Plant bodies are built from immobile cells, making the regulation of cell expansion essential for growth, development, and adaptation. In roots, cell elongation executes the movement of the root tips through the soil. This process is tightly controlled by numerous signaling pathways. Among these, gibberellin and auxin signaling stand out for their contrasting effects on root growth, interacting through complex cross talk at multiple regulatory levels. Here, we reveal the molecular basis of the auxin-gibberellin cross talk in the model plant Arabidopsis thaliana. We show that the auxin signaling pathway steers the expression of GIBBERELLIN 2-OXIDASES (GA2OXs), key gibberellin-deactivating enzymes in the root elongation zone (EZ). GA2OXs are negative regulators of root cell elongation; GA2OX8 overexpression decreases gibberellin levels and inhibits root cell elongation; in contrast, the ga2ox heptuple mutant roots show elevated gibberellin levels in the EZ and grow longer roots. Intriguingly, shoot-derived auxin can regulate GA2OX6 and GA2OX8 expression in roots, linking systemic auxin signaling to local gibberellin level modulation. Together, our findings identify GA2OX6 and GA2OX8 enzymes as key mediators of auxin-gibberellin cross talk, providing insights into their roles in root elongation. These results expand our understanding of how auxin integrates with gibberellin signaling to coordinate root development and growth dynamics.

• Keywords
• Arabidopsis, auxin, gibberellin, root,
• MeSH
• Arabidopsis * growth & development   metabolism   genetics   enzymology   MeSH
• Gibberellins * metabolism   MeSH
• Plant Roots * growth & development   metabolism   genetics   MeSH
• Indoleacetic Acids * metabolism   MeSH
• Mixed Function Oxygenases * metabolism   genetics   MeSH
• Arabidopsis Proteins * metabolism   genetics   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Growth Regulators metabolism   MeSH
• Signal Transduction MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• gibberellin 2-dioxygenase MeSH Browser
• Gibberellins * MeSH
• Indoleacetic Acids * MeSH
• Mixed Function Oxygenases * MeSH
• Arabidopsis Proteins * MeSH
• Plant Growth Regulators MeSH

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

Although angiosperm plants generally react to immunity elicitors like chitin or chitosan by the cell wall callose deposition, this response in particular cell types, especially upon chitosan treatment, is not fully understood. Here we show that the growing root hairs (RHs) of Arabidopsis can respond to a mild (0.001%) chitosan treatment by the callose deposition and by a deceleration of the RH growth. We demonstrate that the glucan synthase-like 5/PMR4 is vital for chitosan-induced callose deposition but not for RH growth inhibition. Upon the higher chitosan concentration (0.01%) treatment, RHs do not deposit callose, while growth inhibition is prominent. To understand the molecular and cellular mechanisms underpinning the responses to two chitosan treatments, we analysed early Ca2+ and defence-related signalling, gene expression, cell wall and RH cellular endomembrane modifications. Chitosan-induced callose deposition is also present in the several other plant species, including functionally analogous and evolutionarily only distantly related RH-like structures such as rhizoids of bryophytes. Our results point to the RH callose deposition as a conserved strategy of soil-anchoring plant cells to cope with mild biotic stress. However, high chitosan concentration prominently disturbs RH intracellular dynamics, tip-localised endomembrane compartments, growth and viability, precluding callose deposition.

• Keywords
• arabidopsis, cell wall, defence, gene expression, signalling,
• MeSH
• Arabidopsis * growth & development   drug effects   metabolism   physiology   MeSH
• Cell Membrane metabolism   MeSH
• Cell Wall * metabolism   MeSH
• Chitosan * pharmacology   MeSH
• Glucans * metabolism   MeSH
• Glucosyltransferases metabolism   MeSH
• Plant Roots * growth & development   metabolism   drug effects   MeSH
• Arabidopsis Proteins * metabolism   genetics   MeSH
• Gene Expression Regulation, Plant drug effects   MeSH
• Calcium metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• callose MeSH Browser
• Chitosan * MeSH
• Glucans * MeSH
• Glucosyltransferases MeSH
• Arabidopsis Proteins * MeSH
• Calcium 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

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

Potassium ion (K+) plays a critical role as an essential electrolyte in all biological systems. Genetically-encoded fluorescent K+ biosensors are promising tools to further improve our understanding of K+-dependent processes under normal and pathological conditions. Here, we report the crystal structure of a previously reported genetically-encoded fluorescent K+ biosensor, GINKO1, in the K+-bound state. Using structure-guided optimization and directed evolution, we have engineered an improved K+ biosensor, designated GINKO2, with higher sensitivity and specificity. We have demonstrated the utility of GINKO2 for in vivo detection and imaging of K+ dynamics in multiple model organisms, including bacteria, plants, and mice.

• MeSH
• Biosensing Techniques * methods   MeSH
• Potassium MeSH
• Ions MeSH
• Mice MeSH
• Fluorescence Resonance Energy Transfer * methods   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Potassium MeSH
• Ions 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