Aux/IAA proteins are well-known as key components of the nuclear auxin signaling pathway, repressing gene transcription when present and enabling gene activation upon their degradation. In this review, we explore the additional roles of Aux/IAA proteins in the known auxin perception pathways-the TIR1/AFBs nuclear as well as in the emerging cytoplasmic and apoplastic pathways. We summarize recent advances in understanding the regulation of Aux/IAA protein stability at the post-translational level, a critical factor in auxin-regulated transcriptional output. We further highlight the roles of auxin-nondegradable non-canonical Aux/IAAs in auxin-mediated transcription and their involvement in apoplastic auxin signalling. Additionally, we discuss the importance of Aux/IAAs for the adenylate cyclase activity of TIR1/AFB receptors and speculate on their involvement in the cytoplasmic auxin pathway. Using Arabidopsis root as a model, this work underscores the central role of Aux/IAA proteins in mediating auxin-driven developmental processes and environmental responses. Key questions for future research are proposed to further unravel the dynamic roles of Aux/IAAs in auxin signaling networks.

• MeSH
• Arabidopsis * metabolism   genetics   MeSH
• F-Box Proteins metabolism   genetics   MeSH
• Plant Roots metabolism   MeSH
• Indoleacetic Acids * metabolism   MeSH
• Arabidopsis Proteins * metabolism   genetics   MeSH
• Receptors, Cell Surface metabolism   genetics   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Growth Regulators * metabolism   MeSH
• Plant Proteins * metabolism   genetics   MeSH
• Signal Transduction MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• F-Box Proteins MeSH
• Indoleacetic Acids * MeSH
• Arabidopsis Proteins * MeSH
• Receptors, Cell Surface MeSH
• Plant Growth Regulators * MeSH
• Plant Proteins * MeSH

PIN proteins establish the auxin concentration gradient, which coordinates plant growth. PIN1-4 and 7 localized at the plasma membrane (PM) and facilitate polar auxin transport while the endoplasmic reticulum (ER) localized PIN5 and PIN8 maintain the intracellular auxin homeostasis. Although an antagonistic activity of PIN5 and PIN8 proteins in regulating the intracellular auxin homeostasis and other developmental events have been reported, the membrane topology of these proteins, which might be a basis for their antagonistic function, is poorly understood. In this study we optimized digitonin based PM-permeabilizing protocols coupled with immunocytochemistry labeling to map the membrane topology of PIN5 and PIN8 in Arabidopsis thaliana root cells. Our results indicate that, except for the similarities in the orientation of the N-terminus, PIN5 and PIN8 have an opposite orientation of the central hydrophilic loop and the C-terminus, as well as an unequal number of transmembrane domains (TMDs). PIN8 has ten TMDs with groups of five alpha-helices separated by the central hydrophilic loop (HL) residing in the ER lumen, and its N- and C-terminals are positioned in the cytoplasm. However, the topology of PIN5 comprises nine TMDs. Its N-terminal end and the central HL face the cytoplasm while its C-terminus resides in the ER lumen. Overall, this study shows that PIN5 and PIN8 proteins have a divergent membrane topology while introducing a toolkit of methods for studying membrane topology of integral proteins including those localized at the ER membrane.

• Keywords
• Arabidopsis thaliana, Auxin homeostasis and transport, Digitonin PM-permeabilizing protocols, Endoplasmic reticulum, Immunocytochemistry, Membrane topology determination methods, PIN auxin efflux carriers,
• Publication type
• Journal Article MeSH

The plant-signaling molecule auxin triggers fast and slow cellular responses across land plants and algae. The nuclear auxin pathway mediates gene expression and controls growth and development in land plants, but this pathway is absent from algal sister groups. Several components of rapid responses have been identified in Arabidopsis, but it is unknown if these are part of a conserved mechanism. We recently identified a fast, proteome-wide phosphorylation response to auxin. Here, we show that this response occurs across 5 land plant and algal species and converges on a core group of shared targets. We found conserved rapid physiological responses to auxin in the same species and identified rapidly accelerated fibrosarcoma (RAF)-like protein kinases as central mediators of auxin-triggered phosphorylation across species. Genetic analysis connects this kinase to both auxin-triggered protein phosphorylation and rapid cellular response, thus identifying an ancient mechanism for fast auxin responses in the green lineage.

• Keywords
• RAF kinase, auxin, plant evolution, protein phosphorylation,
• MeSH
• Arabidopsis genetics   metabolism   MeSH
• Algal Proteins metabolism   MeSH
• Phosphorylation MeSH
• Indoleacetic Acids metabolism   MeSH
• Protein Kinases metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Proteins metabolism   MeSH
• Plants metabolism   MeSH
• Signal Transduction * MeSH
• Embryophyta * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Algal Proteins MeSH
• Indoleacetic Acids MeSH
• Protein Kinases MeSH
• Plant Proteins MeSH

In vivo microscopy of plants with high-frequency imaging allows observation and characterization of the dynamic responses of plants to stimuli. It provides access to responses that could not be observed by imaging at a given time point. Such methods are particularly suitable for the observation of fast cellular events such as membrane potential changes. Classical measurement of membrane potential by probe impaling gives quantitative and precise measurements. However, it is invasive, requires specialized equipment, and only allows measurement of one cell at a time. To circumvent some of these limitations, we developed a method to relatively quantify membrane potential variations in Arabidopsis thaliana roots using the fluorescence of the voltage reporter DISBAC2(3). In this protocol, we describe how to prepare experiments for agar media and microfluidics, and we detail the image analysis. We take an example of the rapid plasma membrane depolarization induced by the phytohormone auxin to illustrate the method. Relative membrane potential measurements using DISBAC2(3) fluorescence increase the spatio-temporal resolution of the measurements and are non-invasive and suitable for live imaging of growing roots. Studying membrane potential with a more flexible method allows to efficiently combine mature electrophysiology literature and new molecular knowledge to achieve a better understanding of plant behaviors. Key features Non-invasive method to relatively quantify membrane potential in plant roots. Method suitable for imaging seedlings root in agar or liquid medium. Straightforward quantification.

• Keywords
• Auxin, In vivo, Microfluidics, Microscopy, Non invasive,
• Publication type
• Journal Article MeSH

Plant roots navigate in the soil environment following the gravity vector. Cell divisions in the meristem and rapid cell growth in the elongation zone propel the root tips through the soil. Actively elongating cells acidify their apoplast to enable cell wall extension by the activity of plasma membrane AHA H+-ATPases. The phytohormone auxin, central regulator of gravitropic response and root development, inhibits root cell growth, likely by rising the pH of the apoplast. However, the role of auxin in the regulation of the apoplastic pH gradient along the root tip is unclear. Here, we show, by using an improved method for visualization and quantification of root surface pH, that the Arabidopsis thaliana root surface pH shows distinct acidic and alkaline zones, which are not primarily determined by the activity of AHA H+-ATPases. Instead, the distinct domain of alkaline pH in the root transition zone is controlled by a rapid auxin response module, consisting of the AUX1 auxin influx carrier, the AFB1 auxin co-receptor, and the CNCG14 calcium channel. We demonstrate that the rapid auxin response pathway is required for an efficient navigation of the root tip.

• Keywords
• A. thaliana, auxin, pH, plant biology, root,
• MeSH
• Adenosine Triphosphatases metabolism   MeSH
• Arabidopsis * metabolism   MeSH
• Cyclic Nucleotide-Gated Cation Channels metabolism   MeSH
• Hydrogen-Ion Concentration MeSH
• Plant Roots MeSH
• Indoleacetic Acids metabolism   MeSH
• Arabidopsis Proteins * metabolism   MeSH
• Soil MeSH
• Gene Expression Regulation, Plant MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphatases MeSH
• AUX1 protein, Arabidopsis MeSH Browser
• CNGC14 protein, Arabidopsis MeSH Browser
• Cyclic Nucleotide-Gated Cation Channels MeSH
• Indoleacetic Acids MeSH
• Arabidopsis Proteins * MeSH
• Soil MeSH

The phytohormone auxin triggers root growth inhibition within seconds via a non-transcriptional pathway. Among members of the TIR1/AFB auxin receptor family, AFB1 has a primary role in this rapid response. However, the unique features that confer this specific function have not been identified. Here we show that the N-terminal region of AFB1, including the F-box domain and residues that contribute to auxin binding, is essential and sufficient for its specific role in the rapid response. Substitution of the N-terminal region of AFB1 with that of TIR1 disrupts its distinct cytoplasm-enriched localization and activity in rapid root growth inhibition by auxin. Importantly, the N-terminal region of AFB1 is indispensable for auxin-triggered calcium influx, which is a prerequisite for rapid root growth inhibition. Furthermore, AFB1 negatively regulates lateral root formation and transcription of auxin-induced genes, suggesting that it plays an inhibitory role in canonical auxin signaling. These results suggest that AFB1 may buffer the transcriptional auxin response, whereas it regulates rapid changes in cell growth that contribute to root gravitropism.

• Keywords
• Arabidopsis, auxin signaling, calcium, gravitropism, lateral root,
• MeSH
• Arabidopsis * metabolism   MeSH
• Cytosol metabolism   MeSH
• F-Box Proteins * metabolism   MeSH
• Plant Roots metabolism   MeSH
• Indoleacetic Acids pharmacology   metabolism   MeSH
• Arabidopsis Proteins * metabolism   MeSH
• Receptors, Cell Surface genetics   metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Growth Regulators metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• F-Box Proteins * MeSH
• Indoleacetic Acids MeSH
• Arabidopsis Proteins * MeSH
• Receptors, Cell Surface MeSH
• Plant Growth Regulators MeSH

Roots are sensors evolved to simultaneously respond to manifold signals, which allow the plant to survive. Root growth responses, including the modulation of directional root growth, were shown to be differently regulated when the root is exposed to a combination of exogenous stimuli compared to an individual stress trigger. Several studies pointed especially to the impact of the negative phototropic response of roots, which interferes with the adaptation of directional root growth upon additional gravitropic, halotropic or mechanical triggers. This review will provide a general overview of known cellular, molecular and signalling mechanisms involved in directional root growth regulation upon exogenous stimuli. Furthermore, we summarise recent experimental approaches to dissect which root growth responses are regulated upon which individual trigger. Finally, we provide a general overview of how to implement the knowledge gained to improve plant breeding.

• Keywords
• ABA, SnRK1, SnRK2, TOR, auxin, green systems biology, metabolomics, panomics,
• Publication type
• Journal Article MeSH
• Review MeSH

The phytohormone auxin triggers root growth inhibition within seconds via a non-transcriptional pathway. Among members of the TIR1/AFBs auxin receptor family, AFB1 has a primary role in this rapid response. However, the unique features that confer this specific function have not been identified. Here we show that the N-terminal region of AFB1, including the F-box domain and residues that contribute to auxin binding, are essential and sufficient for its specific role in the rapid response. Substitution of the N-terminal region of AFB1 with that of TIR1 disrupts its distinct cytoplasm-enriched localization and activity in rapid root growth inhibition. Importantly, the N-terminal region of AFB1 is indispensable for auxin-triggered calcium influx which is a prerequisite for rapid root growth inhibition. Furthermore, AFB1 negatively regulates lateral root formation and transcription of auxin-induced genes, suggesting that it plays an inhibitory role in canonical auxin signaling. These results suggest that AFB1 may buffer the transcriptional auxin response while it regulates rapid changes in cell growth that contribute to root gravitropism.

• Publication type
• Journal Article MeSH
• Preprint MeSH

The phytohormone auxin triggers transcriptional reprogramming through a well-characterized perception machinery in the nucleus. By contrast, mechanisms that underlie fast effects of auxin, such as the regulation of ion fluxes, rapid phosphorylation of proteins or auxin feedback on its transport, remain unclear1-3. Whether auxin-binding protein 1 (ABP1) is an auxin receptor has been a source of debate for decades1,4. Here we show that a fraction of Arabidopsis thaliana ABP1 is secreted and binds auxin specifically at an acidic pH that is typical of the apoplast. ABP1 and its plasma-membrane-localized partner, transmembrane kinase 1 (TMK1), are required for the auxin-induced ultrafast global phospho-response and for downstream processes that include the activation of H+-ATPase and accelerated cytoplasmic streaming. abp1 and tmk mutants cannot establish auxin-transporting channels and show defective auxin-induced vasculature formation and regeneration. An ABP1(M2X) variant that lacks the capacity to bind auxin is unable to complement these defects in abp1 mutants. These data indicate that ABP1 is the auxin receptor for TMK1-based cell-surface signalling, which mediates the global phospho-response and auxin canalization.

• MeSH
• Arabidopsis * genetics   metabolism   MeSH
• Phosphorylation MeSH
• Hydrogen-Ion Concentration MeSH
• Indoleacetic Acids * metabolism   MeSH
• Mutation MeSH
• Protein Serine-Threonine Kinases * genetics   metabolism   MeSH
• Arabidopsis Proteins * genetics   metabolism   MeSH
• Proton-Translocating ATPases metabolism   MeSH
• Cytoplasmic Streaming MeSH
• Plant Growth Regulators metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• AT1G66150 protein, Arabidopsis MeSH Browser
• auxin-binding protein 1 MeSH Browser
• Indoleacetic Acids * MeSH
• Protein Serine-Threonine Kinases * MeSH
• Arabidopsis Proteins * MeSH
• Proton-Translocating ATPases MeSH
• Plant Growth Regulators MeSH

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.

• Keywords
• RALF1, auxin, biphasic regulation, cross-talk, root growth inhibition,
• MeSH
• Arabidopsis * metabolism   MeSH
• Phosphotransferases MeSH
• Plant Roots * growth & development   MeSH
• Indoleacetic Acids * metabolism   MeSH
• Peptide Hormones * metabolism   MeSH
• Arabidopsis Proteins * metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• At3g51550 protein, Arabidopsis MeSH Browser
• Phosphotransferases MeSH
• Indoleacetic Acids * MeSH
• Peptide Hormones * MeSH
• Arabidopsis Proteins * MeSH
• RALF1 protein, Arabidopsis MeSH Browser