Biology subjects

Laufs, P.

Publications and source records attributed to Laufs, P..

11 recordsLinked to original sources

bioRxiv2026

The CUP-SHAPED COTYLEDON 2 - MIR164A regulatory feedback loop directs leaf morphogenesis

Gene regulatory networks (GRNs) establish the spatio-temporal gene expression patterns necessary for reproducible development of organisms. MicroRNAs have emerged as important regulators of gene expression controlling either target gene levels, spatial patterns or timing. These molecular functions may vary depending on how they are integrated into GRNs. Here we used the Arabidopsis leaf as a model to study the genetic and molecular interactions between the CUP-SHAPED COTYLEDON 2 (CUC2) transcription factor and its negative regulator, miR164A. Quantitative morphometric analyses showed that miR164A acts from early stages during leaf development regulating CUC2 expression dynamic rather than the spatial distribution of CUC2. At the molecular level, CUC2 directly activates MIR164A expression, forming a single negative feedback loop. Further, using a ratiometric approach, we identified a cis-regulatory element (CRE) sufficient to drive CUC2-dependent MIR164A expression at the leaf sinus. Ordinary differential equation (ODE) modelling suggests that CUC2-CRE may control the timely accumulation of CUC2. Accordingly, quantification of CUC2 protein accumulation dynamics using a spatio-temporal mean cartography strategy in CRISPR cis-regulatory variants at the MIR164A locus revealed that CUC2-dependent MIRA164A expression is required to maintain both the precise timing of CUC2 expression and CUC2 levels. Altogether, our work provides experimental evidence for a specific role of the CUC2-MIR164A module in shaping leaf development, highlighting the contribution of network topology to GRN functions.

plant biology↗

bioRxiv2026

Sugar metabolism drives shoot branching by repressing BRC1 through miR319-targeted TCP4 in Rosa.

The coupling between sugar availability and shoot branching is well established, yet the underlying molecular mechanisms remain largely unknown. Here, using genome-wide profiling, we characterized the sugar signalling pathways by identifying 10 miRNAs repressed by glycolysis, the tricarboxylic acid (TCA) cycle, and/or the oxidative pentose phosphate pathway (OPPP) in rose buds. Focusing on RhmiR319, a miRNA repressed under active sugar metabolism, we demonstrate that disruption of both glycolysis/the TCA cycle and the OPPP induces the RhmiR319 accumulation, leading to the concomitant repression of its targets, RhTCP4a and RhTCP4b. We additionally reveal that the miR319/TCP4 module regulates shoot branching in Arabidopsis, as miR319 overexpression impairs shoot branching, whereas TCP4 promotes it. Altered branching patterns in Rose and Arabidopsis were associated with the expression of the branching integrator BRC1. Finally, we provide molecular evidence supporting that RhTCP4a/b directly binds to the RhBRC1 promoter and represses its transcription. Together, these results establish the miR319-TCP4-BRC1 regulatory module as a key component in the regulation of shoot branching in response to sugar status. We propose that the miR319-TCP4-BRC1 module is conserved between annual and perennial species and may integrate multiple hormonal and metabolic signals to shape shoot branching in plants.

plant biology↗

bioRxiv2026

Leaf hydraulics is a core component of plant immunity

Hydathodes at leaf margins mediate guttation of xylem-derived fluids and serve as primary entry sites for adapted vascular bacterial pathogens such as Xanthomonas campestris. Infection of Arabidopsis mutants with fewer hydathodes resulted in spontaneous mesophyll water-soaking and revealed unexpectedly-large pathogen populations explained by direct infection of the mesophyll niche through stomata and subsequent proliferation. Physical blockage of hydathodes also induced mesophyll water-soaking and enhanced bacterial growth in both Arabidopsis and cauliflower leaves. These findings reveal a dual role for hydathodes as primary sites of infection while being essential to restrict pathogen proliferation in nonvascular tissues. More broadly, our study identifies leaf hydraulics and guttation as central components of water immunity in vascular plants.

plant biology↗

bioRxiv2026

Genetic coupling of hydathode formation with leaf morphogenesis maintains water homeostasis

Hydathodes are specialized leaf structures present across vascular plants that allow guttation by connecting the xylem to the external environment through epithem tissue and permanently open water pores. However, the genetic mechanisms controlling their formation and physiological roles remain poorly understood. Here, we identify a genetic regulatory network that controls hydathode formation and links this process to leaf morphogenesis. This network converges on auxin signaling to coordinate the formation of the three hydathode cell types. We further show that epithem development requires sustained cell proliferation with limited endoreduplication. Analysis of hydathode mutants demonstrates that hydathode size and number are required to prevent reversible leaf flooding. Together, these findings establish a genetic framework for hydathode morphogenesis and uncover a central role for hydathodes in maintaining leaf water homeostasis under fluctuating environmental conditions.

plant biology↗

bioRxiv2024

Developmental rewiring of the NGAL/CUC/KLU network associated with pleiotropic roles of NGAL genes

Gene Regulatory Networks (GRNs) play prominent roles in regulating developmental processes, and their modulation across species is a major source for evolutionary innovation. However, it remains poorly understood how GRNs are rewired between different organs within a single species. This question is particularly relevant for pleiotropic genes, which may exhibit organ-specific GRN modulations potentially reflecting their diverse functions. To address this, we investigated the NGATHA-like (NGAL) genes, as a model for pleiotropic genes that regulate growth or patterning in multiple Arabidopsis organs via two distinct pathways involving the CUP-SHAPED COTYLEDON (CUC) and KLUH (KLU) genes. By combining genetic analysis with gene expression characterization, we uncovered significant organ-specific rewiring of the NGAL/CUC/KLU regulatory module. Our findings highlight that changes in gene expression patterns, potentially arising from developmental constraints, play a pivotal role in the organ-specific modulation of GRNs. Furthermore, GRNs at the molecular and functional levels do not always align perfectly, potentially due to the influence of additional regulatory mechanisms. Altogether, our findings reveal significant modulation of the GRNs associated with pleiotropic genes. We propose that this flexibility in GRNs facilitates gene pleiotropy.

plant biology↗

bioRxiv2024

M2WISH: an easy and efficient protocol for whole-mount mRNA in situ hybridization that allows 3D cell resolution of gene expression in Arabidopsis thaliana.

Gene expression analysis is essential for understanding the mechanisms involved in plant development. Here, we developed M2WISH, a protocol based on MicroWave treatment for Wholemount mRNA In Situ Hybridization in Arabidopsis. By permeabilizing tissues without damaging cellular organisation this protocol results in high and homogeneous hybridization yields that enables systematic analysis of gene expression dynamics. Moreover, when combined with cellular histochemical staining, M2WISH provides a cellular resolution of gene expression on roots, aerial meristems, leaves and embryos in the seed. We applied M2WISH to study the spatial dynamics of WUSCHEL (WUS) and CLAVATA3 (CLV3) expression during in vitro meristematic conversion of roots into shoot apical meristems. Thus, we showed that shoot apical meristems could arise from two different types of root structures that differed by their CLV3 gene expression patterns. We constructed 3D cellular representations of WUS and CLV3 gene co-expression pattern, and stressed the variability inherent to meristem conversion. Thus, this protocol generates a large amount of data on the localization of gene expression, which can be used to model complex systems.

plant biology↗

bioRxiv2023

A fully sequenced collection of homozygous EMS mutants for forward and reverse genetic screens in Arabidopsis thaliana

Genetic screens are powerful tools for biological research and are one of the reasons for the success of the thale cress Arabidopsis thaliana as a model species. Here, we describe the whole-genome sequencing of 871 Arabidopsis lines from the Homozygous EMS Mutant (HEM) collection as a novel resource for forward and reverse genetics. With an average 576 high-confidence mutations per HEM line, over three independent mutations altering protein sequence are found on average per gene in the collection. Pilot reverse genetics experiments on reproductive, developmental and physiological traits confirmed the efficacy of the tool for identifying both null and knockdown alleles. The possibility of conducting subtle repeated phenotyping of HEM lines and the immediate availability of the mutations will empower forward genetic approaches. The sequence resource is searchable with the ATHEM web interface (https://lipm-browsers.toulouse.inra.fr/pub/ATHEM/), and the biological material is distributed by the Versailles Arabidopsis Stock Center.

plant biology↗

bioRxiv2023

Compensation of compromised PRC2 regulation by a miRNA ensures robustness of Arabidopsis leaf development

Robustness is pervasive throughout biological systems, enabling them to maintain persistent outputs despite perturbations in their components. Here, we reveal a novel mechanism contributing to leaf morphology robustness in the face of genetic perturbations. In Arabidopsis, leaf shape is established during early development through the quantitative action of the CUP-SHAPED COTYLEDON2 (CUC2) gene that is negatively regulated by the co-expressed MICRORNA164A (MIR164A) gene. Compromised epigenetic regulation due to defective Polycomb Repressive Complex 2 (PRC2) function results in the transcriptional derepression of CUC2 but has no impact on CUC2 protein dynamics or early morphogenesis. We solve this apparent paradox by showing that compromised PRC2 function simultaneously activates a compensatory mechanism involving another member of the MIR164 gene family, the MIR164B gene. This mechanism dampens CUC2 protein levels, thereby compensating for compromised PRC2 function and canalizing early leaf morphogenesis. Furthermore, we show that this compensation mechanism is active under different environmental conditions. Our findings shed light on how the interplay between different types of transcriptional regulation can contribute to developmental robustness.

plant biology↗

bioRxiv2022

Arabidopsis hydathodes are sites of intense auxin metabolism and nutrient scavenging

Hydathodes are small organs located on the leaf margins of all vascular plants. They release excess xylem sap through guttation when stomata are closed or when the humidity level is high. Many promoter analyses have suggested other hydathode functions in metabolite transport and auxin metabolism, but experimental demonstration is still lacking. Here, we compared the transcriptomic and metabolomic features of mature Arabidopsis hydathodes to the leaf blade. 1460 differentially-expressed genes were identified revealing that genes related to auxin metabolism, transport, stress, DNA, plant cell wall, RNA or wax were on average more expressed in hydathodes. On the other hand, genes involved in glucosinolate metabolism, sulfation pathway, metal handling or photosynthesis were downregulated in hydathodes. In hydathodes, there are an increased expression of auxin transcriptional regulators and biosynthetic genes, a lower expression of auxin transport genes and a differential expression of genes related to its vacuolar storage that is consistent with increased contents of free and conjugated auxin. We also found that ca. 78% of the total content of 52 xylem sap metabolites were removed from guttation fluid at the hydathode level. Using reverse genetics, we showed that the capture of nitrate and phosphate in the guttation fluid relies on the NRT2.1 and PHT1;4 transporters, respectively. Thus, hydathodes absorb a significant part of xylem sap nutrients, limiting the loss of valuable chemicals during guttation. Our transcriptomic and metabolomic analyses reveal an organ with its own transcriptomic and physiological identity and highlight hydathode biological processes that may impact the whole plant. One sentence summaryTranscriptome and physiological analysis of mature and healthy hydathodes of Arabidopsis demonstrates that those organs are sites of intense auxin metabolism and nutrient scavenging

plant biology↗

bioRxiv2022

De novo stem cell establishment in meristems requires repression of organ boundary cell fate

Stem cells play important roles in animal and plant biology as they sustain morphogenesis and tissue replenishment following aging or injuries. In plants, stem cells are embedded in multicellular structures called meristems and the formation of new meristems is essential for the plastic expansion of the highly branched shoot and root systems. In particular, axillary meristems that produce lateral shoots arise from the division of boundary domain cells at the leaf base. The CUP-SHAPED COTYLEDON (CUC) genes are major determinants of the boundary domain and are required for axillary meristem initiation. However, how axillary meristems get structured and how stem cells become established de novo remains elusive. Here, we show that two NGATHA-LIKE transcription factors, DPA4 and SOD7, redundantly repress CUC expression in the initiating axillary meristem. Ectopic boundary fate leads to abnormal growth and organisation of the axillary meristem and prevents de novo stem cell establishment. Floral meristems of the dpa4 sod7 double mutant show a similar delay in stem cell de novo establishment. Altogether, while boundary fate is required for the initiation of axillary meristems, our work reveals how it is later repressed to allow proper meristem establishment and de novo stem cell niche formation.

plant biology↗

bioRxiv2021

A cell wall-associated gene network shapes leaf boundary domains

Boundary domains delimit and organize organ growth throughout plant development almost relentlessly building plant architecture and morphogenesis. Boundary domains display reduced growth and orchestrate development of adjacent tissues in a non-cell autonomous manner. How these two functions are achieved remains elusive despite the identification of several boundary-specific genes. Here, we show using morphometrics at the organ and cellular levels that leaf boundary domain development requires SPINDLY (SPY), an O-fucosyltransferase, to act as cell growth repressor. Further we show that SPY acts redundantly with the CUP-SHAPED COTYLEDON transcription factors (CUC2 and CUC3), which are major determinants of boundaries development. Accordingly at the molecular level, CUC2 and SPY repress a common set of genes involved in cell wall loosening providing a molecular framework for the growth repression associated with boundary domains. Atomic force microscopy (AFM) confirmed that young leaf boundary domain cells have stiffer cell walls than marginal outgrowth. This differential cell wall stiffness was reduced in spy mutant. Taken together our data reveal a concealed CUC2 cell wall associated gene network linking tissue patterning with cell growth and mechanics. Summary statementDecreased cell-wall loosening gene expression contributes to the coordination of cell growth and mechanics with tissue patterning thus driving boundary development.

plant biology↗