwei and Li, Qian and Wang, Shuwei and Wang, Yalu and Wang, Shao-Shuai and Wang, Yi and Xuan, Wei and Li, Zhen and Guo, Yan and Gong, Zhizhong and Friml, Jiří and Zhang, Jing}, issn = {1534-5807}, journal = {Developmental Cell}, keywords = {Developmental Biology, Cell Biology, General Biochemistry, Genetics and Molecular Biology, Molecular Biology}, number = {23}, pages = {2638--2651.e6}, publisher = {Elsevier}, title = {{Nitrate availability controls translocation of the transcription factor NAC075 for cell-type-specific reprogramming of root growth}}, doi = {10.1016/j.devcel.2022.11.006}, volume = {57}, year = {2022}, }