TY - JOUR AB - 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. AU - Li, Lanxin AU - Chen, Huihuang AU - Alotaibi, Saqer S. AU - Pěnčík, Aleš AU - Adamowski, Maciek AU - Novák, Ondřej AU - Friml, Jiří ID - 11723 IS - 31 JF - Proceedings of the National Academy of Sciences KW - Multidisciplinary SN - 0027-8424 TI - RALF1 peptide triggers biphasic root growth inhibition upstream of auxin biosynthesis VL - 119 ER - TY - JOUR AB - 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. AU - Tian, Z AU - Zhang, Yuzhou AU - Zhu, L AU - Jiang, B AU - Wang, H AU - Gao, R AU - Friml, Jiří AU - Xiao, G ID - 12053 IS - 12 JF - The Plant Cell SN - 1040-4651 TI - Strigolactones act downstream of gibberellins to regulate fiber cell elongation and cell wall thickness in cotton (Gossypium hirsutum) VL - 34 ER - TY - JOUR AB - 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. AU - Konstantinova, N AU - Hörmayer, Lukas AU - Glanc, Matous AU - Keshkeih, R AU - Tan, Shutang AU - Di Donato, M AU - Retzer, K AU - Moulinier-Anzola, J AU - Schwihla, M AU - Korbei, B AU - Geisler, M AU - Friml, Jiří AU - Luschnig, C ID - 12052 JF - Nature Communications SN - 2041-1723 TI - WAVY GROWTH Arabidopsis E3 ubiquitin ligases affect apical PIN sorting decisions VL - 13 ER - TY - JOUR AB - 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. AU - Yang, Z AU - Xia, J AU - Hong, J AU - Zhang, C AU - Wei, H AU - Ying, W AU - Sun, C AU - Sun, L AU - Mao, Y AU - Gao, Y AU - Tan, S AU - Friml, Jiří AU - Li, D AU - Liu, X AU - Sun, L ID - 12054 IS - 7927 JF - Nature SN - 0028-0836 TI - Structural insights into auxin recognition and efflux by Arabidopsis PIN1 VL - 609 ER - TY - JOUR AB - 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. AU - Zhao, Jierui AU - Bui, Mai Thu AU - Ma, Juncai AU - Künzl, Fabian AU - Picchianti, Lorenzo AU - De La Concepcion, Juan Carlos AU - Chen, Yixuan AU - Petsangouraki, Sofia AU - Mohseni, Azadeh AU - García-Leon, Marta AU - Gomez, Marta Salas AU - Giannini, Caterina AU - Gwennogan, Dubois AU - Kobylinska, Roksolana AU - Clavel, Marion AU - Schellmann, Swen AU - Jaillais, Yvon AU - Friml, Jiří AU - Kang, Byung-Ho AU - Dagdas, Yasin ID - 12121 IS - 12 JF - Journal of Cell Biology KW - Cell Biology SN - 0021-9525 TI - Plant autophagosomes mature into amphisomes prior to their delivery to the central vacuole VL - 221 ER - TY - JOUR AB - 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. AU - Huang, Jian AU - Zhao, Lei AU - Malik, Shikha AU - Gentile, Benjamin R. AU - Xiong, Va AU - Arazi, Tzahi AU - Owen, Heather A. AU - Friml, Jiří AU - Zhao, Dazhong ID - 12130 JF - Nature Communications KW - General Physics and Astronomy KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Chemistry KW - Multidisciplinary SN - 2041-1723 TI - Specification of female germline by microRNA orchestrated auxin signaling in Arabidopsis VL - 13 ER - TY - JOUR AB - 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. AU - Johnson, Alexander J AU - Kaufmann, Walter AU - Sommer, Christoph M AU - Costanzo, Tommaso AU - Dahhan, Dana A. AU - Bednarek, Sebastian Y. AU - Friml, Jiří ID - 12239 IS - 10 JF - Molecular Plant KW - Plant Science KW - Molecular Biology SN - 1674-2052 TI - Three-dimensional visualization of planta clathrin-coated vesicles at ultrastructural resolution VL - 15 ER - TY - JOUR AB - 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. AU - Bilanovičová, V AU - Rýdza, N AU - Koczka, L AU - Hess, M AU - Feraru, E AU - Friml, Jiří AU - Nodzyński, T ID - 11489 IS - 11 JF - International Journal of Molecular Sciences SN - 1422-0067 TI - The hydrophilic loop of Arabidopsis PIN1 auxin efflux carrier harbors hallmarks of an intrinsically disordered protein VL - 23 ER - TY - JOUR AB - 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. AU - Qi, Linlin AU - Kwiatkowski, Mateusz AU - Chen, Huihuang AU - Hörmayer, Lukas AU - Sinclair, Scott A AU - Zou, Minxia AU - del Genio, Charo I. AU - Kubeš, Martin F. AU - Napier, Richard AU - Jaworski, Krzysztof AU - Friml, Jiří ID - 12144 IS - 7934 JF - Nature SN - 0028-0836 TI - Adenylate cyclase activity of TIR1/AFB auxin receptors in plants VL - 611 ER - TY - JOUR AB - 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. AU - Xiao, Huixin AU - Hu, Yumei AU - Wang, Yaping AU - Cheng, Jinkui AU - Wang, Jinyi AU - Chen, Guojingwei AU - Li, Qian AU - Wang, Shuwei AU - Wang, Yalu AU - Wang, Shao-Shuai AU - Wang, Yi AU - Xuan, Wei AU - Li, Zhen AU - Guo, Yan AU - Gong, Zhizhong AU - Friml, Jiří AU - Zhang, Jing ID - 12120 IS - 23 JF - Developmental Cell KW - Developmental Biology KW - Cell Biology KW - General Biochemistry KW - Genetics and Molecular Biology KW - Molecular Biology SN - 1534-5807 TI - Nitrate availability controls translocation of the transcription factor NAC075 for cell-type-specific reprogramming of root growth VL - 57 ER -