Biology subjects

Novak, O.

Publications and source records attributed to Novak, O..

At least 19 recordsLinked to original sources

bioRxiv2026

Longitudinal Imaging in a Mouse Model of Focal Cortical Dysplasia Reveals Lesion Expansion and Loss of Pre-Discharge Neuronal Recruitment

ABSTRACT Objective: Focal cortical dysplasia type II (FCD II) is a leading cause of drug-resistant focal epilepsy, yet how its lesional circuits behave and change over time in vivo has not been directly observed. Methods: We used a mouse model of FCD II carrying the patient-derived gain-of-function mTORp.L2427P variant introduced by in utero electroporation. Through a chronic cranial window over the lesion, we performed repeated in vivo two-photon calcium imaging of identified dysmorphic neurons, pyramidal cells, and VIP interneurons at defined time points, combined with video-EEG monitoring, to determine seizure status and relate cellular activity to interictal discharges. Results: The lesion was not static in adulthood. Dysplastic cortex expanded progressively. This expansion occurred without the tenascin R/C accumulation reported in human FCD II, and was partly explained by continued somatic growth of dysmorphic neurons. Dysmorphic neurons were generally hypoactive; in seizing mice, both dysmorphic and pyramidal cell activity progressively declined, whereas VIP interneuron activity increased slightly in non-seizing animals. In non-seizing lesional cortex, neurons were reliably recruited immediately before interictal discharges. By late disease stages, pre-discharge recruitment had weakened to the point that lesional layer 2/3 neurons were no more active before discharges than expected by chance. Significance: These findings recast FCD II as a lesion that continues to remodel structurally and functionally throughout adulthood, and does so most markedly in animals with seizures. Growth, a cell-type-specific shift in spontaneous activity, and a progressive loss of the lesion's temporal lead over interictal discharges together suggest that FCD II is a dynamically evolving lesion whose relationship to epileptiform activity changes over time. If the lesion progressively loses its generative role, the window in which removing it can arrest the disease is finite, consistent with clinical evidence favoring earlier surgery.

neuroscience↗

bioRxiv2026

Cytokinin regulates the gravitropic setpoint angle of seminal roots in barley and wheat

Plant root growth angle determines the spatial distribution of roots in soil and thereby influences access to water and nutrients when these resources are heterogeneously distributed. In barley, multiple seminal roots emerge at distinct gravitropic setpoint angles (GSAs) and they reliably re-establish their characteristic growth angles following reorientation, indicating robust intrinsic control. However, the molecular basis underlying GSA variation among roots within a single root system remains unknown. Here, we combined individual root type-specific transcriptomics with laser-capture microdissection to profile gravity-responsive zones (root cap, meristem, and elongation zone). We identified two CYTOKININ OXIDASE/DEHYDROGENASE (CKX) genes whose expression in the root cap was associated with root-specific GSA. Functional analysis showed that ckx mutants, which show higher cytokinin levels, exhibit significantly steeper seminal root GSAs, implicating cytokinin catabolism in root angle regulation. Consistent with this, exogenous cytokinin treatment induced a dose-dependent steepening in root angle in barley. Analyses of gravitropic curvature after gravistimulation further showed that ckx mutants exhibit altered gravitropic responses, indicating that CKX-mediated regulation of the root angle depends on gravity-responsive pathways. Notably, GSA-associated CKX expression patterns and cytokinin-dependent root angle responses were also observed in wheat, suggesting a conservation of this mechanism across cereals. Together, these findings identify cytokinin as a key regulator of root specific setpoint angles within a root system architecture, with potential implications for optimizing crop resource acquisition and yield.

plant biology↗

bioRxiv2026

BpLAZY1A Mediates Transcriptional Polarity and Drives Adaxial Tension Wood Like Tissue Formation in Silver Birch (Betula pendula) Branches

A defining feature of most trees is the architectural distinction between a vertically growing main stem and laterally growing branches. This growth habit increases fitness by increasing photosynthetically active surface area and shading competitors. Here, we studied the weeping birch cultivar Betula pendula Youngii to uncover the mechanisms that maintain lateral branch growth. We identified a loss-of-function mutation in BpLAZY1A, a core component of the gravitropic signalling pathway, as the cause of the weeping phenotype. Forward genetic analysis demonstrated that the weeping phenotype is recessive in silver birch. Transgenic BpLAZY1A RNAi lines phenocopied Youngii, confirming the functional role of BpLAZY1A. Reporter analysis revealed that BpLAZY1A is expressed predominantly in gravity-sensing starch sheath cells, with occasional expression in the main stem phloem. Time lapse imaging revealed two distinct gravitropic responses during branch development - an early response associated with establishment of the branch apex gravitropic set-point angle, and a later response associated with polar reinforcement at the branch base. The main stem retained normal gravitropic responses in BpLAZY1A RNAi lines, indicating a branch-specific role for BpLAZY1A. Integrative analyses combining transcriptomics, chemical profiling, and histochemistry indicated that BpLAZY1A establishes adaxial-abaxial polarity during early branch development. In wild-type branches, pectin-rich tension wood-like tissue formed preferentially in the adaxial xylem, accompanied by adaxial expression of pectin- and tension wood-associated genes, including BpRRT1 and BpCOBRA-LIKE4. Auxin-related genes, including BpIAA29 and BpSAURs, were preferentially upregulated in the abaxial side. This transcriptional asymmetry was reduced in BpLAZY1A RNAi line 1, where tension wood-like tissue formed in both adaxial and abaxial sides of the xylem, indicating a loss of polarity. Together, these findings demonstrate that BpLAZY1A functions specifically in branch gravitropism and links spatially asymmetric gene expression with branch growth orientation and biomechanical reinforcement. Our results identify BpLAZY1A as a key regulator coordinating gravitropic signalling, tissue polarity, and the developmental biomechanics underlying lateral branch growth.

plant biology↗

bioRxiv2026

High-frequency oscillations reveal progressive recruitment of remote cortex into the epileptic network in a mouse model of focal cortical dysplasia type II

Interictal epileptiform discharges (IEDs) and pathological high-frequency oscillations (HFOs) are established biomarkers of epileptogenic tissue, but how their spatiotemporal evolution reflects epileptic network reorganization in neocortical epilepsy remains unclear. We investigated longitudinal IED and HFO dynamics throughout epileptogenesis and chronic epilepsy in a mouse model of focal cortical dysplasia type II (FCD II). Long-term bilateral cortical recordings were obtained before and after spontaneous seizure onset. IEDs and HFOs (gamma, ripples, and fast ripples) were quantified in the dysplastic lesion and contralateral cortex during pre-epileptic, early, and late epileptic stages. The dysplastic lesion remained the principal seizure onset zone throughout disease progression and showed stable HFO activity after epilepsy onset. In contrast, the contralateral cortex exhibited progressive increases in IEDs and HFOs, demonstrating continuous recruitment into the epileptic network. Fast ripples were the earliest marker of this process, emerging within the first week after the first seizure and preceding increases in ripples, gamma, and IEDs. Propagation analysis showed that this contralateral increase was driven by independently generated HFOs rather than propagation from the lesion, indicating emergence of autonomous epileptogenic activity outside the primary focus. Spatiotemporal HFO analysis thus captures dynamic epileptic network remodeling beyond epileptogenic lesion. Fast ripples may serve as an early signature of network expansion and epileptogenicity emerging outside the primary lesion. Widespread structural and connectivity abnormalities extending beyond the lesion, combined with intense recurrent epileptic activity, may underlie the high endogenous epileptogenicity of FCD II, enabling small dysplastic lesions to recruit extensive neuronal networks across both hemispheres.

neuroscience↗

bioRxiv2026

Spatially-restricted metabolic delivery of auxin for selective adventitious root induction

Auxin impacts on nearly every aspect of plant growth and development. Its exogenous application therefore results in pleiotropic growth responses. Exploiting this activity for plant propagation requires avoiding or minimizing such off-target effects and is generally achieved as a trade-off between toxicity and organogenetic efficacity. We recently identified the compound HYSPARIN (HYS) with potent, and uniquely selective adventitious root inductive activity. Unlike other root-inducing compounds, HYS preferentially activates auxin responses in the shoot via an unknown mechanism. Here, we show that HYS acts as a shoot-specific proauxin. Rather than acting through auxin homeostasis, we found that HYS is hydrolysed in planta independently of ILR1/ILL amidohydrolases to release the synthetic auxin MCPA. Structure-activity relationship analysis confirmed a strong dependence on its MCPA moiety for activating auxin responses, and identified its promoiety as a determinant of shoot-specificity and activity. Selective application of MCPA also potently induces AR is consistent with a model in which HYS metabolism produces a spatially restricted, AR inductive auxin signal. The activation mechanism of HYS thus provides a conceptual framework for tissue-specific metabolic delivery of auxin and may enable the programmable delivery of other xenobiotics in plants.

plant biology↗

bioRxiv2026

Clade III HIPP genes encode plasmodesmata-targeted proteins with pleiotropic functions in regulating plant development.

Heavy metal-associated isoprenylated plant proteins (HIPPs) are encoded by large gene families, which have diversified specifically in vascular plants. Their physiological functions and molecular mode of activity are currently largely unknown. In this study, we characterize a group of phylogenetically closely related genes HIPP32, HIPP33, and HIPP34 in Arabidopsis thaliana, revealing their essential roles in controlling diverse developmental pathways. Through comprehensive genetic analyses, we demonstrate that these genes exhibit partially overlapping pleiotropic functions, influencing multiple aspects of plant growth such as embryogenesis, maintenance of apical meristems, root architecture, shoot branching, leaf morphogenesis and floral organ formation. Transcriptomic profiling of hipp mutants identified significant deregulation in several regulatory pathways involved in plant hormone responses, with a specific impact on auxin signaling processes. Interestingly, we show that the analyzed HIPP proteins localize very specifically to plasmodesmata, suggesting their potential function in regulating intercellular communication in shaping plant development.

plant biology↗

bioRxiv2026

A tonoplast cytokinin riboside transporter gates intracellular hormone availability at the plant-microbe interface

Cytokinin ribosides are major mobile and precursor forms of cytokinins, plant hormones whose transport and subcellular distribution shape developmental and stress responses. Here, we identify Arabidopsis thaliana EQUILIBRATIVE NUCLEOSIDE TRANSPORTER1 (ENT1) as a tonoplast-localized cytokinin riboside transporter. Tissue-specific subcellular analysis under native regulatory elements localized ENT1 predominantly to the tonoplast of root epidermal and lateral root cap cells, where it gates intracellular cytokinin riboside availability. Accordingly, ENT1 overexpression enhanced cytokinin riboside sensitivity and signalling, whereas loss of ENT1 altered adenosine metabolism and disrupted cytokinin homeostasis, leading to the accumulation of multiple zeatin-type cytokinins. ENT1-dependent cytokinin riboside compartmentalization was required for beneficial microbe-induced protection, as ent1 mutants failed to acquire protection against the fungal pathogen Botrytis cinerea and the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. These findings reveal a vacuolar gatekeeping mechanism that controls intracellular cytokinin riboside availability and links hormone compartmentalization to beneficial microbe-dependent plant defence.

plant biology↗

bioRxiv2026

Dasatinib-Quercetin May Reduce Senescence Markers, Without Senolysis or Seizure Modification, in a Mouse Model of Focal Cortical Dysplasia

Mounting evidence from surgical type II focal cortical dysplasia (FCD) tissues and mouse models have recently shown that dysmorphic neurons carrying MTOR mutations (DNs) in FCD exhibit hallmarks of cellular senescence. Building on pioneering work from the Baulac group identifying cellular senescence as a feature of mTOR-pathway FCD, a recent study by Ribierre et al. (2024) [1] proposed oral dasatinib and quercetin (DQ) as a therapy that partially decreases the load of mutant, senescent neurons and thus reduces seizure occurrence in FCD mice. Using a different mouse strain and a different gain-of-function mutation in MTOR, our data confirm the presence of senescence hallmarks in FCD mice, but do not support one of the conclusions of Ribierre et al.--that DQ acts as a senolytic in an FCD mouse model--and we propose an alternative interpretation. We longitudinally tracked individual cell fate using two-photon microscopy and complemented these data with EEG monitoring and immunohistochemistry. Immunohistochemical analyses were performed within the same sections using multiple markers, allowing direct identification of mutant neurons and assessment of senescence-associated labeling. While we observed a detectable reduction in a senescence-associated marker, consistent with a senomorphic effect, it did not translate into a change in seizure phenotype, despite treatment timing and dosing matching those in the original study. For detailed materials and methods, see Extended Methods.

neuroscience↗

bioRxiv2026

Cytokinin N-conjugate Form Activity, Metabolism, and Signaling During Leaf Senescence

Cytokinin (CK) N-glucosides are the most abundant CK metabolites in Arabidopsis and most angiosperms, yet their role in cytokinin activity and response is unclear. Here, we examined metabolomic, transcriptomic, and proteomic profiles of seven CK N-glucoside conjugates in detached Arabidopsis leaves across a 144-hour dark-induced senescence (DIS) timecourse. All tested N-glucosides were found to undergo a slow conversion to their corresponding base forms at position-dependent rates, with N9-glucosides releasing base faster than their corresponding N7-glucosides. Conversion during DIS was strictly isoform-specific and not accompanied by coordinated induction of CK biosynthesis genes, arguing against de novo synthesis as the source of accumulated base. Despite progressive base accumulation, N-glucoside-treated leaves produced substantially fewer Differentially Expressed Genes than direct base application at comparable base concentrations, revealing a disconnect between hormone presence and transcriptional output. Unbiased model comparison identified the base:glucoside ratio as a stronger predictor of CK-Two Component Signaling (TCS) gene expression than absolute base concentration, though modulated by base-type-specific receptor affinities. Early proteomic profiling further revealed a coordinated response shared across N-glucosides but largely absent from base treatments. Together, these findings support that CK N-glucosides as kinetically slow, position-dependent reservoirs whose presence in abundance modulate activation of CK-TCS elicited by bioactive forms. HighlightsPhysiology, metabolomic, transcriptomic, and proteomic findings here support CK N-glucosides as kinetically slow, position-dependent reservoirs whose presence in abundance modulate activation of CK-TCS elicited by bioactive forms.

plant biology↗

bioRxiv2026

miR160 controls class III peroxidase via StARF10/17 and couples salicylic acid and ROS signaling networks in potato hypersensitive response to potato virus Y

O_LIHypersensitive response (HR) is an effector-triggered immune response leading to pathogen restriction and local cell death. Small RNAs (sRNAs), mediating RNA silencing, have a well-established role in antiviral immunity, however, their role in HR has not been addressed to date. C_LIO_LIWe applied a spatially resolved sRNAomics approach to uncover changes at the sRNA level and the dynamics of sRNA-gene regulatory networks in potato HR response to potato virus Y (PVY). C_LIO_LIWe show that miR160 is repressed in cells adjacent to HR lesions in the resistant cultivar, but not in PVY-sensitive, salicylic acid (SA)-depleted plants. Beyond its canonical regulation of StARF10 and StARF17, miR160 controls a broader regulatory network spanning auxin signaling, cell-wall remodeling, redox homeostasis, and tuberization. Elevated miR160 levels promote susceptibility-associated traits, reduce thermotolerance, and suppress genes strongly activated in HR. C_LIO_LIMechanistically, we identify a novel miR160-StARF10/StARF17-StPRX28 module that links miR160 repression to the induction of a class III apoplastic peroxidase during HR, with SA acting upstream. Together, these findings establish miR160 as a crucial integrator of SA-auxin-ROS signaling pathways that tunes development, defense, and heat resilience, and highlight miR160 as a promising target for fine-tuning tolerance to multiple stresses with minimal yield loss. C_LI

molecular biology↗

bioRxiv2026

Rewiring vascular patterning through translational control in Arabidopsis

Plant vascular systems exhibit a wide developmental spectrum, from rigid woody tissues to soft, fleshy storage tissues. We show that increasing polyamine thermospermine transport into wild-type rootstocks, together with cytokinin, reprograms xylem identity from woody to fleshy in Arabidopsis. Our findings establish thermospermine as a mobile developmental signal and suggest a strategy for engineering plant vascular architecture.

plant biology↗

bioRxiv2026

Comprehensive characterisation of IAA inactivation pathways reveals the impact of glycosylation on auxin metabolism and plant development

Together with biosynthesis and transport, inactivation regulates the concentration of indole-3-acetic acid (IAA), a key auxinic compound with a myriad of functions in plant development. Main inactive IAA metabolites are categorised into oxidised forms and ester- or amide-linked conjugates. DIOXYGENASE FOR AUXIN OXIDATION1 (DAO1) and DAO2, 2-oxoglutarate and iron-dependent dioxygenases, contribute to IAA oxidative inactivation in collaboration with group II GRETCHEN HAGEN3 (GH3) IAA-amido synthetases, while a group of UDP-glycosyltransferases (UGTs) conjugate IAA to sugars. To study the IAA inactivation routes, we generated combinatorial mutants between all group II GH3s (gh3oct) and DAO1 or DAO2, as well as between the DAOs and main UGTs. In vivo [13C6]IAA feeding experiments traced the metabolic fate of the exogenously applied IAA, supporting the main IAA inactivation pathway, in which DAO acts downstream of GH3s. They also indicated that UGT-mediated IAA glycosylation is more important than previously assumed for modulating IAA levels and plant development. Our metabolic and transcriptomic data further revealed that gh3oct may still produce some GH3 activity, explaining previous reported phenotypic inconsistencies. Our data additionally suggest that other not yet identified metabolic activities might play a role in IAA overproducing plants, and that the premature downregulation of flowering time integrators like FLOWERING LOCUS C (FLC) likely underlies the early flowering of gh3oct and gh3oct dao1 plants.

plant biology↗

bioRxiv2026

Cytokinin Senescence Delay Is Shaped by Receptor Specificity and Metabolic Stability

Each of the four different cytokinin (CK) base forms, trans Zeatin (tZ), isopentenyladenine (iP), dihydrozeatin (DHZ), and cis Zeatin (cZ) have distinct chemical metabolism and affinity to the CK Histidine Kinase (CHK) receptors. However, it remains unclear how specific biochemical features of each form such receptor specificity or metabolic differences drives distinct tissue-specific physiological hormone output in response to application of these CK bases. Here, we show that CK receptor preference and metabolic persistence together shape isoform-specific CK signaling strength, including tissue-dependent hormone responses in Arabidopsis leaf versus root assays. Physiological, genetic, and multi-omics integration was used to show that tZ and iP anti senescence activity is matched by DHZ through a distinct receptor metabolic mechanism. DHZ requires Arabidopsis Histidine Kinase 3 (AHK3) signaling to be fully effective in a leaf Dark Induced Senescence (DIS) assay and where it overcomes its lower receptor affinity through higher metabolic persistence, accumulating at levels [~]2.5-fold above tZ and iP early in a senescence time course. Together, these findings provide a framework for integration of receptor preference and metabolic stability to determine CK isoform activity.

plant biology↗

bioRxiv2025

The plant longevity gene AHL15 delays leaf senescence by repressing ORESARA1 and cytokinin degradation

The Arabidopsis thaliana (Arabidopsis) AT-HOOK MOTIF NUCLEAR LOCALIZED 15 (AHL15) gene is associated with various longevity phenotypes and extends the life span of plants when overexpressed. In this study, we show that, in addition to previously described longevity phenotypes, constitutive overexpression of AHL15 in Arabidopsis delays leaf senescence, whereas ahl15 loss-of-function accelerates this process. Dexamethasone-induced nuclear localization of AHL15-GR during dark-triggered senescence results in a stay-green phenotype and represses the expression of several early senescence-associated genes. Among these, the ORESARA1 (ORE1) locus is directly bound by AHL15, suggesting a direct repressive effect of AHL15 on senescence. Furthermore, we demonstrate that AHL15 acts by directly repressing the expression of several CYTOKININ OXIDASE (CKX) genes involved in cytokinin inactivation, resulting in a delayed degradation of cytokinins during dark-induced senescence. Cytokinins are known to delay senescence, and together with the downregulation of ORE1 expression, this explains the repressive effect of AHL15 on senescence. SignificanceHere, we show that in addition to its previously reported effects on aging processes, the AT-HOOK MOTIF NUCLEAR LOCALIZED-family protein AHL15 also represses leaf senescence. Our results demonstrate that AHL15 delays the senescence program in two ways: by directly repressing the senescence master regulator ORESARA1, and by transcriptional repression of CYTOKININ OXIDASE genes, resulting in a delayed breakdown of the senescence-inhibiting hormone cytokinin, which together explain the strong stay-green phenotype of AHL15-overexpressing plants.

plant biology↗

bioRxiv2025

SEIZURE OCCURRENCE IN FCD TYPE II IS PREDICTED BY LESION POSITION AND LINKED TO CYTOARCHITECTURAL ALTERATIONS

Focal cortical dysplasia (FCD) is a common malformation of cortical development and a major cause of early-onset, drug-resistant epilepsy. FCD type II is defined by abnormal lamination, altered cellular composition, and pathological cells, notably dysmorphic neurons (DNs) and balloon cells. DNs are thought to drive epileptogenicity through both cell-autonomous and non-cell-autonomous mechanisms, the latter including not only aberrant connectivity but also indirect modulation of excitability in local cell populations. We performed a multiscale structural and morphological analysis to elucidate the basis of FCD epileptogenicity and the impact of somatic mTOR mutations during brain development. Using a mouse model of FCD type II, we show that lesions in frontal and motor cortical regions are the strongest predictors of spontaneous seizure occurrence. This localization-dependent epileptogenicity offers an experimental explanation for the higher clinical epileptogenicity of frontal FCDs and suggests that posterior lesions may remain silent--an open question in human pathology. In our model, FCD tissue displayed considerable expansion, with cortical thickness up to [~]20% in seizure-bearing animals. This expansion coincided with an overall [~]40% reduction in neuronal density, consistent with tissue hypertrophy. DN density did not differ between seizure and non-seizure animals, challenging the notion that higher DN load directly predicts epileptogenesis. At the microscopic level, we describe DN axonal pathologies, including giant varicosities. In the cortex, these appeared as vesicle-filled boutons, whereas along callosal axons they were frequent but largely empty. Bouton density was markedly reduced in FCD cortex. Together, these findings leave the net synaptic effect of dysmorphic neurons unresolved, challenging the assumption that axonal hypertrophy translates into increased excitatory drive. While morphological abnormalities in FCD type II are well documented, their functional consequences remain incompletely understood. Here, we used macro- and microscopic structural features of FCDII to assess seizure susceptibility, providing new insights into epileptogenesis.

neuroscience↗

bioRxiv2025

Tomato leaf transcriptomic changes promoted by long-term water scarcity stress can be largely prevented by a fungal-based biostimulant

Water availability is by far the leading environmental factor limiting crop productivity worldwide. The use of cell-free microbial culture filtrates (CF) as biostimulant is gaining ground as a safe and ecologically sound approach to improving crop yields while reducing anthropogenic pressure. However, their action mechanisms remain unknown. Here we found that foliar application of Trichoderma harzianum CF enhanced fruit yield, root growth and photosynthesis in plants of a commercial tomato cultivar grown under long-term water deficit in Mediterranean greenhouse conditions. To investigate the biochemical and molecular mechanisms underlying this phenomenon, we adopted an integrative and systems biology approach to characterize plants grown under optimal and suboptimal irrigation conditions (OIC and SOIC, respectively) with or without the fungal CF treatment. Water shortage promoted changes in the levels of drought stress-related signalling molecules, and in the transcriptome of leaves that potentially accounted for the physiochemical differences recorded between OIC- and SOIC-grown plants. Notably, many of these changes were largely prevented by the foliar application of fungal CF to SOIC-grown plants, including the expression of ca. 50% of the expression of water scarcity-responsive genes. These genes did not respond to CF in OIC-grown plants, indicating that the transcriptomic response to CF is strongly determined by the water status of the plant. Taken together, the data provided evidence that foliar application of fungal CF improves yield and long-term water scarcity tolerance by preventing a large portion of the stress-induced transcriptional response, rendering plants "blind", or less sensitive to water stress.

plant biology↗

bioRxiv2025

The CYP71A, NIT, AMI, and IAMH gene families are dispensable for indole-3-acetaldoxime-mediated auxin biosynthesis in Arabidopsis

Indole-3-acetic acid (IAA) is a crucial auxin governing plant development and environmental responses. While the indole-3-pyruvic acid (IPyA) pathway is the predominant IAA biosynthesis route, other pathways, like the indole-3-acetaldoxime (IAOx) pathway, have been proposed. The IAOx pathway has garnered attention due to its supposed activation in auxin-overproducing mutants (e.g., sur1, sur2, ugt74b1) and the auxin-like responses triggered by exogenous application of its proposed intermediates: IAOx, indole-3-acetonitrile (IAN), and indole-3-acetamide (IAM). However, despite supporting evidence for individual steps, conclusive physiological relevance of the IAOx pathway remains unproven. Using a comprehensive genetic approach combined with metabolic and phenotypic profiling, we demonstrate that mutating gene families proposed to function in the IAOx pathway does not result in prominent auxin-deficient phenotypes, nor are these genes required for high-auxin production in the sur2 mutant. Our findings also challenge the previously postulated linear IAOx pathway. While exogenously provided IAOx, IAN, and IAM can be converted to IAA in vivo, they do not act as precursors for each other. Finally, our findings question the physiological relevance of IAM and IAN as IAA precursors in plants and suggest the existence of a yet uncharacterized auxin biosynthetic route, likely involving IAOx as an intermediate, for the production of IAA in the sur2 mutant. Future identification of the metabolic steps and the corresponding genes in this new pathway may uncover the previously unknown way of synthesizing IAA in plants.

genetics↗

bioRxiv2025

Interictal activity fluctuations follow rather than precede seizures on multiple time scales in a mouse model of focal cortical dysplasia

The unpredictability of seizure occurrence is a major debilitating factor for people with epilepsy. A seizure forecasting system would greatly improve their quality of life. Successful seizure forecasting necessitates a comprehensive understanding of the factors influencing seizure timing at multiple temporal scales. In this study, we investigated multiscale properties of interictal epileptiform discharges (IEDs) and seizure parameters in a highly realistic mouse model of focal cortical dysplasia-related epilepsy. We analyzed the properties evolution at four timescales, ranging from epilepsy progression and seizure clusters to circadian and peri-ictal changes. We discovered that the FCD-related epilepsy syndrome was progressive in terms of interictal activity rate and seizure characteristics. Sixty percent of seizures occurred in clusters. During the clusters, the seizure duration, seizure power, and IED rate were increasing. Circadian rhythm influenced seizure occurrence with the peak seizure probability at 4 p.m. under a standard 12/12 light dark cycle with lights-on at 6 a.m. Peri-ictal analysis revealed no significant change in IED rate preceding individual seizures; however, a consistent two-peak pattern of IED elevation was observed following seizures. Specifically, an initial peak in IED rate emerged 5-10 minutes post-seizure, returning to baseline within two hours, followed by a secondary peak 6-12 hours later, which again subsided to baseline levels in 24-48 hours. This pattern could be fitted with a sum of three exponentials. Using the three-exponential pattern, we simulated IED rate fluctuations in each animal. The smoothed simulated IED rates showed good agreement with the smoothed real recorded IED rates, suggesting that the cumulative effect of post-ictal IED patterns can account for long-term fluctuations in IED rate. Our results indicate that, in our model of FCD-related epilepsy, consistent IED rate fluctuations follow rather than precede individual seizures. Therefore, fluctuations in IED rate can be viewed as a reflection of cyclic seizure occurrence. This implies that either IED rate fluctuations or accurate seizure records may be equally valuable for seizure risk forecasting. HighlightsO_LIFCD-related epilepsy model displays a progressive nature and fluctuations between high and low seizure risk. C_LIO_LISeizures occur with higher probability in the day time which corresponds to sleep-related seizures commonly occurring in human patients with FCD. C_LIO_LIIED rate increases significantly after seizures, indicating a postictal effect rather than a preictal one, with the post-ictal phase displaying a two-peak pattern of fast and slow IED rate increase. C_LIO_LILong-term changes in the IED rate could be attributed to the time-dependent cumulative effect of the two-peak seizure-related increase in IED rate. C_LI

neuroscience↗