Auxin, primarily indole-3-acetic acid (IAA), is a central regulator of growth and development in land plants, but its physiological role in chlorophyte algae remains unclear. Here, we show that exogenous IAA modulates growth in Chlorella sorokiniana, Chlorella variabilis, and Chlamydomonas reinhardtii in a concentration-dependent manner. Low IAA concentrations promoted growth by accelerating the onset of cell division without affecting cell size, whereas higher concentrations inhibited proliferation. Radiotracer assays showed that all three species take up and release IAA across the plasma membrane through a combination of passive diffusion and energy-dependent, saturable processes. Competition by excess unlabeled natural and synthetic auxins further supported the presence of carrier-mediated transport with broad substrate recognition. Phylogenetic analyses identified potential PIN-like auxin exporters in chlorophytes and other non-plant eukaryotes, and structural modeling supported conservation of the overall PIN fold and predicted auxin-binding residues. However, functional assays in Xenopus laevis oocytes, tobacco BY-2 cultured cells, and Arabidopsis thaliana did not support a role for these proteins in directional auxin export. Instead, non-plant PIN homologs localized predominantly to the endoplasmic reticulum and showed limited or no transport activity in heterologous systems. Together, these findings indicate that auxin responsiveness and basic cellular auxin transport predate canonical PIN-mediated directional auxin export, which appears to be a later innovation of the streptophyte lineage.

Reactive oxygen species (ROS) have been implicated in multiple signaling processes in plants, but the underlying mechanisms and roles remain enigmatic. In this study, we developed a method of live imaging of apoplastic ROS at the root surface. Distinct signals, including auxin, extracellular adenosine triphosphate, and rapid alkalinization factor 1 peptide, induce cytosolic calcium transients and apoplastic ROS bursts. Genetic and optogenetic manipulations of Arabidopsis identified calcium transients as necessary and sufficient for ROS bursts through activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases RBOHC and RBOHF. Apoplastic ROS bursts are not required, but they do limit gravity-induced root bending. Root bending is sensed by the stretch-activated calcium channel MCA1, leading to NADPH oxidase activation. The resulting ROS production stiffens cell walls to facilitate soil penetration. Apoplastic ROS thus provides a means to balance tissue flexibility and stiffness to navigate soil.

The versatile and pivotal roles of the phytohormone auxin in regulating plant growth and development are typically linked to its directional transport, relying on the polarized PIN-FORMED (PIN) auxin exporters at the plasma membrane (PM). For decades, auxin has been proposed to promote PIN polarization, generating self-regulatory feedback mediating much of plant development, but mechanistic insight into this regulation is lacking. Here, we uncover an auxin-induced protein complex at the PM, containing auxin co-receptors transmembrane kinases (TMKs) and PIN1 auxin exporter, as the core machinery that underlies this feedback regulation. Auxin promotes PIN1 phosphorylation by TMKs, modulating PIN1 polarization and transport activity. We also provide evidence that PIN1-exported extracellular auxin is crucial for TMK activation and cell elongation, thus forming the simplest two-element self-regulatory feedback circuit. Thus, these findings offer direct mechanistic insights into a potential self-organizing circuit for auxin signaling and transport to ensure proper plant development in Arabidopsis.

Embryogenesis in the model plant Arabidopsis thaliana provides a framework for understanding how cell polarity and patterning coordinate with hormonal signalling to establish the plant body plan. Following fertilisation, the zygote divides asymmetrically to generate apical and basal lineages, establishing the apical-basal axis that defines future shoot and root poles. Genetic and molecular analyses of classical mutants including gnom, monopteros (mp), bodenlos (bdl) and topless revealed that localised auxin biosynthesis, directional transport and downstream transcriptional responses are central to apical-basal axis establishment and organ initiation. The main components of this regulation are polarly localised PIN auxin transporters and downstream modules involving MONOPTEROS and WUSCHEL-RELATED HOMEOBOX transcription factors. Advances in microscopy have transformed the study of Arabidopsis embryogenesis: fluorescence-compatible clearing reagents and three-dimensional reconstructions now permit quantitative analyses of cell geometry, division orientation, and cytoskeletal dynamics. Live ovule imaging setups with confocal laser scanning and multiphoton microscopes enable real-time observation of embryo development, while laser-assisted cell ablation can be used to probe cell-to-cell communication and fate plasticity. Together, these methodological breakthroughs position Arabidopsis embryos as a prime model for dissecting the chemical and biophysical cues that shape plant development.

Ginseng (Panax ginseng) derives its renowned therapeutic properties from ginsenoside metabolites. However, the long cultivation cycle and susceptibility to diseases hinder the advancement of the ginseng industry. Here, we demonstrate that the embryonic protoderm of ginseng can efficiently produce ginsenosides. Single-cell transcriptome and mass spectrometry imaging analyses reveal that ginsenosides accumulate in the protoderm of ginseng embryonic callus (EC) at levels comparable to those in forest ginseng. Epigenetic analyses indicate that elevated histone acetylation and enhanced chromatin accessibility at regeneration- and ginsenoside metabolism-related gene loci are associated with the ginsenoside-producing capacity of EC. Increasing histone acetylation levels or overexpressing the regeneration-related WUSCHEL-RELATED HOMEOBOX11 (WOX11) gene further enhances ginsenoside production in EC. Our findings suggest that the protoderm of EC could serve as an in situ biological compartment for high-efficiency ginsenoside producion, offering a complementary approach to traditional ginseng cultivation.

Gravitropism is a fundamental adaptive response in plants that enables directional growth to optimize resource acquisition. In this study, we employed forward genetic screening to identify Arabidopsis mutants with defective hypocotyl gravitropism and isolated the short and agravitropic hypocotyl in dark1 (sad1) mutant, which carries a point mutation (G110E) in the SAC1 gene encoding a phosphoinositide phosphatase. Deficiency of SAC1 disrupted gravity-induced polar localization of PIN3 in endodermal cells, impairing auxin redistribution and leading to hypocotyl gravitropism defects. Subcellular localization analysis revealed that SAC1 is partially localized to the PVC/tonoplast and participates in late endosomal trafficking. The sac1 mutation leads to abnormal vacuolar morphology, which is associated with defects in amyloplast sedimentation during the gravitropic response in Arabidopsis shoots. We further revealed that SAC1 interacts with GRV2, a key regulator of the late endocytic pathway, and that both proteins cooperatively regulate shoot gravitropism. In summary, this study identified SAC1 as a regulator of shoot gravitropism, revealing its important role in modulating vacuolar homeostasis, amyloplast sedimentation, PIN3 trafficking, and auxin distribution. These findings provide insights into the molecular mechanisms linking membrane transport to environmental adaptation in plants.

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 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. TOW promotes PIN interaction with these kinases and stabilizes PINs at the cell surface. 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.

Cyclic adenosine monophosphate (cAMP) is a fundamental second messenger involved in diverse signaling pathways across both animals and plants. While the role of 3 ',5 '-cAMP has been extensively characterized, the biological significance of its structural isomer, 2 ',3 '-cAMP, remains largely unexplored, particularly in plants. Here, we show that 2 ',3 '-cAMP and 3 ',5 '-cAMP represent parallel signaling systems in Arabidopsis thaliana, with different enzymatic origins and largely distinct downstream effects. In vitro enzymatic assays show that plant adenylate cyclases (ACs), including AFB5 and HpAC1, produce specifically 3 ',5 '-cAMP from ATP, whereas the TIR domain of protein L7 also catalyzes the formation of 2 ',3 '-cAMP from RNA. Comprehensive multiomics analyses reveal that two isomers elicit distinct yet partially overlapping metabolic, proteomic, and transcriptional response: 2 ',3 '-cAMP activates broad, stress-adaptive gene expression reprogramming, while 3 ',5 '-cAMP fine-tunes responses related to nutrient status and cellular homeostasis. Our findings establish the existence of dual cAMP signaling systems in plants, each with specialized functions and provide insights into the complex regulatory networks governing plant physiology.

Within the plant endomembrane system, the vesicle coat protein clathrin localizes to the plasma membrane (PM) and the trans-Golgi Network/early endosome (TGN/EE). While the role of clathrin in endocytosis at the PM is well established, its function at TGN/EE, presumably in late secretion (trafficking from the TGN/EE to the cell surface) or en route to the vacuole, is debated. Similarly debated are potential homeostatic mechanisms balancing the trafficking routes, especially endocytosis and late secretion. We address these questions in Arabidopsis thaliana using conditional silencing of CLATHRIN HEAVY CHAIN (CHC), conditional overexpression of the clathrin uncoating factor AUXILIN-LIKE1, and secretory mutants. CHC silencing interferes with trafficking of cargoes destined for the apoplast and the PM, supporting a function of clathrin in late secretion. The secretory cargoes become abnormally rerouted from the TGN/EE to the vacuole. Unlike CHC silencing, overexpression of AUXILIN-LIKE1 selectively inhibits clathrin-mediated endocytosis while secretion continues normally at early points of induction. Conversely, secretory mutants exhibit a reduced PM recruitment of clathrin, and variably, of the TPLATE endocytic component. Together, our data show a role of clathrin in secretion and suggest secretion as a fundamental trafficking process to which endocytosis is adjusted by a weak homeostatic mechanism.

Auxin and calcium (Ca2+) are central plant signals that coordinate growth, development, and responses to environmental cues. Typically studied as largely parallel pathways, they are now increasingly recognized as interconnected networks whose activities converge at multiple regulatory levels. Recent advances in live imaging, biosensors, optogenetics, and structural approaches have revealed that auxin can rapidly trigger Ca2+ influx from the apoplast via TRANSPORT INHIBITOR RESPONSE1 (TIR1)/AUXIN-SIGNALING F-BOX (AFB)-dependent signaling mechanisms and Ca2+ channels, presumably involving cyclic nucleotides produced by TIR1/AFB auxin receptors. Conversely, Ca2+ signals arising from mechanosensing, wounding, and environmental stress can modulate auxin transport, signaling, and transcriptional outputs. Together, these discoveries highlight a dynamic, reciprocal signaling axis where Ca2+ acts both downstream and upstream of auxin to shape growth, development, tropisms, and regeneration.

Abstract The triterpenoid-rich Panax spp. possess notable allelopathic capabilities, yet current knowledge lacks comprehensive understanding of the biological mechanisms underlying these inhibitory effects. Here, we show that Zingibroside R1 (ZR1), a newly identified allelochemical from Panax spp., inhibits the adventitious root growth in ginseng and primary root elongation in Arabidopsis. Mechanistic analyses revealed that ZR1 disrupts auxin transport, as evidenced by monitoring DR5::GFP and PINs markers. The effect of ZR1 on auxin transport was further elucidated through synergistic effects observed with the auxin transport inhibitor NPA and in the auxin transport mutants aux1 and pin2. Additionally, ZR1 downregulates key root development regulators, including stem cell maintenance regulators PLT1/2, WOX5, SHR and SCR. Transcriptomic and biochemical data indicate that ZR1 triggers a ROS response in plants, which disrupts ROS scavenging—via altered peroxidase activities and GSH/GSSG balance—thereby impairing detoxification function and resulting in root shortening by regulation of auxin distribution and stem cell gene expression. Our results establish a mechanistic framework for understanding how ginsenosides mediate allelopathic effects on root development.

Plant tropisms enable roots to navigate complex soils by responding to directional environmental cues. Biological decay, although central to nutrient cycling, also creates microbially active and potentially hostile niches. In this work, we identified "saprotropism," a previously unrecognized growth response that enables roots to actively bend away from decaying plant-derived matter. Fungal-driven microbial decomposition released organic acids and formed stable pH gradients in surrounding soil, allowing roots to pinpoint decay without direct contact. Root epidermal cells sensed this acidic gradient through the root meristem growth factor peptide-receptor module, converting external pH asymmetry into asymmetric abscisic acid (ABA) distribution. ABA asymmetry drove microtubule reorganization, which was decoded into decay-avoidant root bending. Together, these findings establish microbial decay-derived chemical gradients as an instructive signal for root navigation and expand the framework of microbe-soil-plant communication.

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 auxin sensitivity in real time remain unknown. Here, we used the light-gated, Ca 2+ -permeable ChannelRhodopsin 2 variant XXM2.0 to optogenetically impose defined Ca 2+ signatures on Arabidopsis root cells. Repetitive light activation triggered cytosolic Ca 2+ signals that in turn suppressed auxin-induced membrane depolarization and Ca 2+ transients. Moreover, persistent optogenetic Ca 2+ stimulation affects auxin-responsive transcriptional reprogramming. As phenotypic output, reversible inhibition of cell division and elongation leading to root inhibition was observed. Our study thus introduces a new tool to decompose calcium–auxin crosstalk in plant cells, and demonstrates that optogenetically imposed cytosolic Ca 2+ signals act as dynamic regulators of auxin susceptibility in roots.

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. ### Competing Interest Statement The authors have declared no competing interest.

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. ### Competing Interest Statement The authors have declared no competing interest. FWF Austrian Science FundFWF Austrian Science Fund, https://ror.org/013tf3c58, 6123-B European Research CouncilEuropean Research Council, , 101142681 National Science CenterNational Science Center, , 2021/43/I/NZ1/01835 ERDF Programme Johannes Amos ComeniusERDF Programme Johannes Amos Comenius, , CZ.02.01.01/00/22_008/0004581

The plant hormone auxin regulates growth and development through at least two distinct signaling pathways. The nuclear pathway, involving TIR1/AFB receptors, mediates transcription; whereas the cell surface ABP1-TMK1 auxin perception triggers global ultrafast phosphorylation response. Here, we revisit the rich history of the disputed ABP1 auxin receptor, highlighting recent findings of the involvement of TMKs and other molecular components and focusing on their role in auxin canalization-mediated development.

This study demonstrates that Marchantia non‐canonical PINs are predominantly localized to the plasma membrane, with MpPINX and MpPINW exhibiting asymmetric distribution. A newly identified miniW domain within the MpPINW hydrophilic loop governs subcellular trafficking and asymmetric PM localization of non‐canonical PINs in Marchantia .

Very long-chain fatty acids (VLCFAs), being constituents of different types of lipids, are critical factors in plant development, presumably due to their impact on the endomembrane system. The VLCFAs are synthesized in the endoplasmic reticulum by a heterotetrameric enzymatic complex including β-ketoacyl CoA reductase 1 (KCR1), whose mutant is lethal. Here, we describe the ectopic shoot meristems (esm) mutant, a viable kcr1 allele presumably affecting surface properties of the KCR1 protein. This kcr1-2 mutant shows reduced fatty acyl elongation that impacts VLCFAs. The kcr1-2 plants show severe defects during different stages of development, which all correlate with defects in polar localization and subcellular trafficking of PIN auxin transporters and resulting asymmetric auxin distribution. Detailed analysis of KCR1 expression and patterning defects in kcr1-2 suggests that KCR1 plays a role in delineating boundaries around meristematic and specialized differentiating tissues, including root and shoot meristems, initiating lateral roots, lateral root primordia, and trichomes. In these contexts, KCR1-produced VLCFAs may act in a non-cell-autonomous manner. Viable kcr1-2 represents a useful tool to study VLCFA roles in plant development and highlights VLCFAs as critical developmental factors at the interface of cell polarity and tissue development.

The phytohormone auxin (Aux) is a principal endogenous developmental signal in plants. It mediates transcriptional reprogramming by a well-established canonical signalling mechanism. TIR1/AFB auxin receptors are F-box subunits of an ubiquitin ligase complex; after auxin perception, they associate with Aux/IAA transcriptional repressors and ubiquitinate them for degradation, thus enabling the activation of auxin response factor (ARF) transcription factors1-3. Here we revise this paradigm by showing that without TIR1 adenylate cyclase (AC) activity4, auxin-induced degradation of Aux/IAAs is not sufficient to mediate the transcriptional auxin response. Abolishing the TIR1 AC activity does not affect auxin-induced degradation of Aux/IAAs but renders TIR1 non-functional in mediating transcriptional reprogramming and auxin-regulated development, including shoot, root, root hair growth and lateral root formation. Transgenic plants show that local cAMP production in the vicinity of the Aux/IAA-ARF complex by unrelated AC enzymes bypasses the need for auxin perception and is sufficient to induce ARF-mediated transcription. These discoveries revise the canonical model of auxin signalling and establish TIR1/AFB-produced cAMP as a second messenger essential for transcriptional reprograming.