Biology subjects

Arnaud, D.

Publications and source records attributed to Arnaud, D..

6 recordsLinked to original sources

bioRxiv2025

Papaver S-determinants trigger an integrated network of mitochondrially derived ROS and disruption of energy metabolism in incompatible pollen tubes

Many plants use self-incompatibility (SI) mechanisms to prevent inbreeding by inhibiting pollen tube growth in "self" pistils. In Papaver rhoeas, SI is triggered by allele-specific interaction between the pollen and pistil S-determinants, activating a Ca2+-dependent signalling network that leads to rapid reactive oxygen species (ROS) production and eventual programmed cell death (PCD) in incompatible pollen tubes. Expression of the Papaver pollen S-determinant (PrpS) in Arabidopsis thaliana recapitulates Papaver SI when challenged with the cognate pistil ligand (PrsS). Using roGFP2-Orp1, a genetically encoded hydrogen peroxide (H2O2) sensor, combined with measurements of mitochondrial metabolism we have revealed a complex SI response. Within minutes, elevated cytosolic Ca2+ ([Ca2+]cyt) and cytosolic acidification converge to trigger mitochondrial H2O2 production (with consequent cytosolic and plastid H2O2 increase), membrane depolarisation, decreased respiration rate, and ATP depletion. In parallel, oxidative inactivation of cytosolic GAPDH inhibits glycolytic flux, resulting in decreased TCA cycle intermediates and providing a feedback loop to enhance mitochondrial disruption. Preceding production of mitochondrially-sourced ROS, we show that SI rapidly arrests pollen tube growth via inactivation of plasma membrane localised NADPH oxidase (RBOH) mediated superoxide production. Our findings provide new insights into how different ROS signatures from NADPH oxidase and mitochondria drive distinct processes. We demonstrate that mitochondrial disruption is an early and central event in the SI-response, likely driven by positive feedback loops involving altered Ca2+ signalling, cytosolic pH and ROS. Our findings provide novel insights into how SI rapidly disrupts energy metabolism in pollen tubes through interconnected signalling networks centred on mitochondria prior to PCD.

plant biology↗

bioRxiv2025

Arabidopsis lines with modified ascorbate concentrations reveal a link between ascorbate and auxin biosynthesis

Ascorbate is the most abundant water-soluble antioxidant in plants, and it is an essential molecule for normal plant development. It is present in all green plants, with very different concentrations in different plant species. While ascorbate accumulation is a trait of nutritional, and therefore, agronomical interest, the impact of different concentrations over cellular homeostasis remains elusive. In order to shed light over this question, we leveraged Arabidopsis lines with very low ascorbate (vtc2 mutant with 20% of WT ascorbate levels), and low ascorbate concentration (vtc4 mutant with 65% of WT levels), and we generated a line that accumulates 165% of WT levels (vtc2/OE-VTC2). An 80% reduction of ascorbate increased the expression of genes implicated in defense against pathogens, but repressed genes associated with abiotic stress responses. Unexpectedly, lines with increased (165% of WT) and decreased (65% of WT) ascorbate levels shared 85% of induced transcription factors and the GO terms associated with their transcriptional programs. Among the group of genes whose expression is positively correlated with ascorbate content, we identified TAA1/WEI8, a gene encoding a tryptophan aminotransferase that catalyzes the first step of auxin biosynthesis. Using a combination of genetic and pharmacological approaches in fluorescent and histochemical reporter lines for auxin biosynthesis and signaling activity, we revealed that TAA1- and TAA1-RELATED2 (TAR2)-mediated auxin biosynthesis is necessary for plants to cope with increased ascorbate concentration in a light-dependent manner, revealing a new layer of complexity in the regulatory landscape of redox homeostasis.

plant biology↗

bioRxiv2024

Cytokinin-inducible DIRIGENT13 involved in lignan synthesis and ROS accumulation promotes root growth and abiotic stress tolerance in Arabidopsis

Dirigent (DIR) proteins mediate regio- and stereoselectivity of phenoxy radical coupling of monolignols for (neo)lignan or lignin synthesis. However, the role of DIR proteins and lignans in plant development and response to environmental stresses remains elusive. Here, we provide a functional characterization of the cytokinin-responsive DIRIGENT13 (DIR13) in Arabidopsis thaliana. DIR13 was localized to the root endodermis and at the margins of lateral root primordia and lateral roots. While not necessary for Casparian strip formation, DIR13 promoted both main and lateral root growth. Untargeted metabolomics and imaging mass spectrometry analyses unveiled the role of DIR13 in facilitating (neo)lignan synthesis in primary and lateral roots. Interestingly, DIR13 activated the production of putative oxomatairesinol and matairesinol-cysteine which are oxidative derivatives of the lignan matairesinol. Our data also identified DIR13 as an enhancer of salt and drought tolerance. Particularly, DIR13 attenuated salt stress-mediated inhibition of germination and root growth. Moreover, DIR13 activated reactive oxygen species (ROS) accumulation both under control and salt stress conditions, and cytokinin further enhanced the salt-induced ROS production specifically in the DIR13 overexpressing line. Our results uncover a role for DIR13-produced lignans in the priming of ROS accumulation, mediating both abiotic stress tolerance and the control of root architecture.

plant biology↗

bioRxiv2023

Cytokinins control secondary cell wall formation in the inflorescence stem of Arabidopsis

Spatiotemporal control over developmental programs is vital to all organisms. Here we show that cytokinin (signaling) deficiency leads to early secondary cell wall (SCW) formation in Arabidopsis inflorescence stem that associates with precocious upregulation of a SCW transcriptional cascade controlled by NAC TFs (NSTs). We demonstrate that cytokinin signaling through the AHK2/3 and the ARR1/10/12 suppresses the expression of several NSTs and SCW formation in the apical portions of stems. Exogenous cytokinin application reconstituted both proper development and apical-basal gradient of NST1 and NST3 in a cytokinin biosynthesis-deficient mutant. We show that AHK2 and AHK3 required functional NST1 or NST3 to control SCW initiation in the interfascicular fibers, further evidencing that cytokinins act upstream of NSTs transcription factors. The premature onset of a rigid SCW biosynthesis and altered expression of NST1/3 and VND6/7 due to cytokinin deficiency led to the formation of smaller tracheary elements (TEs) and impaired hydraulic conductivity. We conclude that cytokinins downregulate NSTs to inhibit premature SCW formation in the apical part of the inflorescence stem, facilitating thus the development of fully functional TEs and interfascicular fibers. Summary statementCytokinins attenuate premature secondary cell wall (SCW) formation via downregulating the expression of NAC TFs, the master switches of SCW transcriptional cascade, thus affecting the tracheary elements size and conductivity.

plant biology↗

bioRxiv2021

Differences between apoplastic and cytosolic reactive oxygen species production in Arabidopsis during pattern-triggered immunity

Despite an ever-increasing interest in reactive oxygen species (ROS) signalling during plant-microbe interactions, very little information exists, mainly for technical reasons, on the molecular mechanisms regulating intracellular hydrogen peroxide (H2O2) signalling during PAMP-triggered immunity. Here, we used a sensitive fluorimetry method and the H2O2 sensor roGFP2-Orp1, which revealed unsuspected features on the regulation of cytoplasmic H2O2 and thiol redox dynamics upon pathogen-associated molecular patterns (PAMPs) perception by Arabidopsis thaliana. Extended PAMP-induced cytosolic roGFP2-Orp1 oxidation was distinct from the transient oxidative burst in the apoplast measured by luminol oxidation. Pharmacological and genetic analyses indicate that the prolonged PAMP-induced H2O2 increase in the cytoplasm was largely independent on NADPH oxidases and apoplastic peroxidases. By contrast, the NADPH oxidase mutant rbohF was hyper-sensitive to roGFP2-Orp1 oxidation by H2O2 and PAMP indicating a lower antioxidant capacity. Unlike previous reports, the rbohF mutant, but not rbohD, was impaired in PAMP-triggered stomatal closure and ROS production measured by a fluorescein-based probe in guard cells resulting in defects in stomatal defences against bacteria. However, stomatal closure was not correlated with an increase in roGFP2-Orp1 oxidation in guard cells. Interestingly, RBOHF also participated in PAMP-induced apoplastic alkalinisation. Altogether, our results provide novel insights on the interplay between apoplastic and cytosolic ROS dynamics and highlight the importance of RBOHF in plant immunity. Significance statementPlants mount defence responses to pathogens by detecting pathogen-associated molecular patterns (PAMPs). One response is a rapid and transient burst of reactive oxygen species (ROS, e.g. superoxide and hydrogen peroxide) in the cell wall (apoplast) produced by NADPH oxidases and cell wall peroxidases. Using a genetically-encoded hydrogen peroxide sensor roGFP2-Orp1, we found that, in contrast to the transient apoplastic ROS burst, there is also prolonged hydrogen peroxide production in the cytosol upon PAMP perception which is independent of NADPH oxidase and cell wall peroxidases. Our results suggest that apoplastic ROS rather than intracellular hydrogen peroxide is a signal triggering stomatal closure during PAMP-triggered immunity. Additionally, we re-address the relative contribution of the NADPH oxidases D and F in stomatal immunity.

plant biology↗

bioRxiv2020

Organization and control of the ascorbate biosynthesis pathway in plants

The enzymatic steps involved in O_SCPLOWLC_SCPLOW-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff-Wheeler, SW pathway) has been well established and here we comprehensively analyze the subcellular localization, potential physical interactions of SW pathway enzymes and assess their role in control of ascorbate synthesis. Transient expression of GFP-fusions in Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana) mutants complemented with genomic constructs showed that while GME is cytosolic, VTC1, VTC2, VTC4, and O_SCPLOWLC_SCPLOW-GalDH have cytosolic and nuclear localization. While transgenic lines GME-GFP, VTC4-GFP and O_SCPLOWLC_SCPLOW-GalDH-GFP driven by their endogenous promoters accumulated the fusion proteins, the functional VTC2-GFP protein is detected at low level using immunoblot in a complemented vtc2 null mutant. This low amount of VTC2 protein and the extensive analyses using multiple combinations of SW enzymes in N. benthamiana supported the role of VTC2 as the main control point of the pathway on ascorbate biosynthesis. Interaction analysis of SW enzymes using yeast two hybrid did not detect the formation of heterodimers, although VTC1, GME and VTC4 formed homodimers. Further coimmunoprecipitation (CoIP) analysis indicted that consecutive SW enzymes, as well as the first and last enzymes (VTC1 and O_SCPLOWLC_SCPLOW-GalDH), associate thereby adding a new layer of complexity to ascorbate biosynthesis. Finally, metabolic control analysis incorporating known kinetic characteristics, showed that previously reported feedback repression at the VTC2 step confers a high flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration. One sentence summaryMetabolic engineering, genetic analysis and functional mutant complementation identify GDP-O_SCPLOWLC_SCPLOW-galactose phosphorylase as the main control point in ascorbate biosynthesis in green tissues.

molecular biology↗