Biology subjects

Sibout, R.

Publications and source records attributed to Sibout, R..

10 recordsLinked to original sources

bioRxiv2026

In vivo validation of predicted fitness effects at single-base resolution in a Brachypodium distachyon mutant population

Computational tools, including biological language models (LMs), show substantial promise in predicting the impact of genetic variants on plant fitness. However, validating variant effect predictions (VEP) requires experimental populations where genetic variation consists of discrete point mutations rather than segregating recombination blocks. In this study, we generated a novel population of Brachypodium distachyon mutant lines to evaluate the accuracy of VEP at single-base resolution. These lines were advanced through single-seed descent for five generations (M1 to M5), with whole-genome sequencing performed at M2 and M5 and phenotypic measurements recorded at M3 and M4. Using state-of-the-art VEP models, we predicted the functional impact of missense protein-coding variants and gene-proximal non-coding variants. We validated these predictions by estimating the effect of mutations on whole-plant measurements (burden tests) and their probability of fixation from M2 to M5 (purging tests). Among missense variants, the protein LM ESM showed superior predictive accuracy compared to the bioinformatic standard SIFT and the genomic LM PlantCAD. Notably, the relationship between VEP scores and allele fixation suggested a log-linear relationship between VEP scores and variant fitness. Among gene-proximal variants, PlantCAD appeared more accurate than supervised models of regulatory activity, such as chromatin accessibility (a2z) and RNA abundance (PhytoExpr). Collectively, our findings highlight the utility of state-of-the-art VEP tools as predictors of fitness and demonstrate the potential of mutant populations to evaluate computational tools for precision breeding applications.

genomics↗

bioRxiv2025

Exodermis lignification impacts lateral root emergence in Brachypodium distachyon

RationaleThe mechanisms controlling lateral root emergence in monocots, particularly the role of the exodermis, are poorly understood. We investigated how natural variation in the Brachypodium distachyon stress response shapes root system architecture by modulating cell wall dynamics. MethodsWe used root tip excision to synchronize lateral root development across natural accessions. The resulting phenotypes were analysed using comparative transcriptomics, biochemical lignin quantification, confocal Raman spectroscopy, and chemical inhibition of lignin biosynthesis. Key ResultsTwo distinct root system architectures, pine tree and fishbone, were identified. The fishbone phenotype results from an emergence-specific defect caused by the premature and intense lignification of the exodermis. This was driven by the transcriptional upregulation of lignin biosynthesis genes and was rescued by a lignin inhibitor. Main ConclusionStress-induced exodermal lignification acts as a mechanical brake on lateral root emergence. This positions the exodermis as a key regulatory hub that integrates environmental cues with developmental programs to control RSA plasticity in grasses.

plant biology↗

bioRxiv2024

Comparative transcriptomics in ferns reveals key innovations and divergent evolution of secondary cell wall

Despite ferns being crucial to understanding plant evolution, their large and complex genomes has kept their genetic landscape largely uncharted, with only a handful of genomes sequenced and sparse transcriptomic data. Addressing this gap, we generated extensive RNA-sequencing data for multiple organs across 22 representative species over the fern phylogeny, assembling high-quality transcriptomes. These data facilitated the construction of a time-calibrated fern phylogeny covering all major clades, revealing numerous whole-genome duplications and highlighting the uniqueness of fern genetics, with half of the uncovered gene families being fern-specific. Our investigation into fern cell walls through biochemical and immunological analyses identified occurrences of the lignin syringyl unit and its independent evolution in ferns. Moreover, the discovery of an unusual sugar in fern cell walls hints at a divergent evolutionary path in cell wall biochemistry, potentially driven by gene duplication and sub-functionalization. We provide an online database preloaded with genomic and transcriptomic data for ferns and other land plants, which we used to identify an independent evolution of lignocellulosic gene modules in ferns. Our data provide a framework for the unique evolutionary path that ferns have navigated since they split from the last common ancestor of euphyllophytes more than 360 million years ago.

plant biology↗

bioRxiv2024

A new group of LysM-RLKs involved in symbiotic signal perception and arbuscular mycorrhiza establishment

Lipo-chitooligosaccharides (LCO) and short-chain chitooligosaccharides (CO) are produced by arbuscular mycorrhizal fungi (AMF) and activate the plant symbiosis signalling pathway, which is essential for mycorrhiza formation. High affinity LCO receptors belonging to the LysM receptor-like kinase (LysM-RLK) phylogenetic group LYR-IA play a role in AM establishment, but no plant high affinity short-chain CO receptors have yet been identified. Here we studied members of the uncharacterized LYR-IB group, and found that they show high affinity for LCO, short- and long-chain CO, and play a complementary role with the LYR-IA LCO receptors for AM establishment. While LYR-IB knock out mutants had a reduced AMF colonization in several species, constitutive/ectopic expression in wheat increased AMF colonization. LYR-IB function is conserved in all tested angiosperms, but in most japonica rice a deletion creates a frameshift in the gene, explaining differences in AM phenotypes between rice and other monocot single LYR-IA mutants. In conclusion, we identified a class of LysM-RLK receptors in angiosperms with new biochemical properties and a role in both LCO and CO perception for AM establishment.

plant biology↗

bioRxiv2023

Forward Genetic Stomatal CO2 Response Screen in Grass Brachypodium distachyon Reveals Central Function of a MAP Kinase in Early Stomatal CO2 Signal Transduction

Plants respond to increased CO2 concentrations through rapid stomatal closure which can contribute to increased water use efficiency. Grasses display faster stomatal responses than eudicots due to dumbbell-shaped guard cells flanked by subsidiary cells working in opposition. However, forward genetic screening for stomatal CO2 signal transduction mutants in grasses has not been reported. The grass model Brachypodium distachyon is closely related to agronomically important cereal crops, sharing largely collinear genomes. To gain insights into CO2 control mechanisms of stomatal movements in grasses, we developed a forward genetics screen with an EMS-mutagenized Brachypodium distachyon M5 generation population using infrared imaging to identify plants with altered canopy leaf temperature at elevated CO2. Among isolated mutants, a "chill1" mutant exhibited cooler leaf temperatures than wildtype Bd21-3 parent control plants after exposure to increased [CO2]. chill1 plants showed strongly impaired high CO2-induced stomatal closure, despite retaining a robust abscisic acid-induced stomatal closing response. Through bulked segregant whole-genome-sequencing analyses followed by analyses of further backcrossed F4 generation plants and generation and characterization of CRISPR-cas9 mutants, chill1 was mapped to a protein kinase, BdMPK5. The chill1 mutation impaired BdMPK5 protein-mediated CO2/HCO3- sensing in vitro. Furthermore, AlphaFold2-directed structural modeling suggests that the identified BdMPK5-D90N chill1 mutant residue is located at the interface with the HT1 Raf-like kinase. BdMPK5 is a key signaling component involved in CO2-induced stomatal movements, potentially functioning as a component of the CO2 sensor in grasses.

plant biology↗

bioRxiv2023

Transposition of HOPPLA in siRNA-deficient plants suggests a limited effect of the environment on retrotransposon mobility in Brachypodium distachyon

Long terminal repeat retrotransposons (LTR-RTs) are powerful mutagens regarded as a major source of genetic novelty and important drivers of evolution. Yet, the uncontrolled and potentially selfish proliferation of LTR-RTs can lead to deleterious mutations and genome instability, with large fitness costs for their host. While population genomics data suggest that an ongoing LTR-RT mobility is common in many species, the understanding of their dual role in evolution is limited. Here, we harness the genetic diversity of 320 sequenced natural accessions of the Mediterranean grass Brachypodium distachyon to characterize how genetic and environmental factors influence plant LTR-RT dynamics in the wild. When combining a coverage-based approach to estimate global LTR-RT copy number variations with mobilome-sequencing of nine accessions exposed to eight different stresses, we find little evidence for a major role of environmental factors in LTR-RT accumulations in B. distachyon natural accessions. Instead, we show that loss of RNA polymerase IV (Pol IV), which mediates RNA-directed DNA methylation in plants, results in high transcriptional and transpositional activities of RLC_BdisC024 (HOPPLA) LTR-RT family elements, and that these effects are not stress-specific. This work supports findings indicating an ongoing mobility in B. distachyon and reveals that host RNA-directed DNA methylation rather than environmental factors controls their mobility in this wild grass model. Author summaryLong terminal repeat retrotransposons (LTR-RTs) are major components of plant genomes. Their copy- and-paste replication mechanism allows them to rapidly increase in copy number, with potentially negative effects on host fitness. On the other hand, because they can rewire transcriptional networks and alter phenotypes, their mobility is an important driver of evolution. Ever since their discovery, LTR-RT activity has been linked to stress exposure, suggesting that LTR-RTs modulate the pace of evolution in response to the environment. In this study, we test this hypothesis by harnessing the genetic variation in a set of 320 natural accessions of the Mediterranean grass Brachypodium distachyon originating from diverse habitats. We find little evidence for the importance of stresses in activating B. distachyon LTR-RTs. Instead, we show that the loss of RNA polymerase IV, a component of plant retrotransposon silencing, leads to the activation and transposition of an LTR-RT family that we name HOPPLA. HOPPLA is the first LTR-RT family in B. distachyon shown to transpose in real-time. These findings open up new avenues for studying retrotransposon-mediated evolution in this close relative of staple crops, such as rice and wheat.

genomics↗

bioRxiv2023

Cell type-specific control of secondary cell wall formation by Musashi-type translational regulators in Arabidopsis

Deciphering the mechanism of secondary cell wall/SCW formation in plants is key to understanding their development and the molecular basis of biomass recalcitrance. Although transcriptional regulation is essential for SCW formation, little is known about the implication of post-transcriptional mechanisms in this process. Here we report that two bonafide RNA-binding proteins homologous to the animal translational regulator Musashi, MSIL2 and MSIL4, function redundantly to control SCW formation in Arabidopsis. MSIL2/4 interactomes are similar and enriched in proteins involved in mRNA binding and translational regulation. MSIL2/4 mutations alter SCW formation in the fibers, leading to a reduction in lignin deposition, and an increase of 4-O-glucuronoxylan methylation. In accordance, quantitative proteomics of stems reveal an overaccumulation of glucuronoxylan biosynthetic machinery, including GXM3, in the msil2/4 mutant stem. We showed that MSIL4 immunoprecipitates GXM mRNAs, suggesting a novel aspect of SCW regulation, linking post-transcriptional control to the regulation of SCW biosynthesis genes.

plant biology↗

bioRxiv2022

PHYTOCHROME C regulation of PHOTOPERIOD1 is mediated by EARLY FLOWERING 3 in Brachypodium distachyon

Daylength sensing in many plants is critical for coinciding the timing of flowering with the appropriate season. Temperate-climate-adapted grasses such as Brachypodium distachyon flower during the spring when days are becoming longer. The photoreceptor PHYTOCHROME C is essential for long-day (LD) flowering in B. distachyon. PHYC is required for the LD activation of a suite of genes in the photoperiod pathway including PHOTOPERIOD1 (PPD1) that, in turn, result in the activation of FLOWERING LOCUS T (FT1)/FLORIGEN, which causes flowering. Thus, phyC mutants are extremely delayed in flowering. Here we show that PHYC-mediated activation of PPD1 occurs via EARLY FLOWERING 3 (ELF3), a component of the evening complex in the circadian clock. The extreme delay of flowering of the phyC mutant disappears when combined with an elf3 loss-of-function mutation. Moreover, the dampened PPD1 expression in phyC mutant plants is elevated in phyC/elf3 mutant plants consistent with the rapid flowering of the double mutant. We show that loss of PPD1 function also results in reduced FT1 expression levels and extremely delayed flowering consistent with reports from wheat and barley. Additionally, elf3 mutant plants have elevated expression levels of PPD1 and we show that overexpression of ELF3 results in delayed flowering, which is associated with a reduction of PPD1 and FT1, demonstrating ELF3 represses PPD1 transcription, consistent with previous studies showing that ELF3 binds to the PPD1 promoter. Indeed, PPD1 is the main target of ELF3-mediated flowering as elf3/ppd1 double mutant plants are delayed flowering. Our results indicate that ELF3 operates downstream from PHYC and acts as a repressor of PPD1 in the photoperiod flowering pathway of B. distachyon. AUTHOR SUMMARYDaylength is an important environmental cue that plants and animals use to coincide important life history events with a proper season. In plants, timing of flowering to a particular season is an essential adaptation to many ecological niches. Perceiving changes in daylength starts with the perception of light via specific photoreceptors such as phytochromes. In temperate grasses, how daylength perception is integrated into downstream pathways to trigger flowering is not fully understood. However, some of the components involved in the translation of daylength perception into the induction of flowering in temperate grasses have been identified from studies of natural variation. For example, specific alleles of two genes called EARLY FLOWERING 3 (ELF3) and PHOTOPERIOD1 (PPD1) have been selected during breeding of different wheat and barley varieties to modulate the photoperiodic response to maximize reproduction in different environments. Here, we show in the temperate grass model Brachypodium distachyon that the translation of the light signal perceived by phytochromes into a flowering response is mediated by ELF3, and that PPD1 is genetically downstream of ELF3 in the photoperiodic flowering pathway. These results provide a genetic framework for understanding the photoperiodic response in temperate grasses that include agronomically important crops such as wheat, oats, barley, and rye.

plant biology↗

bioRxiv2022

A hair cell-specific peroxidase coordinates stomatal and prickle hair cell size to optimize epidermal cell functionality in grasses

The leaf epidermis is the outermost cell layer forming the interface between plants and the atmosphere that must both provide a robust barrier against (a)biotic stressors and facilitate carbon dioxide uptake and leaf transpiration 1. To achieve these opposing requirements, the plant epidermis developed a wide range of specialized cell types such as stomata and hair cells. While factors forming these individual cell types are known 2-5, it is poorly understood how their number and size is coordinated. Here, we identified a role for BdPRX76/BdPOX, a class III peroxidase, in regulating hair cell and stomatal size in the model grass Brachypodium distachyon. In bdpox mutants prickle hair cells were smaller and stomata were longer. Because stomatal density remained unchanged, the negative correlation between stomatal size and density was disrupted in bdpox and resulted in higher stomatal conductance and lower intrinsic water-use efficiency. BdPOX was exclusively expressed in hair cells suggesting that BdPOX cell-autonomously promotes hair cell size and indirectly restricts stomatal length. Cell wall autofluorescence and lignin stainings indicated a role for BdPOX in lignification or crosslinking of related phenolic compounds at the hair cell base. Ectopic expression of BdPOX in the stomatal lineage increased phenolic autofluorescence in guard cell walls and restricted stomatal elongation in bdpox. Together, we highlight a developmental interplay between hair cells and stomata that optimizes epidermal functionality. We propose that cell-type-specific changes disrupt this interplay and lead to compensatory developmental defects in other epidermal cell types.

plant biology↗

bioRxiv2021

The PMT-driven p-coumaroylation of poplar lignins impacts lignin structure and improves wood saccharification

Transgenic poplars (Populus tremula x Populus alba, clone INRA 717-1B4) were produced by introducing the Brachypodium distachyon Bradi2g36910 (BdPMT1) gene driven by the Arabidopsis (Arabidopsis thaliana) Cinnamate 4-Hydroxylase (AtC4H) promoter in the wild-type (WT) line and in a line overexpressing the Arabidopsis Ferulate 5-Hydroxylase (AtF5H). BdPMT1 encodes a transferase which catalyzes the acylation of monolignols by p-coumaric acid (CA). Several BdPMT1- OE/WT and BdPMT1-OE/AtF5H-OE transgenic lines were grown in the greenhouse and BdPMT1 expression in xylem was confirmed by RT-PCR. The analysis of the cell walls (CW) of poplar stems and of corresponding purified dioxan lignins (DL) revealed that the BdPMT1-OE lignins were as p-coumaroylated as the lignins of C3 grass straws. For some transformants, CA levels even reached about 11 mg/g CW and 66 mg/g DL, which by far exceeds those of Brachypodium or wheat samples. This unprecedentedly high p-coumaroylation of poplar lignins affected neither the poplar growth, nor the stem lignin content. By contrast, the transgenic lignins were structurally modified, with an increase of terminal units with free phenolic groups. Relative to controls, this increase argues for a reduced polymerization degree of BdPMT1-OE lignins and makes them more soluble in cold NaOH solution. The p-coumaroylation of poplar samples, up to the levels of C3 grasses, improved the saccharification yield of alkali-pretreated poplar CW. These results establish that the genetically-driven p-coumaroylation of lignins is a promising strategy to make wood lignins more susceptible to the alkaline treatments that can be used during the industrial processing of lignocellulosics. One-sentence summaryThe expression of a grass p-coumaroyl-CoA:monolignol transferase induces a high p-coumaroylation of poplar lignins and a better saccharification of alkali-pretreated poplar wood without growth penalty

plant biology↗