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bioRxiv2019

Biochemical properties and in vitro biological activities of extracts from seven folk medicinal plants growing wild in southern Tunisia

Recently, much attention has been paid to the extracts obtained from plant species in order to analyse their biological activities. Due to the climate diversity in Tunisia, the traditional pharmacopoeia consists of a wide arsenal of medicinal plant species since long used in folk medicine, in foods as spices, and in aromatherapy. Although many of these species are nearly facing extinction, only a small proportion of them have been scientifically studied. Therefore, this study explores the biochemical properties of seven spontaneous plants, which were harvested in the arid Tunisian desert: Marrubium vulgare L., Rhus tripartita (Ucria) D.C., Thymelaea hirsute (L.) Endl., Plantago ovata Forsk., Herniaria fontanesii J. Gay., Ziziphus lotus and Hyoscyamus albus. Extracts from these plants were found to contain different types of secondary metabolites (polyphenols, flavonoids, condensed tannins, crude saponins, carotenoids and alkaloids) that are involved in important biological activities. The biological activity of the extracts obtained from each Tunisian plant was assessed: first of all, leukaemia and colon cancer cell lines (K-562 and CaCo-2 respectively) were treated with different concentrations of extracts, and then the anti-proliferative activity was observed. The results showed, in particular, how the plant extract from Rhus tripartita significantly inhibits cell proliferation, especially on the K-562 tumour cell line. Subsequently, the anti-inflammatory activity was also assessed, and the results showed that Herniaria fontanesii and Marrubium vulgare possess the highest activity in the group of analysed plants. Finally, the greatest acetylcholinesterase inhibitory effect was exhibited by the extract obtained from Rhus tripartita. In conclusion, all the Tunisian plants we analysed were shown to contain a remarkable amount of different bio-active compounds, thus confirming their involvement in several biological activities. Rhus tripartita and Ziziphus lotus were shown to be particularly effective in anti-proliferative activity, while Herniaria fontanesii were shown to have the best anti-inflammatory activity.

plant biology↗

bioRxiv2021

Calpain DEK1 acts as a developmental switch gatekeeping cell fate transitions

Calpains are cysteine proteases that control cell fate transitions. Although calpains are viewed as modulatory proteases displaying severe, pleiotropic phenotypes in eukaryotes, human calpain targets are also directed to the N-end rule degradatory pathway. Several of these destabilized targets are transcription factors, hinting at a gene regulatory role. Here, we analyze the gene regulatory networks of Physcomitrium patens and characterize the regulons that are deregulated in DEK1 calpain mutants. Predicted cleavage patterns of regulatory hierarchies in the five DEK1-controlled subnetworks are consistent with the genes pleiotropy and the regulatory role in cell fate transitions targeting a broad spectrum of functions. Network structure suggests DEK1-gated sequential transition between cell fates in 2D to 3D development. We anticipate that both our method combining phenotyping, transcriptomics and data science to dissect phenotypic traits and our model explaining the calpains role as a switch gatekeeping cell fate transitions will inform biology beyond plant development.

developmental biology↗

bioRxiv2026

PSoup: an R package for simulating biological networks from a qualitative perspective

Mathematical modelling is essential for understanding how complex biological systems respond to genetic, physiological, and environmental changes. Existing approaches, however, often require trade-offs between mechanistic detail, model size, parameter uncertainty, and interpretability. Ordinary differential equation (ODE) models capture biochemical processes with quantitative precision but can demand extensive parameterisation. In contrast, large statistical and machine-learning models rely on substantial datasets and frequently lack mechanistic transparency. Qualitative approaches such as Boolean networks improve scalability but may oversimplify biological behaviour. To address some of these limitations, we present PSoup, an R package that automatically converts knowledge graphs into transparent, parameter-free, qualitative models. PSoup uses algebraic update rules designed around a fixed, biologically interpretable baseline, enabling predictions of relative change across diverse perturbations without requiring kinetic parameters. This design allows PSoup to integrate information across biological scales and from heterogeneous experimental sources. We evaluated PSoup using the well-studied shoot branching network of Bertheloot et al. (2019), which incorporates hormonal (auxin, strigolactone, cytokinin) and metabolic (sucrose) regulation. Across 78 experimental conditions, PSoup correctly predicted 88.5%of perturbation outcomes, including 89.5%accuracy for unique, biologically consistent comparisons. We further demonstrate how PSoup can distinguish among alternative plausible network topologies, revealing how structural differences influence emergent system behaviour. PSoup offers an intuitive, accessible, and mathematically transparent framework for exploring biological networks. Its capacity to integrate diverse knowledge and test alternative hypotheses positions it as a powerful tool for biological discovery and a valuable complement to existing modelling approaches.

plant biology↗

bioRxiv2021

Automated imaging of duckweed growth and development

Duckweeds are some of the smallest angiosperms, possessing a simple body architecture and high rates of biomass accumulation. They can grow near-exponentially via clonal propagation. Understanding their reproductive biology, growth, and development is essential to unlock their potential for phytoremediation, carbon capture, and nutrition. However, there is a lack of non-laborious and convenient methods for spatially and temporally imaging an array of duckweed plants and growth conditions in the same experiment. We developed an automated microscopy approach to record time-lapse images of duckweed plants growing in 12-well cell culture plates. As a proof-of-concept experiment, we grew duckweed on semi-solid media with and without sucrose and monitored its effect on their growth over 3 days. Using the PlantCV toolkit, we quantified the thallus area of individual plantlets over time, and showed that L. minor grown on sucrose had an average growth rate four times higher than without sucrose. This method will serve as a blueprint to perform automated high-throughput growth assays for studying the development patterns of duckweeds from different species, genotypes, and conditions.

plant biology↗

bioRxiv2021

The Arabidopsis thaliana PeptideAtlas; harnessing world-wide proteomics data for a comprehensive community proteomics resource

We developed a new resource, the Arabidopsis PeptideAtlas (www.peptideatlas.org/builds/arabidopsis/), to solve central questions about the Arabidopsis proteome, such as the significance of protein splice forms, post-translational modifications (PTMs), or simply obtain reliable information about specific proteins. PeptideAtlas is based on published mass spectrometry (MS) analyses collected through ProteomeXchange and reanalyzed through a uniform processing and metadata annotation pipeline. All matched MS-derived peptide data are linked to spectral, technical and biological metadata. Nearly 40 million out of [~]143 million MSMS spectra were matched to the reference genome Araport11, identifying [~]0.5 million unique peptides and 17858 uniquely identified proteins (only isoform per gene) at the highest confidence level (FDR 0.0004; 2 non-nested peptides [≥] 9 aa each), assigned canonical proteins, and 3543 lower confidence proteins. Physicochemical protein properties were evaluated for targeted identification of unobserved proteins. Additional proteins and isoforms currently not in Araport11 were identified, generated from pseudogenes, alternative start, stops and/or splice variants and sORFs; these features should be considered for updates to the Arabidopsis genome. Phosphorylation can be inspected through a sophisticated PTM viewer. This new PeptideAtlas is integrated with community resources including TAIR, tracks in JBrowse, PPDB and UniProtKB. Subsequent PeptideAtlas builds will incorporate millions more MS data. One sentence summaryA new web resource providing the global community with mass spectrometry-based Arabidopsis proteome information and its spectral, technical and biological metadata integrated with TAIR and JBrowse

plant biology↗

bioRxiv2025

Chemical modulation of chloroplast de- and re-differentiation reveals a role for the SAL1-PAP retrograde pathway in facilitating plastid transitions

Plastids are dynamic organelles that remodel their composition, ultrastructure, and function according to developmental and environmental demands. The synthetic molecule X57 induces the conversion of leaf chloroplasts into tocopherol-rich plastids lacking thylakoids and containing proliferating plastoglobules. Removal of X57 triggers chloroplast re-differentiation, enabling precise spatial-temporal dissection of these transitions. X57 directly binds and inhibits the phosphatase SAL1, causing accumulation of its substrate 3'-phosphoadenosine 5'-phosphate (PAP), a retrograde signal that modulates nuclear gene expression. SAL1 inhibition and subsequent PAP accumulation activate a cascade that depletes cytokinins and down-regulates GOLDEN2-LIKE1 (GLK1) and other transcription factors involved in chloroplast biogenesis. SAL1-defective mutants fail to undergo this signaling pathway. The SAL1-PAP-mediated weakening of chloroplast identity preconditions plastids for their eventual conversion into storage-type organelles upon X57-promoted accumulation of tocopherols. After X57 withdrawal, photosynthetic gene expression and chloroplast functions are restored. This framework identifies key molecular mechanisms underlying chloroplast plasticity, a central process in biology.

plant biology↗

bioRxiv2025

Single-cell transcriptomic atlas reveals that vascular tissues orchestrate cell fate transitions to initiate adventitious root formation in tomato

O_LIAdventitious root (AR) formation enables vegetative propagation critical for agriculture, conservation, and breeding, yet the cellular basis of AR competence in crop species remains poorly characterized. Most mechanistic studies rely on Arabidopsis, which exhibits constrained AR capacity compared to species dependent on clonal propagation. C_LIO_LIWe constructed a single-cell transcriptomic atlas of tomato (Solanum lycopersicum) AR development comprising 7,920 cells across 12 cell types. Regulatory networks were inferred using hdWGCNA and MINI-EX, validated by RNA in situ hybridization and qPCR. Cross-dataset integration with shoot-borne root initiation data enabled developmental staging, while SATURN-based integration of 118,730 vascular cells from eight species permitted evolutionary comparison. C_LIO_LICytoTRACE analysis revealed that vascular tissues--root stele and phloem--possess the highest developmental potential, supporting a model of latent stem cell-like identity rather than dedifferentiation-based reprogramming. Network analysis identified LEA3 as a central hub gene constitutively expressed throughout AR development, regulated by the DOF transcription factor DOF11. Cross-species integration demonstrated that tomato AR-initiating cells share transcriptomic similarity with woody dicots (Liriodendron, r = 0.46; Eucalyptus, r = 0.42) but not Arabidopsis (r = -0.02), with LEA3-containing macrogene weights highest in basal angiosperms and woody species. C_LIO_LIOur findings establish the DOF11-LEA3 axis as a conserved vascular identity module and suggest that AR competence represents an ancestral program retained in species with active cambial dynamics. These results reframe Arabidopsis as a derived rather than representative model for AR biology and identify molecular targets for enhancing vegetative propagation. C_LI

plant biology↗

bioRxiv2020

Spike development inhibition in the ftin mutant is associated with multiple phenotypes and regulated by multiple biological pathways

Spike development of wheat line 3558M was strongly inhibited by low temperature stress in spring. The fertile tiller inhibition (ftin) gene in the wheat line 3558M is associated with multiple phenotypes, including the production of fewer tillers, delayed floral transition, and death of the shoot apical meristem. We systematically investigated the genes and pathways underlying the differences using ITRAQ proteomics and RNA-sequencing technologies and found multiple biological pathways including to the cold acclimation pathway and multiple defence responses (e.g. reactive oxygen species-mediated hypersensitive response, salicylic acid-mediated systemic acquired resistance) are activated and led to tillers death of the wheat line 3558M under cold stress. Meanwhile, the cold acclimation pathway inhibited the SVP-SCO1-LFY flowering pathway and led to delayed floral transition. Particularly, two TaPIN proteins were significantly downregulated, and multiple auxin signalling genes were also differentially expressed. Knocking down the two TaPIN genes using RNAi technology significantly reduced the tiller number. The cold stress inhibited the auxin transport to reduce the tillers of 3558M. Taken together, the ftin gene might be a cold-sensitive mutation and that is the cause of multiple biological pathways and phenotypic changes.

plant biology↗

bioRxiv2022

The phase separated CO2-fixing pyrenoid proteome determined by TurboID

Phase separation underpins many biologically important processes such as RNA metabolism, signaling and CO2 fixation. However, determining the composition of a phase separated organelle is often challenging due to their sensitivity to environmental conditions which limits the application of traditional proteomics techniques like organellar purification or affinity purification mass spectrometry to understand their composition. In Chlamydomonas reinhardtii, Rubisco is condensed into a crucial phase separated organelle called the pyrenoid that improves photosynthetic performance by supplying Rubisco with elevated concentrations of CO2. Here, we developed a TurboID based proximity labeling technique in Chlamydomonas chloroplasts, where proximal proteins are labeled by biotin radicals generated from the TurboID-tagged protein. Through the expression of two core pyrenoid components fused with the TurboID tag, we have generated a high confidence pyrenoid proxiome that contains the majority of known pyrenoid proteins plus a number of novel pyrenoid candidates. Fluorescence protein tagging of 8 previously uncharacterized TurboID-identified proteins showed 7 were localized to a range of sub-pyrenoid regions. The resulting proxiome also suggests new secondary functions for the pyrenoid in RNA-associated processes and redox sensitive iron-sulfur cluster metabolism. This developed pipeline opens the possibility of investigating a broad range of biological processes in Chlamydomonas especially at a temporally resolved sub-organellar resolution.

plant biology↗

bioRxiv2024

Small heat shock proteins (sHSPs) identified in nodules of tropical woody legumes.

Biological nitrogen fixation (BNF) is a primary input of nitrogen to natural and agricultural systems globally. BNF is a temperature-dependant enzymatic process and can be conducted by microbes (including Rhizobia) hosted symbiotically in root nodules of some plants. Heat shock proteins (Hsps) have been implicated in the process of acquiring thermotolerance or acclimating to elevated temperature, as they play a vital role in maintaining cell integrity and homeostasis during heat stress. Although the BNF response to temperature may crucially impact future ecosystem productivity in the face of global climate change, little is known about Hsp expression in nodules of N-fixing non-agricultural species, such as tropical N-fixing trees in the Fabaceae family. This project aimed to characterize small (15-20 kDa) Hsp (sHsp) expression in nodule tissue to examine the biochemical mechanisms of heat response in these tissues. To first identify Hsps in nodule tissues, Vachellia farnesiana and Acacia confusa nodules were excised, heat shock was induced, and protein content was isolated via chemical treatment before separation of protein species and analysis with SDS-PAGE. Two polyacrylamide gels yielded bands in the 15-20 kDa region that displayed differential Coomassie staining, which were sent for further characterization by HPLC-MS analysis for protein sequencing. Ten rhizobial sHsps were detected in these samples in addition to seven Acacia sHsps when compared independently to reference rhizobial and plant proteome databases. In an attempt quantify relative expression of Hsps in nodule and root tissue, we performed western blot experiments using Anti-Hsp20 antibodies raised against human and mouse Hsp proteins, with anti-beta actin loading control. While nonuniform beta-actin expression across tissue types (A. confusa nodules versus root control) prevented quantitative analysis, the experiments validated that Hsp20s are expressed in Acacia nodules as well as in root tissue. These experiments provide a foundation for future studies to determine variation in responses to key stressors predicted to increase with global climate change and help determine the implications of warming across the tropics and beyond. Proteomics data are available via ProteomeXchange with identifier PXD055599.

plant biology↗

bioRxiv2019

Dynamic measurement of cytosolic pH and uncovers the role of the vacuolar transporter AtCLCa in the control of cytosolic pH.

Ion transporters are key players of cellular processes. The mechanistic properties of ion transporters have been well elucidated by biophysical methods. Meanwhile the understanding of their exact functions in the whole cell homeostasis is limited by the difficulty to monitor their activity in vivo. The development of biosensors to track subtle changes in intracellular parameters provides an invaluable key to tackle this challenging issue. Here, we adapted the use of a dual biosensor using guard cells as experimental model to visualize the impact on the cytosol of anion transport from intracellular compartments. To image the activity of AtCLCa, a vacuolar NO3-/H+ exchanger regulating stomata aperture in Arabidopsis thaliana, we expressed a genetically encoded biosensor, ClopHensor allowing monitoring the dynamics of cytosolic anion concentration and pH. We first show that ClopHensor is not only a Cl- but also a NO3- sensor. We were then able to unravel and quantify the variations of NO3- and pH in the cytosol. Our data show that AtCLCa activity modifies cytosolic pH and NO3-, demonstrating that the transport activity of a vacuolar exchanger has a profound impact on cytosolic homeostasis. We propose that a major function of this endomembrane transporter is to adjust cytosolic conditions to cellular needs. This opens a novel perspective on the function of intracellular transporters of the CLC family in eukaryotes: not only controlling the intra organelle lumen but also actively modifying cytosolic conditions.\n\nSignificanceIntracellular transporters are key actors in cell biological processes. Their disruption causes major physiological defects. The role of intracellular ion transporters is usually seen through an \"intra organelle\" lens, meanwhile their potential action on cytosolic ion homeostasis is still a black box. The case of a plant CLC is used as a model to uncover the missing link between the regulation of conditions inside the vacuole and inside the cytosol. The development of an original live imaging workflow to simultaneously measure pH and anion dynamics in the cytosol reveals the role of an Arabidopsis thaliana CLC, AtCLCa, in the modification of cytosolic pH. Our data highlight an unsuspected function of endomembrane transporters in the regulation of cytosolic pH.

plant biology↗

bioRxiv2023

Grove-level analysis of titer and prevalence of Candidatus Liberibacter asiaticus and Wolbachia in Diaphorina citri, vector of citrus Huanglongbing

Huanglongbing (HLB, or citrus greening disease) affects all citrus varieties world-wide. In the USA, Asia, and South America the causal agent is "Candidatus Liberibacter asiaticus" (CLas), a phloem-limited, uncultured, alphaproteobacterium. The hemipteran insect vector, Diaphorina citri (Asian citrus psyllid) acquires and transmits CLas in a circulative, propagative manner. In addition to CLas, D. citri hosts multiple symbiotic bacterial species including Wolbachia (wDi). In D. citri, wDi has been sequenced and studied but specific roles in D. citri biology are unknown. Using well established quantitative PCR methods we measured CLas titer in D. citri collected from four groves in central Florida with distinct HLB management strategies and tested whether CLas and wDi titer were correlated in a sub-set of these insects. Grove site had the largest effect on CLas titer. Sex had no effect on CLas titer, while higher wDi titer was correlated with non-infected insects. Our results suggest that more directed follow-up research is necessary and important to clarify whether field management tactics influence CLas titer in D. citri and to better understand gene-by-environment interactions among D. citri, wDi and CLas. Now that millions of trees in Florida have been treated with injectable formulations of oxytetracycline, which is likely to decrease bacterial populations in D. citri, this study may represent the last biologically meaningful snapshot of grove-level vector-pathogen ecology in the state during the HLB epidemic.

plant biology↗

bioRxiv2026

Simultaneous triple staining for detecting cell-type specific spatio-temporal distribution of cell wall materials in monocot roots

O_LIAnatomical and histochemical imaging of grass root systems relies on tissue sectioning and cell wall staining dyes because molecular reporter lines are limited for most organisms. C_LIO_LIDistinct staining dyes require variable incubation time and concentration across different tissues and organisms. As a result, staining with multiple dyes becomes time consuming or challenging. Here, we report a rapid method to perform simultaneous triple staining on a glass slide. The entire protocol requires [~]4 hours and a smaller volume of stain than traditional methods. C_LIO_LIWe tested this method using the roots of two economically important crops, Triticum aestivum (wheat) and Zea mays (maize), as proof of concept. We have also demonstrated the presence of exodermis in wheat roots. Additionally, we identified the formation of polar lignin caps in maize exodermis using our simultaneous triple staining method. C_LIO_LIThis method empowers a quantitative approach to cell biology by elucidating cell-type specific spatio-temporal distribution of cell wall materials in monocot root systems. C_LI

plant biology↗

bioRxiv2026

MozClo: An Expanded MoClo Toolset for Large Multigene Assembly and Plant Transformations

The Modular Cloning (MoClo) and PhytoBrick standards have revolutionized plant synthetic biology by establishing a standardized, hierarchical assembly grammar. However, as the engineering of complex metabolic pathways, multi-trait stacks, and synthetic gene circuits expands, existing toolkits hit practical boundaries in assembly capacity and fixed grammars. To overcome these bottlenecks, we present MozClo, an expansion of the MoClo/PhytoBrick architecture. MozClo expands the standard Level 1 assembly framework to 10 positions using new L1 acceptors, end-linkers and dummy parts. We also identify and resolve a critical, sticky-end collision at L1 position 7 that has caused assembly failures during L2 cloning of large plasmids. To address commercial DNA synthesis length constraints and to lower cloning costs, we designed a universal 5-in-1 gene fragment multiplexing system. This architecture embeds up to five distinct parts flanked by orthogonal pairs of BpiI restriction sites into a single synthesized fragment, allowing them to sort independently into their respective L0 acceptor plasmids while maintaining complete modular flexibility of part types. Finally, we provide Level 2 cloning backbones with built in selection genes for common soybean transformation methods to facilitate downstream plant selection. Together, these advancements reduce DNA synthesis overhead and accelerate the construction of complex multigene payloads for plant biotechnology.

synthetic biology↗

bioRxiv2017

Increased pericarp cell length underlies a major QTL for grain weight in hexaploid wheat

O_LICrop yields must increase to address food insecurity. Grain weight, determined by grain length and width, is an important yield component, but our understanding of the underlying genes and mechanisms is limited.\nC_LIO_LIWe used genetic mapping and near isogenic lines (NILs) to identify, validate and fine map a major quantitative trait loci (QTL) on wheat chromosome 5A associated with grain weight. Detailed phenotypic characterisation of developing and mature grains from the NILs was performed.\nC_LIO_LIWe identified a stable and robust QTL associated with a 6.9 % increase in grain weight. The positive interval leads to 4.0 % longer grains, with differences first visible twelve days post fertilization. This grain length effect was fine-mapped to a 4.3 cM interval. The locus also has a pleiotropic effect on grain width (1.5 %) during late grain development that determines the relative magnitude of the grain weight increase. Positive NILs have increased maternal pericarp cell length, an effect which is independent of absolute grain length.\nC_LIO_LIThese results provide direct genetic evidence that pericarp cell length affects final grain size and weight in polyploid wheat. We propose that combining genes which control distinct biological mechanisms, such as cell expansion and proliferation, will enhance crop yields.\nC_LI

plant biology↗

bioRxiv2024

A rapid and efficient in vivo inoculation method for introducing tree stem canker pathogens onto leaves, suitable for large-scale assessment of resistance in poplar breeding progeny

Hybrid breeding, a direct and efficient strategy for disease control and management in tree species, is currently limited by the selection method of resist clones: the "in vitro stem segment inoculation method." This method, constrained by the availability of inoculating materials, cannot rapidly, efficiently, and cost-effectively screen the resistance of all hybrid clones. To overcome these limitations, we introduce a novel pathogen inoculation method for the resistance assessment of hybrid clones in the poplar-Valsa sordida pathosystem. This method involves inoculating the stem canker pathogen on the host leaf, a unique and promising approach we have successfully validated. Results showed that stem canker pathogen V. sordida induced the extended necrotic lesion and even induced the formation of pycnidium structure and conidia on the leaf surface five days after mycelium inoculation; 1) the upper 5-7th leaves exhibited higher resistance than the middle 18-20th leaves; 2) the shading conditions induced more severe symptoms on the leaves than lighting conditions; 3) the juvenile mycelium inoculums (4-day-cultured) were more susceptible to poplar leaves than the old ones (7-day-cultured). Our results demonstrate the robustness of the "in vivo leaf inoculation method" in revealing the resistance differentiation in poplar hybrid clones. According to the leaf necrotic area disease index, we divided these poplar clones into seven different resistance groups. The resistance assessed by leaf assessment was validated in 15 selected poplar clones using the "in vitro stem segment inoculation method." Results showed that the effectiveness of these two methods was consistent. Moreover, the leaf inoculation method can be used to detect the pathogenicity diversity of the pathogen population of tree species. Compared to the conventional "in vitro stem segment inoculation method," the leaf method has the advantages of abundant inoculation materials, easy operation, rapid disease onset, and almost no adverse effect on the host. It is particularly suitable for the resistance screening of all progeny and the early (seedling) phenotypic selection of resistant poplar clones in poplar stem disease resistance breeding. The "in vivo leaf inoculation method" holds significant promise in poplar breeding, tree pathology, and molecular biology research on tree stem diseases.

plant biology↗

bioRxiv2026

Recurrent Hermaphroditism and Sex-Biased ABCDE Gene Expression Reveal Latent Floral Plasticity in the Pedunculate Oak Lineage (Q.robur s.l.)

Background and AimsThe development of unisexual flowers relies on the tight coordination of flower organ identity and sex determination. The genus Quercus is typically considered strictly monoecious, bearing fully segregated male and female flowers within the same individual tree. However, several reports of atypical flowering across the genus challenge this canonical view, suggesting that flowering in oaks may be more flexible than traditionally assumed. In this work, the dynamics of flower development in Quercus orocantabrica were examined to correlate contrasting floral morphologies with divergent molecular profiles. MethodsThe flowering phenology of Q. orocantabrica trees was closely monitored over several individuals and years, together with a detailed floral morphological analysis of male, female and atypical flowers. Key floral homeotic gene homologues were identified, and their expression assayed in the development of different flowers. Key ResultsRecurrent and widespread hermaphroditic flowering was detected in several Q. orocantabrica trees, frequently associated with unseasonal flowering events. Gene expression analysis of male, female and hermaphroditic flowers revealed a sex-biased expression of Q. orocantabrica B- and C-class genes, with the B-class gene QoPI in particular being tightly associated with the presence of fully-developed stamens. In addition, the expression of the C-class gene QoSHP contrasted with reports in other Fagaceae, highlighting a potential functional divergence of the C/D-class lineage within the family. ConclusionsThe results here depicted indicate that the dynamics of floral sex identity in oaks are more plastic than traditionally assumed, supporting a reinterpretation of oak reproductive biology based on a versatile and resilient framework responsive to different developmental contexts.

plant biology↗

bioRxiv2025

Combined generalist and host-specific transcriptional strategies enable host generalism in the fungal pathogen Botrytis cinerea

How generalist pathogens infect phylogenetically diverse hosts remains a central question in plant-pathogen biology. In particular, the extent to which broad host range is enabled by genetic variation versus transcriptional plasticity is unclear. To investigate how variation and plasticity contribute to generalism, we studied the generalist necrotrophic fungus Botrytis cinerea that infects more than 1,500 plant species. Using a cross-infection matrix of 72 B. cinerea isolates infected on 57 plant genotypes distributed across 15 eudicot species, we identified general and host-dependent fungal components of lesion formation. Transcriptome profiling at 48 hours post-inoculation revealed two distinct pathogen gene modules: (1) a set of general lesion-associated genes enriched in primary metabolism, showing similar expression across hosts but varied among isolates; and (2) a set of high-entropy, host specific-inducible genes, organized into distinct co-regulated modules that respond dynamically to specific host cues. Both gene sets were genomically dispersed, lacking structural clustering, and were under different levels of selective constraints. Our results demonstrate that B. cinerea employs a modular transcriptional strategy that integrates a core metabolic program along with a plastic, host-responsive regulatory network to achieve broad host colonization. This study presents the most comprehensive cross-species co-transcriptomic dataset to date for any fungal phytopathogen, highlighting transcriptional plasticity as a key mechanism underlying generalism in plant-fungal interactions. Moreover, the identification of conserved fungal gene targets across diverse hosts offers a foundation for developing broad-spectrum resistance strategies in multiple crops.

genomics↗