Biology subjects

Boutet, S.

Publications and source records attributed to Boutet, S..

18 recordsLinked to original sources

bioRxiv2026

Ultrafast X-Rays Capture Retinal Traversing Conical Intersection in Rhodopsin

G-protein-coupled receptors of the Rhodopsin family are crucial medicinal targets, transmitting signals across biomembranes. While light absorption by visual rhodopsin is well studied, its activation via retinal cofactor dynamics remains unclear. Here we use a free-electron laser to show that time-resolved X-ray solution scattering captures the retinal cis-trans isomerization as it passes the conical intersection of excited and ground-state photoproduct energy surfaces. Femtosecond-scale nuclear changes occur due to resonant photon absorption, with all-atom simulations revealing ultrafast amino acid movements that initiate transmembrane helix shifts. Ligand-free opsin measurements confirm that light activation is unaffected by non-resonant processes, showing the photonic energy is directly transmitted within the protein. Our method unveils how cofactor dynamics activate rhodopsin, free of constraints from crystal lattice packing or cryotrapping photointermediates.

biophysics↗

bioRxiv2026

Elevated temperature drives the biosynthesis of novel acylated glucosinolates in Arabidopsis thaliana seeds

Glucosinolates (GSLs) are major defensive compounds massively accumulated in Brassicaceae seeds, including that of the model plant Arabidopsis thaliana. While most studies have focused on the role of GSL in responses to biotic stress, the potential regulation and function of GSLs in responses to abiotic stresses have been neglected, particularly in seeds. In this study, multi-omic analyses revealed a previously uncharacterized GSL modification pathway induced by elevated temperature (ET) during A. thaliana seed development. Activation of this pathway leads to the production of several novel thioglucose-acylated GSLs, including sinapoylated and benzoylated derivatives. A reverse genetics approach demonstrated that the SERINE CARBOXYPEPTIDASE-LIKE 17 (SCPL17) and BENZOYLOXYGLUCOSINOLATE 1 (BZO1) enzymes are required for the acylation of GSL thioglucose moieties. Furthermore, the accumulation of acylated GSLs in seeds of 85 A. thaliana accessions grown under standard condition was shown to correlate with the average annual temperature of their origin site, suggesting that thioglucose-acylated GSLs production may reflect long-term thermal adaptation across natural populations. Taken together, these results demonstrate that thioglucose acylation by SCPL17 and BZO1 represents a new layer of GSL diversification in A. thaliana seeds that contributes to both ET response and long-term environmental adaptation.

plant biology↗

bioRxiv2026

Apoplast multi-omics profiling during fungal infection uncovers new players of basal and early induced immunity

The extracellular space in plant tissues, known as the apoplast, remains one of the least characterized cellular compartments. The apoplastic fluid (APF), akin to mammalian extracellular fluid, serves as the primary interface between pathogens and their host. It allows the exchange of signalling molecules, coordinates host cell responses, and enables the circulation of pathogen effectors that modulate the immune response. We describe here the first multi-omics analysis of the APF content just 6 h after the onset of A. thaliana infection with B. cinerea, a fast-killing necrotrophic fungus. By varying plant nitrogen nutrition, known to affect both plant defenses and pathogen virulence, we identify candidates that do not stand out under optimal conditions. Our analysis uncovers novel nitrogen-dependent mechanisms that regulate both basal and early induced apoplastic immunity, revealing the presence of previously unidentified, potentially protective apoplastic metabolites as well as the intercellular transport of nuclear proteins, thereby offering new insights into early apoplastic immune responses.

plant biology↗

bioRxiv2024

Innovative mutant screening identifies TRANSPARENT TESTA7 as a player in seed oil/protein partitioning

Brassicaceae species mainly accumulate oil and protein in their seeds, which are essential to human life as a source of food, but also as animal feed and resources for green chemistry. To date, Brassicaceae crops such as rapeseed have been selected mainly for their oil content. However, there is a growing interest in their seed protein content. A strong negative correlation between oil and protein content makes it difficult to increase both compounds simultaneously. In this study, an Arabidopsis thaliana homozygous EMS mutant library was screened by near-infrared spectroscopy for seed oil and protein content, with the aim of identifying mutants with impaired oil-protein correlation. The mutant most affected in this correlation was found to be in the TRANSPARENT TESTA7 gene, which is involved in the flavonoid biosynthetic pathway. Analysis of different mutants in the flavonoid pathway revealed that the tt7 mutants were the only ones to show such a significant reduction in seed oil content, highlighting a phenotype never described before for the tt7 mutants and suggesting a specific role for TT7 in the interplay between the oil and flavonoid biosynthetic pathways. Untargeted metabolomic analysis allowed the identification of metabolic features that are highly accumulated and specific to tt7 seeds compared to the other genotypes and genetic analysis established that the accumulation of kaempferol-3-O-rhamnoside seems to be responsible for the seed oil reduction of tt7 mutants. Significance StatementBrassicaceae species accumulate oil and protein in their seeds and understanding how the partitioning of these compounds is regulated is necessary to engineer seeds for specific purposes. By screening an Arabidopsis EMS mutant library, we identified mutants affected in seed oil/protein partitioning, including tt7, highlighting a link between oil and flavonoid biosynthetic pathways, that we explore further in this paper.

plant biology↗

bioRxiv2024

Specific redox and iron homeostasis responses in the root tip of Arabidopsis upon zinc excess

O_LIZinc (Zn) excess negatively impacts primary root growth in Arabidopsis. Yet, the effects of Zn excess on specific growth processes in the root tip remain largely unexplored. C_LIO_LITranscriptomics, ionomics and metabolomics were used to examine the specific impact of Zn excess on the root tip (RT) compared to the remaining root (RR). C_LIO_LIZn excess exposure resulted in shortened root apical meristem and elongation zone, with differentiation initiating closer to the tip of the root. Zn accumulated at a lower concentration in the RT than in RR. This pattern was associated with lower expression of Zn homeostasis and Fe deficiency response genes. C_LIO_LIA distinct distribution of Zn and Fe in RT and RR was highlighted by Laser Ablation ICP-MS analysis. C_LIO_LISpecialized Trp-derived metabolism genes, typically associated with redox and biotic stress responses, were specifically up-regulated in the RT upon Zn excess, among those Phytoalexin Deficient 3 (PAD3) encoding the last enzyme of camalexin synthesis. In roots of wild-type seedlings, camalexin concentration increased by 6-fold upon Zn excess and a pad3 mutant displayed increased Zn sensitivity and an altered ionome. C_LIO_LIOur results indicate that distinct redox and iron homeostasis mechanisms are key elements of the response to Zn excess in the RT. C_LI

plant biology↗

bioRxiv2024

A multi-omics analysis of Arabidopsis thaliana root tips under Cd exposure: A role of HY5 in limiting accumulation

O_LICadmium (Cd) is a major environmental pollutant with high toxicity potential. Even though a reduction of growth, including the primary root, is a clear consequence of Cd exposure, a profound understanding of the impact of Cd on the root apical meristem (RAM) and the elongation/differentiation zone (EDZ) is still lacking. C_LIO_LIIn this study, Arabidopsis thaliana roots were subjected to Cd and divided into root tips (RT) and remaining roots (RR) to separately assess the effect of Cd using transcriptomics, ionomics and metabolomics. C_LIO_LIElemental profiling revealed lower Cd accumulation in RT and differences in mineral contents between RT and RR. Transcriptomic analysis demonstrated distinct gene expression patterns in RT and RR, with Cd having less impact in RT. Functional enrichment analysis revealed genes associated with iron and sulfur homeostasis as well as the response to light in both RR and RT. RT-specific responses to Cd included several genes regulated by the transcription factor ELONGATED HYPOCOTYL 5 (HY5) and notably, the hy5 mutant showed increased Cd sensitivity and accumulation compared to the wild type. C_LIO_LIThis study provides comprehensive insights into the inhibitory effects of Cd on primary root growth, elucidating molecular mechanisms involved, particularly highlighting the role of HY5 in Cd accumulation. C_LI

plant biology↗

bioRxiv2024

Differential induction of Medicago truncatula defence metabolites in response to rhizobial symbiosis and pea aphid infestation

O_LILegumes symbiosis with rhizobial nitrogen-fixing bacteria enable them to grow in nitrate-depleted soils. Rhizobial symbioses also induces systemic plant defence against bioagressors. C_LIO_LIWe investigate how nitrogen-fixing symbiosis (NFS) in the legume Medicago truncatula can prime plant defence against the pea aphid Acyrthosiphon pisum. We analysed metabolite modification both by LC-MS and GC-MS and defence pathway gene expression by qPCR in leaves of both NFS and nitrate-fed (non-inoculated; NI) plants after aphid infestation (Amp). C_LIO_LIThe accumulation of primary and secondary metabolites was modulated by both NFS and aphid infestation. 62 defense-related metabolites such as salicylate, pipecolate, gentisic acid and several soluble sugars were differentially regulated by aphid infestation in both NFS and NI conditions. 19 metabolites, including triterpenoid saponins, accumulated specifically in NFS_Amp conditions. Gene expression analysis showed that aphid-infested plants exhibited significantly higher expression of Chalcone isomerase, flavonol synthase, hydroxyisoflavone-O-methyl transferase and Pterocarpan synthase, while D-pinitol dehydrogenase was only significantly induced in NI infested leaves. C_LIO_LIOur data suggest that NFS, in addition to being a plant nitrogen provider, stimulates specific legume defenses upon pest attack and should also be considered as a potential tool in Integrated Pest Management strategy. C_LI

plant biology↗

bioRxiv2024

Multi-omic analyses unveil temporal and spatial distribution of specialized metabolites in seeds of Camelina sativa

Seeds of Brassicaceae produce a large diversity of beneficial and antinutritional specialized metabolites (SMs) that influence their quality and provide resistance to stresses. While the distribution of these compounds has been described in leaves and roots tissues, limited information is available about their spatio-temporal accumulation in seeds. Camelina sativa (camelina) is an oilseed Brassicaceae cultivated for human and animal nutrition, and for industrial uses. While we previously explored SM diversity and plasticity, no information is available about SM distribution and expression of related proteins and genes in camelina seeds. In this study, we used a multi-omic approach, integrating untargeted metabolomics, data-independent acquisition proteomics, and transcriptomics to investigate the synthesis, modifications and degradations of SMs accumulated in the different seed tissues (i.e. seed coat, endosperm, and embryo) at 6 developmental and 2 germination stages. Our results showed distinct patterns of SMs and their related pathways, highlighting significant contrasts in seed composition and spatial distribution for the defence-related and antinutritional glucosinolate (GSL) compounds among camelina, Arabidopsis thaliana, and Brassica napus, three closely-related Brassicaceae species. Notably, the variation in GSL spatial distributions was primarily driven by differences in their structure and transport mechanisms. Long chain C8-C11 methylsulfinylalkyl GSLs were predominantly accumulated in the seed coat and endosperm, while mid- and short-chain C3-C7 methylsulfinylalkyl GSLs were accumulated in the embryo. Characterizing the spatial dynamics of seed SMs provides valuable insights that can guide the development of crops with optimized distribution of beneficial and toxic metabolites, improving seed nutritional profiles for feed and food.

plant biology↗

bioRxiv2024

Cytokinin-induced immunity enhances rice blast resistance

Plant cytokinins (CKs) affect the outcome of plant-pathogen interactions. However, despite many examples their role remains ambiguous. In rice, CKs act synergistically with salicylic acid (SA) to induce defense genes in vitro but no effect on resistance against the blast fungus, Magnaporthe oryzae, was further observed in planta. Here, we demonstrate that exogenous CKs treatment triggers rice blast resistance in a molecule-, dose- and time-dependent manner by affecting defense- and CK-responsive genes. Similar enhanced resistance and gene expression patterns were confirmed in rice insertion mutant lines impaired for a gene that encodes a putative CK inactivation enzyme, supporting that endogenous CKs affect rice immunity. Together, our work brings insights on the CK-induced resistance of rice to M. oryzae.

plant biology↗

bioRxiv2023

Changes in an Enzyme Ensemble During Catalysis Observed by High Resolution XFEL Crystallography

Enzymes populate ensembles of structures with intrinsically different catalytic proficiencies that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL) to observe catalysis in a designed mutant (G150T) isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations and formation of the thioimidate catalytic intermediate selects for catalytically competent substates. A prior proposal for active site cysteine charge-coupled conformational changes in ICH is validated by determining structures of the enzyme over a range of pH values. A combination of large molecular dynamics simulations of the enzyme in crystallo and time-resolved electron density maps shows that ionization of the general acid Asp17 during catalysis causes additional conformational changes that propagate across the dimer interface, connecting the two active sites. These ionization-linked changes in the ICH conformational ensemble permit water to enter the active site in a location that is poised for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

biochemistry↗

bioRxiv2023

Structural basis for functional properties of cytochrome c oxidase

Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes, thereby establishing the proton gradient required for ATP synthesis1. The full turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized by molecular oxygen to the metastable oxidized OH state, and a reductive phase, in which OH is reduced back to the R state. During each of the two phases, two protons are translocated across the membranes2. However, if OH is allowed to relax to the resting oxidized state (O), a redox equivalent to OH, its subsequent reduction to R is incapable of driving proton translocation2,3. How the O state structurally differs from OH remains an enigma in modern bioenergetics. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX)4, we show that the heme a3 iron and CuB in the active site of the O state, like those in the OH state5,6, are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, a residue covalently linked to one of the three CuB ligands and critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishes O from OH, where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide new insights into the proton translocation mechanism of CcO.

biophysics↗

bioRxiv2022

Heterogeneity in the M. tuberculosis β-lactamase inhibition by Sulbactam

For decades, researchers have been determined to elucidate essential enzymatic functions on the atomic lengths scale by tracing atomic positions in real time. Our work builds on new possibilities unleashed by mix-and-inject serial crystallography (MISC)1-5 at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals6. Here, we report in atomic detail and with millisecond time-resolution how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam (SUB). Our results reveal ligand binding heterogeneity, ligand gating7-9, cooperativity, induced fit10,11 and conformational selection11-13 all from the same set of MISC data, detailing how SUB approaches the catalytic clefts and binds to the enzyme non-covalently before reacting to a trans-enamine. This was made possible in part by the application of the singular value decomposition14 to the MISC data using a newly developed program that remains functional even if unit cell parameters change during the reaction.

molecular biology↗

bioRxiv2022

Metabolite profiling and cytotoxic activity of Andean potatoes: polyamines and glycoalkaloids as potential anticancer agents in human neuroblastoma cells in vitro

Andean potatoes (Solanum tuberosum L. ssp. andigena) are a good source of dietary antioxidant polyphenols. We have previously demonstrated that polyphenol extracts from Andean potato tubers exerted a dose-dependent cytotoxic effect in human neuroblastoma SH-SY5Y cells, being skin extracts more potent than flesh ones. In order to gain insight into the bioactivities of potato phenolics, we investigated the composition and the in vitro cytotoxic activity of total extracts and fractions of skin and flesh tubers of three Andean potato cultivars (Santa Maria, Waicha, and Moradita). Potato total extracts were subjected to liquid-liquid fractionation using ethyl acetate solvent in organic and aqueous fractions. We analyzed both fractions by HPLC-DAD, HPLC-ESI-MS/MS, and HPLC-HRMS. Results corroborated the expected composition of each fraction. Organic fractions were rich in hydroxycinnamic acids (principally chlorogenic acid isomers), whereas aqueous fractions contained mainly polyamines conjugated with phenolic acids, glycoalkaloids, and flavonoids. Organic fractions were not cytotoxic against SH-SY5Y cells, and indeed, some increased cellular metabolism compared to controls. Aqueous fractions were cytotoxic and even more potent than their respective total extracts. Treatment with a combination of both fractions showed a similar cytotoxic response to the corresponding extract. According to correlation studies, it is tempting to speculate that polyamines and glycoalkaloids are crucial in inducing cell death. Our findings indicate that the activity of Andean potato extracts is a combination of various compounds and contribute to the revalorization of potato as a functional food.

cell biology↗

bioRxiv2022

ULTRAFAST STRUCTURAL CHANGES DIRECT THE FIRST MOLECULAR EVENTS OF VISION

Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs). A photon is absorbed by the 11-cis retinal chromophore of rhodopsin which isomerises within 200 femtoseconds to the all-trans conformation, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains elusive. In this work, we use ultrafast time-resolved crystallography at room temperature to determine how an isomerised twisted all-trans retinal stores the photon energy required to initiate protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at 1 ps time-delay of photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Strikingly, the very early structural motions in the protein side chains of rhodopsin appear in regions involved in later stages of the conserved Class A GPCR activation mechanism. Our work sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.

biochemistry↗

bioRxiv2021

Rational control of structural off-state heterogeneity in a photoswitchable fluorescent protein provides switching contrast enhancement

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, a key parameter that largely dictates the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

biophysics↗

bioRxiv2021

Untargeted metabolomic analyses reveal the diversity and plasticity of the specialized metabolome in seeds of different Camelina sativa varieties

Despite the essential role of Specialized Metabolites (SMs) in the interaction of plants with the environment, studying the ability of crop seeds to produce these protective compounds has been neglected. Furthermore, seeds produce a myriad of SMs providing an interesting model to investigate their diversity and plasticity. Camelina sativa gained a lot of interest in the past few years as rustic oil seed crop. A characterization of seed SM landscapes in six camelina genotypes grown in the field and harvested during five growing seasons has been undertaken in this work. This allowed a comprehensive annotation of seed SMs combining analyses that cluster SMs based on their chemical structures and co-accumulation patterns. These data showed broad effects of the environment on the stimulation of the seed-specialized metabolome. Among well annotated compounds, flavonols were identified as the metabolic class characterised by high plasticity, revealing significant variable accumulation according to the year and/or the genotype. Lastly, a deeper characterisation of primary metabolites and lipids in two selected genotypes has been performed. We showed that, in addition to flavonols, alkaloids and glucosinolates displayed a higher phenotypic plasticity with respect to most of the primary metabolites, including some sugars and major storage compounds such as fatty acids, proteins and most lipid classes (e.g. DAG, TAG), but similar plasticity compared to free aminoacids and carboxylic acids. This work highlighted major and unexplored effects of the environment on the seed specialized metabolome demonstrating that seeds exhibit a dynamic and plastic metabolism, with an impact on seed quality. Significance statementSeeds produce a myriad of Specialized Metabolites (SMs) with an essential role in the interaction of plants with the environment. We characterized SM landscapes, primary metabolites and lipid composition in the seeds of camelina genotypes grown in the open field in five consecutive growing seasons. Our results showed the predominant effect of the environment on the regulation of the seed - specialized metabolome, with a potential impact on seed quality of camelina that may also occur in other oilseed crops.

plant biology↗

bioRxiv2019

Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometer to micron scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges not encountered in traditional macromolecular crystallography experiments. Here, we describe XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A (CypA). Our results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample preparation and delivery methods required for each type of experiment effect the crystal structure of the enzyme.

biophysics↗

bioRxiv2019

Benchmarking Single-Cell RNA Sequencing Protocols for Cell Atlas Projects

Single-cell RNA sequencing (scRNA-seq) is the leading technique for charting the molecular properties of individual cells. The latest methods are scalable to thousands of cells, enabling in-depth characterization of sample composition without prior knowledge. However, there are important differences between scRNA-seq techniques, and it remains unclear which are the most suitable protocols for drawing cell atlases of tissues, organs and organisms. We have generated benchmark datasets to systematically evaluate techniques in terms of their power to comprehensively describe cell types and states. We performed a multi-center study comparing 13 commonly used single-cell and single-nucleus RNA-seq protocols using a highly heterogeneous reference sample resource. Comparative and integrative analysis at cell type and state level revealed marked differences in protocol performance, highlighting a series of key features for cell atlas projects. These should be considered when defining guidelines and standards for international consortia, such as the Human Cell Atlas project.

genomics↗