TY - JOUR AB - 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. AU - Yu, Z AU - Zhang, F AU - Friml, Jiří AU - Ding, Z ID - 10719 IS - 2 JF - Journal of Integrative Plant Biology SN - 1672-9072 TI - Auxin signaling: Research advances over the past 30 years VL - 64 ER - TY - JOUR AB - 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. AU - Hajny, Jakub AU - Tan, Shutang AU - Friml, Jiří ID - 10768 IS - 2 JF - Current Opinion in Plant Biology SN - 1369-5266 TI - Auxin canalization: From speculative models toward molecular players VL - 65 ER - TY - JOUR AB - 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. AU - Dahhan, DA AU - Reynolds, GD AU - Cárdenas, JJ AU - Eeckhout, D AU - Johnson, Alexander J AU - Yperman, K AU - Kaufmann, Walter AU - Vang, N AU - Yan, X AU - Hwang, I AU - Heese, A AU - De Jaeger, G AU - Friml, Jiří AU - Van Damme, D AU - Pan, J AU - Bednarek, SY ID - 10841 IS - 6 JF - Plant Cell SN - 1040-4651 TI - Proteomic characterization of isolated Arabidopsis clathrin-coated vesicles reveals evolutionarily conserved and plant-specific components VL - 34 ER - TY - JOUR AB - 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. AU - Lu, Qing AU - Zhang, Yonghong AU - Hellner, Joakim AU - Giannini, Caterina AU - Xu, Xiangyu AU - Pauwels, Jarne AU - Ma, Qian AU - Dejonghe, Wim AU - Han, Huibin AU - Van De Cotte, Brigitte AU - Impens, Francis AU - Gevaert, Kris AU - De Smet, Ive AU - Friml, Jiří AU - Molina, Daniel Martinez AU - Russinova, Eugenia ID - 10888 IS - 11 JF - Proceedings of the National Academy of Sciences of the United States of America TI - Proteome-wide cellular thermal shift assay reveals unexpected cross-talk between brassinosteroid and auxin signaling VL - 119 ER - TY - JOUR AB - 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. AU - Wang, Ren AU - Himschoot, Ellie AU - Chen, Jian AU - Boudsocq, Marie AU - Geelen, Danny AU - Friml, Jiří AU - Beeckman, Tom AU - Vanneste, Steffen ID - 11589 JF - Frontiers in Plant Science TI - Constitutive active CPK30 interferes with root growth and endomembrane trafficking in Arabidopsis thaliana VL - 13 ER -