Biology subjects

Friml, J.

Publications and source records attributed to Friml, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Conserved molecular compatibility for MIZ1 function in cytokinin signaling in land plants

Plant roots adjust their growth direction toward water through hydrotropism, a process that in Arabidopsis thaliana requires the endoplasmic reticulum (ER)-associated protein MIZU-KUSSEI1 (MIZ1). Nonetheless, MIZ1 orthologues occur throughout land plants, including lineages lacking true roots, raising questions about the ancestral function of this protein family. Here, we investigated the evolutionary conservation and functional diversification of MIZ1-like proteins across land plants. MIZ1 orthologues from evolutionarily distant land plants failed to restore hydrotropism or cytokinin sensitivity in the Arabidopsis miz1 mutant, despite conserved protein structure and ER localization. In the liverwort Marchantia polymorpha, loss of MpMIZ1a led to altered cytokinin sensitivity and cytokinin-mediated development in both the sporophyte and gametophyte. These phenotypes and gene expression analyses imply a conserved role of MIZ1 in cytokinin-related processes. Despite this functional similarity, protein-protein docking and bimolecular fluorescence complementation revealed species-specific interactions between MIZ1 proteins and their cognate cytokinin histidine kinase receptors in Arabidopsis and Marchantia. Together, our findings indicate that MIZ1 proteins represent ancient land plant regulators of cytokinin-dependent development whose molecular compatibility and developmental outputs have diverged during evolution. We propose that the role of MIZ1 in root hydrotropism represents a derived recruitment of this ancestral module into root-specific environmental response pathways.

plant biology↗

bioRxiv2025

Non-transcriptional gibberellin DELLA signalling for microtubule dynamics

Plant cell growth is associated with changes in the dynamics and orientation of the microtubule (MT) cytoskeleton. The plant hormone Gibberellic acid (GA), a central regulator of growth also has an impact on MT arrangements, however the causal relationship between these processes and underlying molecular mechanism remain unclear. Here, examining the Arabidopsis thaliana root elongation zone, we show that MT reorientation occurs as a consequence of GA-induced cell elongation and that GA has also a direct effect on the rate of MT polymerization via a non-transcriptional mechanism. Preventing cell elongation blocked GA-induced MT reorganization, whereas GA still promoted elongation in the absence of intact MTs. On the other hand, GA promoted MT dynamics as revealed by the recovery of MTs following depolymerization or photobleaching. Live imaging revealed that GA directly promotes MT polymerization at their plus ends. This GA effect persists even when transcription or translation are inhibited, but still requires the DELLA canonical components of GA signalling and their interactors, the prefoldins (PFDs). Our results support a model where GA-induced DELLA degradation releases in nucleus sequestrated PFDs, enhancing their cytoplasmic function to promote MT polymerization. Hence, GA signalling branches into two mechanistically distinct modules: (i) canonical transcriptional pathway that promotes growth and indirectly drives MT reorientation; and (ii) the non-transcriptional DELLA-PFD axis that directly promotes MT polymerization. Such a dual mechanism enables plants to synchronize growth with cytoskeletal remodelling by coupling rapid, cytoplasmic responses to slower transcriptional programs.

cell biology↗

bioRxiv2025

Nuclear auxin signalling induces autophagy for developmental reprogramming

The phytohormone auxin is a central regulator of plant growth and development, traditionally known for mediating transcriptional reprogramming through the canonical TIR1/AFB-Aux/IAA signalling pathway. In this study, we reveal that auxin rapidly induces macroautophagy, a catabolic process critical for the removal and recycling of superfluous macromolecules. We demonstrate that natural auxin (IAA) triggers autophagy at physiological concentrations. Genetic and pharmacological analyses show that TIR1/AFB receptors and their adenylate cyclase activity are indispensable for autophagy induction. Furthermore, auxin-induced autophagy depends on transcription, highlighting its integration with the broader gene regulatory networks. Functionally, we show that auxin-induced autophagy is required for efficient developmental reprogramming and organogenesis in both root and shoots. Autophagy is induced at places of local auxin maxima and autophagy-deficient mutants exhibit delayed differentiation and retarded organ primordia progression at the meristematic zones. Together, our findings uncover a dual role for auxin in coordinating gene expression and autophagic clearance, thereby facilitating rapid and effective developmental transitions.

plant biology↗

bioRxiv2025

Cytosolic Ca2+ as a universal signal for rapid root growth regulation

Roots must continuously adapt their growth and have evolved the ability to rapidly respond to diverse environmental and hormonal cues, including the phytohormone auxin. Auxin-induced cellular responses involve membrane depolarization, cytosolic calcium (Ca{superscript 2}) elevation, and extracellular pH increase, but how these processes lead to rapid growth regulation remains unclear. Here, we show that cytosolic Ca{superscript 2} acts as a universal signal integrating diverse cues for rapid regulation of root growth. Using live imaging, microfluidics, and optogenetics in Arabidopsis, we demonstrate that a swift rise in cytosolic Ca{superscript 2} is the primary target of auxin signaling affecting growth inhibition. Disruption of Ca{superscript 2} influx abolishes these responses, whereas light-gated Ca{superscript 2} influx from the apoplast or endoplasmic reticulum stores inhibits growth. Multiple unrelated stimuli--including auxin, extracellular ATP, RALF peptides, and hydrogen peroxide--converge on this Ca{superscript 2}-dependent mechanism. Cytosolic Ca{superscript 2} elevation thus represents a necessary and sufficient step for rapid growth inhibition, revealing a unifying principle of root signaling.

plant biology↗

bioRxiv2025

MAKR6 targets TMK and CAMEL-CANAR receptor complexes for auxin canalization in Arabidopsis

Adaptive plant development is orchestrated, among others, by directional, intercellular transport of the phytohormone auxin. Self-organizing development, such as flexible vasculature formation, depends on so-called auxin canalization, manifested by the gradual formation of auxin transport channels through feedback between auxin signalling and transport. Herein, we identify MAKR6 as an important, novel component in this feedback. MAKR6 expression accumulates strongly in vascular cells and is tightly regulated by auxin via the Aux/IAA-ARF-WRKY23 transcriptional network. MAKR6 is required for auxin canalization-dependent processes, including leaf venation, vasculature regeneration, and de novo auxin channel formation from local auxin sources. Mechanistically, MAKR6 interacts with the PIN1 auxin transporter, modulating its trafficking and polarization. MAKR6 also associates with and integrates two key receptor-like kinase complexes involved in canalization, TMK1/4 and the CAMEL-CANAR. Together, our study establishes MAKR6 as a multifaceted regulator that couples transcriptional auxin signalling to PIN1 repolarization and coordinates multiple RLK-mediated signalling pathways during canalization. This provides mechanistic insights into auxin canalization and exemplifies a framework for exploring similar regulatory nodes in other developmental contexts.

plant biology↗

bioRxiv2025

Ca2+ signature-dependent control of auxin sensitivity in Arabidopsis

Plants must continually balance growth with arrest, especially under stress. Auxin signaling acts as a central regulatory hub in this process, yet the mechanisms that dynamically tune early auxin signalling in real time remain unknown. Here, we used the light-gated, Ca2+-permeable Channelrhodopsin 2 variant XXM2.0 to optogenetically impose defined Ca2+ signatures on Arabidopsis root cells. Repetitive light activation triggered cytosolic Ca2+ signals that in turn suppressed auxin-induced membrane depolarization and Ca2+ transients. Moreover, prolonged optogenetic Ca2+ stimulation affects auxin-responsive transcriptional reprogramming. As phenotypic output, reversible inhibition of root growth by suppressing cell division and elongation was observed. We further identify a candidate CaM7-CNGC14 module that likely mediates Ca2+-dependent gating of early auxin signalling. Our study thus introduces a synthetic biology approach to decompose calcium-auxin crosstalk in plant cells, and demonstrates that optogenetically imposed cytosolic Ca2+ signals act as dynamic regulators of auxin susceptibility in roots.

plant biology↗

bioRxiv2025

TOW links TIR1/AFB-mediated signalling with Receptor-Like Kinases in auxin canalization

Auxin canalization is a self-organizing process that governs the flexible formation of vasculature by reinforcing the formation of auxin transport channels. A key prerequisite is the feedback between auxin signaling and directional auxin transport, mediated by PIN transporters. Despite the developmental importance of canalization, the molecular components linking auxin perception to the regulation of PIN auxin transporters remain poorly understood. Here, we identify TOW, a novel and essential component of auxin canalization that links intracellular auxin signaling with cell surface auxin perception. TOW is regulated downstream of TIR1/AFB-Aux/IAA-WRKY23 transcriptional auxin signaling. tow mutants exhibit defects in regeneration and de novo vasculature formation, along with impaired formation of polarized, PIN-expressing auxin channels. At the subcellular level, these mutants display disrupted auxin-induced PIN polarization and altered PIN endocytic trafficking dynamics. TOW localizes to the Golgi, trans-Golgi network, and predominantly to the plasma membrane, where it interacts with receptor-like kinases involved in auxin canalization, including the TMK1 auxin co-receptor and the CAMEL-CANAR complex. Together, our findings identify TOW as a molecular link between intracellular and cell surface auxin signaling mechanisms that converge on PIN trafficking and polarity, providing new insights into how auxin signaling regulates directional auxin transport for the self-organizing formation of vasculature during flexible plant development.

plant biology↗

bioRxiv2025

TMK interacting network of receptor like kinases for auxin canalization and beyond

Receptor-like kinases (RLKs), particularly the Transmembrane Kinase (TMK) family, play essential roles in signaling and development, with TMKs being key components of auxin perception and downstream phosphorylation events. While TMKs involvement in auxin canalization, a process essential for vasculature formation and regeneration, has been established, nonetheless, the additional signaling and regulatory partners remain poorly understood. In this study, we identify and characterize seven leucine-rich repeat RLKs (TINT1-TINT7) as novel interactors of TMK1, revealing their diverse evolutionary, structural, and functional characteristics. Our results show that TINTs interact with TMK1 and highlight their roles in regulating various developmental processes. Majority of TINTs contributes, together with TMK1, to auxin canalization, with TINT5 linking TMK1 to other canalization component CAMEL. Beyond canalization, we also establish the role of TINT-TMK1 interactions in processes such as stomatal movement and the hypocotyls gravitropic response. These findings suggest that TINTs, through their interaction with TMK1, are integral components of various signaling networks, contributing to both auxin canalization and broader plant development.

plant biology↗

bioRxiv2025

Calcium-triggered apoplastic ROS bursts balance gravity and mechanical signals to navigate soil

Reactive oxygen species (ROS) have been implicated repeatedly in multiple signaling processes in plants but the underlying mechanisms and roles remain enigmatic. Here, we developed live imaging of apoplastic ROS at the root surface. Different signals, including auxin, extracellular ATP and RALF1 peptide, all induce cytosolic calcium transients and apoplastic ROS bursts. Genetic and optogenetic manipulations identified calcium transients as necessary and sufficient for ROS bursts via activation of NADPH oxidases RBOHC and RBOHF. Apoplastic ROS bursts are not required but rather limit the gravity-induced root bending. Root bending is sensed by stretch-activated calcium channel MCA1 leading to NADPH oxidase activation at the stretched side. The resulting ROS production stiffens cell wall for better soil penetration. Apoplastic ROS thus provides a means to balance tissue flexibility and stiffness to efficiently navigate soil.

plant biology↗

bioRxiv2025

Armadillo Repeat Only Proteins Are Crucial for the Function of Plant CNGC Channels

The versatile Ca2+ signaling system governs plant responses to a wide array of environmental and developmental cues. CYCLIC NUCLEOTIDE-GATED CHANNELS (CNGCs) trigger cellular responses to diverse signals, including phytohormones, biotic and abiotic stresses; and as such their activity is tightly controlled. Unlike their animal paralogs, plant CNGCs does not seem to be gated by cyclic nucleotides, and the mechanism of their activation remains unresolved. Here we report ARMADILLO REPEAT ONLY (ARO) proteins as novel, plant-specific, and essential activators of plant CNGCs. Reciprocal proximity labeling revealed interactions between all sporophytic CNGCs and AROs. Loss-of-function aro mutants fail to induce Ca2+ transients in response to known CNGC-triggering stimuli. Structural modeling, mutational analysis, and electrophysiological data show that AROs assemble into a complex with CNGC tetramers by interaction with the conserved Calmodulin-binding IQ domain. AROs represent CNGC activators, competing with Calmodulins, showcasing an evolutionarily unique solution to regulation of calcium signaling in plants.

plant biology↗

bioRxiv2024

Auxin fluctuation and PIN polarization in moss leaf cell reprogramming

Auxin and its PIN-FORMED (PIN) exporters are essential for tissue repair and regeneration in flowering plants. To gain insight into the evolution of this mechanism, we investigated their roles in leaves excised from Physcomitrium patens, a bryophyte known for its remarkable cell reprogramming capacity. We used various approaches to manipulate auxin levels, including exogenous application, pharmacological manipulations, and auxin biosynthesis mutants. We observed no significant effect on the rate of cell reprogramming. Rather, our analysis of auxin dynamics revealed a decrease in auxin levels upon excision, which was followed by a local increase before the reprogramming process began. Mutant analysis revealed that PpPINs are required for effective cell reprogramming, and endogenously expressed PpPINA-GFP accumulates polarly at sites that will develop into future filamentous stem cells. In addition, hyperpolarized PpPINA variants carrying mutated phosphorylation sites showed a marked delay in reprogramming, whereas endogenous or non-polar versions do not have this effect. These results underscore that both, the levels and the polarity of PpPINA are important for efficient cell reprogramming. Overall, these findings highlight the pivotal role of PIN polarity in plant regeneration. Furthermore, they suggest that understanding polarity mechanisms could have broader implications for improving regenerative processes across various plant species.

cell biology↗

bioRxiv2024

The role of IAA and its transport in the complex streptophyte algae Chara braunii

The role of auxin indole-3-acetic acid (IAA), a phytohormone with numerous morphogenic functions, is now well-established in land plants. Although the role of IAA and its transport in algae remains unclear, PIN-driven auxin export is probably an ancient and conserved trait within Streptophytes. Among streptophyte algae, Chara represents a genus with a considerable degree of complexity in body arrangement. This study investigates the auxin response and characterizes homologs of land plant PIN auxin efflux carriers in Chara braunii. Through regeneration experiments, we observed that IAA significantly promotes elongation of thallus and side branch development upon thallus tip decapitation, indicating an effect on morphogenesis. We show that IAA is actively uptaken and metabolized by thallus cells and that this process is influenced by N-1-naphthylphthalamic acid (NPA). To elucidate the underlying mechanisms, we cloned and sequenced the most expressed Chara braunii PINs, CbPINa and CbPINc. Using epitope-specific antibodies, we showed their presence in the plasma membrane (PM) of vegetative internodal cells and generative antheridial cells. Functional tests in tobacco BY-2 cells, supported by in silico docking of IAA and NPA to CbPINa and CbPINc, showed that PM-localized CbPINa interferes with auxin transport in contrast to ER-localized CbPINc. Finally, our phosphoproteome analysis indicated that IAA rapidly induces specific phosphorylation events, including RAF-like kinase phosphorylation, highlighting a potential role for IAA in fast signaling processes in Chara braunii. Altogether, we provide new insights into IAA role in Chara braunii morphogenesis and suggest that while the canonical auxin transport mechanism may not be conserved, auxin still may likely play a role in rapid signaling pathways in this close relative of land plants.

plant biology↗

bioRxiv2024

The Draparnaldia genome: alternative mechanisms for multicellularity and terrestrialization in green plants

Green plants contain two algal lineages: Streptophyte algae that diverged into land plants and Chlorophyte algae that are mostly aquatic. Draparnaldia is a Chlorophyte alga morphologically resembling mosses and living in both, the aquatic and terrestrial habitats. Because of its complex morphology and terrestrial adaptations, Draparnaldia can provide new insights into the evolution of multicellularity and terrestrialization in green plants. To develop Draparnaldia into a model, we de novo sequenced its genome and transcriptomes, and profiled its phytohormone repertoire. We found that 1) Expanded gene families in Draparnaldia with respect to unicellular Chlamydomonas are linked to multicellularity and abiotic stresses. 2) Draparnaldias terrestrial adaptations are reflected at both the morphological and molecular levels. 3) Draparnaldia synthesizes most of the phytohormones used by land plants to thrive in terrestrial habitats. All of this makes Draparnaldia a powerful model to uncover and study alternative evolutionary trajectories towards multicellularity and terrestrialization in plants.

evolutionary biology↗

bioRxiv2024

Hierarchical global and local auxin signals coordinate cellular interdigitation in Arabidopsis

The development of multicellular tissues requires both local and global coordination of cell polarization, however, the mechanisms underlying their interplay are poorly understood. In Arabidopsis, leaf epidermal pavement cells (PC) develop a puzzle-piece shape locally coordinated through apoplastic auxin signaling. Here we show auxin also globally coordinates interdigitation by activating the TIR1/AFB-dependent nuclear signaling pathway. This pathway promotes a transient maximum of auxin at the cotyledon tip, which then moves across the leaf activating local PC polarization, as demonstrated by locally uncaged auxin globally rescuing defects in tir1;afb1;afb2;afb4;afb5 mutant but not in tmk1;tmk2;tmk3;tmk4 mutants. Our findings show that hierarchically integrated global and local auxin signaling systems, which respectively depend on TIR1/AFB-dependent gene transcription in the nucleus and TMK-mediated rapid activation of ROP GTPases at the cell surface, control PC interdigitation patterns in Arabidopsis cotyledons, revealing a mechanism for coordinating a local cellular process with the development of whole tissues.

plant biology↗

bioRxiv2024

The Unexpected Membrane Targeting of Marchantia Short PIN Auxin Exporters Illuminates Sequence Determinants and Evolutionary Significance

The plant hormone auxin and its directional transport are crucial for growth and development. PIN auxin transporters, on account of their polarized distribution, are instrumental in guiding auxin flow across tissues. Based on protein length and subcellular localization, the PIN family is classified into two groups: plasma membrane (PM)-localized long PINs and endoplasmic reticulum (ER)-localized short PINs. The origin of PINs was traced to the alga Klebsormidium, with a single PM-localized long KfPIN. Bryophytes, the earliest land plant clade, represent the initial clade harboring the short PINs. We tracked the evolutionary trajectory of the short PINs and explored their function and localization in the model bryophyte Marchantia polymorpha, which carries four short and one long PIN. Our findings reveal that all short MpPINs can export auxin, and they are all PM-localized with MpPINX and MpPINW exhibiting asymmetric distribution. We identified a unique miniW domain within the MpPINW hydrophilic loop region, which is sufficient for its PM localization. Phosphorylation site mutations within the miniW domain abolish the PM localization. These findings not only identify the essential sequence determinant of PINs PM localization but also provide a unique insight into the evolution of ER-localized PINs. Short MpPINW, which is evolutionarily positioned between the ancestral long PINs and contemporary short PINs, still preserves the critical region essential for its PM localization. We propose that throughout land plant evolution, the unique miniW domain has been gradually lost thus converting the PM-localized short PINs in bryophytes to ER-localized short PINs in angiosperms. IMPORTANTO_LIManuscripts submitted to Review Commons are peer reviewed in a journal-agnostic way. C_LIO_LIUpon transfer of the peer reviewed preprint to a journal, the referee reports will be available in full to the handling editor. C_LIO_LIThe identity of the referees will NOT be communicated to the authors unless the reviewers choose to sign their report. C_LIO_LIThe identity of the referee will be confidentially disclosed to any affiliate journals to which the manuscript is transferred. C_LI GUIDELINESO_LIFor reviewers: https://www.reviewcommons.org/reviewers C_LIO_LIFor authors: https://www.reviewcommons.org/authors C_LI CONTACTThe Review Commons office can be contacted directly at: office@reviewcommons.org

plant biology↗

bioRxiv2024

Super-resolution expansion microscopy in plant roots

Super-resolution methods enable spatial resolution far better than the optical diffraction limit of about half the wavelength of light ([~]200-300 nm) but have yet to attain widespread use in plants, owing in large part to plants challenging optical properties. Expansion microscopy improves effective resolution by isotropically increasing physical distances between sample structures while preserving relative spatial arrangements, and clears the sample. However, its application to plants has been hindered by the rigid, mechanically cohesive structure of plant tissues. Here, we report on whole-mount expansion microscopy of Arabidopsis thaliana root tissues (PlantEx), achieving 4-fold resolution increase over conventional microscopy, highlighting microtubule cytoskeleton organization and interaction between molecularly defined cellular constituents. By combining PlantEx with STED microscopy, we increase nanoscale resolution further and visualize the complex organization of subcellular organelles from intact tissues by example of the densely packed COPI-coated vesicles associated with the Golgi apparatus and put these into cellular structural context.

plant biology↗

bioRxiv2024

Transport properties of canonical PIN-FORMED proteins and the role of the loop domain in auxin transport

Indole-3-acetic acid (IAA), the most abundant endogenous auxin is transported in plants in a polar fashion by PIN-FORMED (PIN) transporters and controls virtually all plant growth and developmental processes. Canonical PINs possess a long and largely disordered cytosolic loop domain which is shorter in non-canonical PINs. Auxin transport by canonical PINs is activated loop phosphorylation by kinases. While the structure of the transmembrane domains of these transporters was recently solved, their transport properties remained poorly characterized and particularly the relative roles of the transmembrane and loop domain therein. In this study we used flux studies to obtain quantitative kinetic parameters of IAA transport mediated by canonical PINs as well as of chimeras between transmembrane and loop domains of different PINs upon their activation by D6 PROTEIN KINASE or PINOID. We found that the transporters possess distinct transport properties that are due to both the transmembrane and loop domain. To demonstrate the physiological relevance of these distinct transport properties, we modelled root tip IAA distribution patterns and investigated the potential of different PINs to complement the agravitropic root growth phenotype of the pin2 mutant when expressed in the PIN2 domain. We found a strong correlation between transport parameters and physiological output indicating that in addition to PIN polarity a low transport rate in the PIN2 expression domain is required for gravitropic growth. Overall, the data show that the loop domain is not only required for activation of PIN-mediated auxin transport but has an additional role in the transport cycle by a currently unknown mechanism.

plant biology↗

bioRxiv2023

Guanylate cyclase activity of TIR1/AFB auxin receptorsin rapid auxin responses

The plant hormone auxin is perceived by TIR1/AFB receptors, which mediate transcriptional reprogramming via a ubiquitination- and cAMP-dependent mechanism. Same receptors mediate also rapid, non-transcriptional growth responses that are mechanistically less well understood. Here, we demonstrate that TIR1/AFB receptors possess not only adenylate cyclase but also guanylate cyclase activity. Auxin perception independently activates cAMP and cGMP production by TIR1/AFBs in vitro and elevates both cAMP and cGMP levels in planta. Genetic and synthetic biology approaches revealed that AFB1 guanylate cyclase activity is required for the rapid cytosolic Ca2+ transients and root growth inhibition. This unprecedented combination of adenylate and guanylate cyclase activities within a single hormone receptor establishes a new paradigm for how one perception mechanism can mediate distinct downstream responses. One-Sentence SummaryTIR1/AFB auxin receptors possess both, adenylate and guanylate cyclase activities, mediating transcriptional reprogramming and rapid Ca2+-dependent growth responses, respectively.

plant biology↗