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The small gaseous molecule nitric oxide (NO) is well established as a major ubiquitouscomponent of cell signalling. A key signalling mechanism mediating NO effects is Snitrosation, a post-translational modification by which NO can impact the target proteinactivities, subcellular localizations, and capacities to form protein complexes. The identificationof proteins targeted by NO is of major interest in order to elucidate NO functions. Interestingly,land plants lack NO synthase (NOS), which is the main enzyme for NO synthesis in metazoans,while a few algal species possess it, thus raising many interrogations. Therefore, we focused onthe identification of S-nitrosated proteins during salt stress in Klebsormidium nitens, afreshwater algal species possessing a NOS and established as a model to study plants adaptationto land. We applied the Biotin Switch method followed by mass spectrometry analysis. Thismethod allows the purification of S-nitrosated proteins. We found 43 proteins with significantlyhigher S-nitrosation levels in salt response condition. Orthology analysis were performedagainst the model plant Arabidopsis thaliana, in order to determine the potential function ofthese proteins. Among them, we selected an interesting protein called INOSITOLPOLYPHOSPHATE MULTIKINASE 2 (IPK2), potentially involved in cell signaling andstress response. We produced it successfully in Escherichia coli and measured its activity incollaboration with the Leibniz-Forschungsinstitut für Molekulare Pharmakologie. The Snitrosation of IPK2 was proved to occur on Cys125. We generated 2 mutants, IPK2(C125A)and IPK2(C125S), and will observe the conservation of their activity before proceeding to themeasurement after S-nitrosation of the wild type and mutated proteins. Structure analysis werealso caried in silico with alphafold, giving insights of potential NO effects. The identificationand functional analysis of S-nitrosated proteins in K. nitens, this project will provide a betterunderstanding of the functions of NO in unicellular green algae with NOS.

In the actual situation of decreasing the use of chemicals in agriculture, enhancing our understanding of plant immunity is a critical task to develop more sustainable plant health protection methods. An interesting strategy is to study how plants, and in particular grapevine, perceives and responds to different microorganisms. Microorganisms are notably recognizedby LysM Receptor-like Kinase (LYKs) and previous works have identified 16 LYKs encoded by the grapevine genome (VvLYKs) (Roudaire et al. 2023). Among them, VvLYK1-1, VvLYK1-2 and VvLYK5-1 are involved in chitin perception and thus play a role in the plant immunity. Interestingly, immune responses are also involved during symbiotic interaction but the receptors involved in this process are still unknown. In addition, it has been demonstrated that the Unfolded Protein Response (UPR), which is activated when unfolded proteins accumulate in the endoplasmic reticulum, is also involved during the plant immune response.It’s therefore important to improve our knowledge about the role of the different grapevine LYK receptors involved in the balance between immunity and symbiosis and in addition to determine the role of stress-related signalling of endoplasmic reticulum and the consequent activation of the UPR in these two different pathways.In this context, we aim to characterize new VvLYKs involved in the perception of Myc-factors which allow the down-regulation of the plant immunity during the establishment of mycorrhizal symbiosis. In addition we also intend to characterize the UPR signalling pathways in grapevinewhich is still unknown and finally how

The small gaseous molecule nitric oxide (NO) is well established as a major ubiquitous component of cell signalling. A key signalling mechanism mediating NO effects is S-nitrosation, a post-translational modification by which NO can impact the target protein activities, subcellular localizations and capacities to form protein complexes. The identification of proteins targeted by NO is of major interest in order to elucidate NO functions. Interestingly, land plants lack NO synthase (NOS), which is the main enzyme for NO synthesis in metazoans, while a few algal species possess it, thus raising many interrogations. Therefore, we focused on the identification of S-nitrosated proteins during salt stress in Klebsormidium nitens, a freshwater algal species possessing a NOS and established as a model to study plants adaptation to land. We applied the Biotin Switch method followed by mass spectrometry analysis. This method allows the purification of S-nitrosated proteins. We found 43 proteins with significantly higher S-nitrosation levels in salt response condition. Orthology analysis were performed against the model plant Arabidopsis thaliana, in order to determine the potential function of these proteins. Among them, three were selected for being potentially involved in cell signaling. We aim to produce them in bacteria, in order to measure their activity and observe the impact of the S-nitrosation on it.

The small gaseous molecule nitric oxide (NO) is well established as a major ubiquitous component of cell signalling. A key signalling mechanism mediating NO effects is S-nitrosation, a post-translational modification by which NO can impact the target protein activities, subcellular localizations, and capacities to form protein complexes. The identification of proteins targeted by NO is of major interest in order to elucidate NO functions. Interestingly, land plants lack NO synthase (NOS), which is the main enzyme for NO synthesis in metazoans, while a few algal species possess it, thus raising many interrogations. Therefore, we focused on the identification of S-nitrosated proteins during salt stress in Klebsormidium nitens, a freshwater algal species possessing a NOS and established as a model to study plants adaptation to land. We applied the Biotin Switch method followed by mass spectrometry analysis. This method allows the purification of S-nitrosated proteins. We found 43 proteins with significantly higher S-nitrosation levels in salt response condition. Orthology analysis were performed against the model plant Arabidopsis thaliana, therefore we established a classification of these proteins according to their localization, molecular function and biological process they are involved in. This is done with the PANTHER classification system, based on Gene Ontology terms (GO terms). Then, from this list, we selected an interesting protein called INOSITOL POLYPHOSPHATE MULTIKINASE 2a/b (IPK2a/b), potentially involved in cell signaling and stress response. We produced it in Escherichia coli and are currently measuring its activity. This protein will then be Snitrosated in vitro and we will determine if its activity is impaired. Through the identification and functional analysis of Snitrosated proteins in K. nitens, this project will provide a better understanding of the functions of NO in unicellular green algae with NOS

Objectives: Nitric oxide (NO) is an important cellular signaling molecule regulating various physiologicalprocesses, in both animals and plants. In animals, NO synthesis is mainly catalyzed by NO synthase(NOS) enzymes. In plants, NOS-like activities sensitive to mammalian NOS inhibitors have beenmeasured, although no sequences encoding mammalian NOSs have been found in land plants.Interestingly, we identified NOS-like sequences in 20 algae species. These latter include thefilamentous charophyte green algae Klebsormidium nitens, a biological model to study the earlytransition step from aquatic algae to land plants. In order to understand the mechanisms governingNO synthesis and signaling in green lineage we initiated the functional characterization of K. nitensNOSs (KnNOS) by analyzing their primary sequences as well as their expression levels in response toabiotic stresses.Methods: KnNOSs nucleotide sequences were verified by RACE-PCR and sequencing, and their mRNAlevel were monitored by RT-qPCR and protein abundance by western blot. Protein partners werestudied, firstly in sillico using the BioGrid database and human NOS interaction data, and secondly invivo by immunoprecipitation experiments followed by mass spectrometry analysis.Results: Currently, two NOSs were identified in K. nitens genome: the KnNOS1 which possessesclassical mammalian NOS architecture consisting of oxygenase and reductase domains with somespecificities as lack of conserved residues in binding domain of BH4 cofactors; and the KnNOS2displaying a large C-ter extension containing an ANK motif and a globin domain. The two KnNOSs seemto be regulated in different ways. KnNOS1 exhibited constitutive expression during the conditionstested, whereas KnNOS2 appeared to be transcriptionally regulated during stress. In parallel studies,we also built the in silico protein–protein interaction network of human NOSs. Interestingly, genesencoding orthologs of several of these candidates were found in K. nitens genome. Some of theseconserved partners are known to be involved in mammalian NOSs regulation and represent interestingcandidates for further investigation.Conclusions: Overall these findings open the way for a deeper characterization of KnNOSs and itsprotein partners and will facilitate further investigation of NO signaling in green lineage.Relevant references:Chatelain, P., Astier, J., Wendehenne, D., Rosnoblet, C., and Jeandroz, S. (2021). Identification of Partner Proteinsof the Algae Klebsormidium nitens NO Synthases: Toward a Better Understanding of NO Signaling in EukaryoticPhotosynthetic Organisms. Front. Plant Sci. 12, 3068. doi: 10.3389/fpls.2021.797451.Jeandroz, S., Wipf, D., Stuehr, D. J., Lamattina, L., Melkonian, M., Tian, Z., et al. (2016). Occurrence, structure,and evolution of nitric oxide synthase–like proteins in the plant kingdom. Sci. Signal. 9, re2–re2. doi:10.1126/scisignal.aad4403.

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National audience; Nitric oxide (NO) is an important cellular signaling molecule across kingdoms. During bioticor abiotic stresses, NO burst is detected in both plants and mammals although no sequenceencoding the well described mammalian NO synthase (NOS) is highlighted in plants. Comparedto terrestrial plants, some algae present transcripts encoding the NOS-like enzyme. Amongthem, Klebsormidium nitens the model alga to study the early transition step from aquatic algaeto land plants is found. As mechanisms governing NO synthesis and signaling in green lineageremain unclear, the study of NOS from K. nitens (KnNOS) through (i) the identification ofregulator proteins, (ii) the identification of target proteins, and (iii) their expression levels mightbe an important breakthrough knowledge. To achieve this goal, the identification of proteinpartners will be performed by pull down experiments followed by mass spectrometry analysis.Despite the KnNOS is a challenging protein to produce and purify, we are currently optimizinga promising protocol to obtain recombinant GST-tagged KnNOS protein in Escherichia coli.Since expression level analysis require suitable reference genes for reliable normalization, weselected 13 candidate reference genes based on literature, and performed RT-qPCR to studyexpression stability during 4 abiotic stresses commonly used. Final ranking list of most stablereference genes was obtain based on results from algorithms including, the ΔCt method,geNorm, NormFinder and BestKeeper. This finding opens the way for a deeper characterizationof KnNOS and its protein partners and will facilitate further investigation of gene expressionin K. nitens.

Studies performed in our Agroecology Department show that the immune response of plants is linked to their iron nutrition and is modulated by pyoverdine, a siderophore produced by the plant beneficial rhizobacteria Pseudomonas fluorescens C7R12. Accordingly, Arabidopsis thaliana plantlets exposed to iron deficiency and treated with pyoverdine in its iron non‐chelated structure (apo‐pyo) show an enhanced growth but a decreased immune response capacity. We hypothesize that nitric oxide (NO), a universal signaling molecule, is a key component of the regulation of the immune response in plants exposed to apo‐pyo and to the C7R12 strain. We checked by fluorescence microscopy that NO is actually produced in response to iron deficiency in presence or in absence of apo-pyo. To verify a role of NO in these processes, three tasks will be developed: ‐ identification of S‐nitrosylated proteins in response to apo‐pyo and P. fluorescens C7R12. S-nitrosylation is a major NO‐dependent posttranslational protein modification which targets cysteine residues (Cys). ‐ analysis of the impact of NO on the structure and function of the selected proteins. The influence of NO on the structure and function of the proteins will be investigated in vitro and the Cys residues regulated by NO will be identified by directed mutagenesis. ‐ study of the incidence of the S‐nitrosylation of the proteins of interest in planta. A. thaliana plants impaired in the expression of the proteins of interest or expressing them in a non‐nitrosylable structure will be generated and their interaction with pathogenic or beneficial micro‐organisms will be analyzed.

The existence of a tightly regulated balance between growth and immunity in plants has recently emerged. In this study, we challenged this concept thanks to the biological model pyoverdine-Arabidopsis thaliana. Pyoverdine is a siderophore produced by the plant growth promoting rhizobacteria Pseudomonas fluorescens C7R12. Under iron deficiency, P. fluorescens excretes the iron free form of pyoverdine (apo‐pyo) in the soil. Once chelated with iron (ferri‐pyo), the complex is internalized by the bacteria. We demonstrated that Arabidopsis thaliana plants treated by apo‐pyo in a medium containing or not iron internalize pyoverdine. Interestingly, apo‐pyo-treated plants did not show a typical growth reduction induced by iron deficiency. Accordingly, the expression of genes related to growth, development and iron uptake/transport was induced, these latter being involved in the growth improvement induced by apo‐pyo under iron deficiency. In contrast, a strong down-regulation of the expression of genes related to immunity was observed. Furthermore, the resistance to the fungal pathogen Botrytis cinerea conferred by iron deficiency was partially impaired following apo‐pyo treatment. The overexpression of the HBI1 transcription factor, known to be involved in the growth‐immunity tradeoff, was linked to the above observations. These apo‐pyo effects were not observed after treatment of plants under iron sufficient conditions, indicating that apo‐pyo effects are dependent on the plant iron status. To conclude, this work draws first elements of pyoverdine effects on plant physiology. In a larger view, this work supports the recent concept of the existence of a cross‐regulation between growth, immunity and iron homeostasis in plants.

Stilbenes, especially resveratrol and its derivatives, have become famous for their positive effects on a wide range of medical disorders, as indicated by a huge number of published studies. A less investigated area of research is their antimicrobial properties. A series of 13 trans-resveratrol analogues was synthesized via Wittig or Heck reactions, and their antimicrobial activity assessed on two different grapevine pathogens responsible for severe diseases in the vineyard. The entire series, together with resveratrol, was first evaluated on the zoospore mobility and sporulation level of Plasmopara viticola (the oomycete responsible for downy mildew). Stilbenes displayed a spectrum of activity ranging from low to high. Six of them, including the most active ones, were subsequently tested on the development of Botrytis cinerea (fungus responsible for grey mold). The results obtained allowed us to identify the most active stilbenes against both grapevine pathogens, to compare the antimicrobial activity of the evaluated series of stilbenes, and to discuss the relationship between their chemical structure (number and position of methoxy and hydroxy groups) and antimicrobial activity.

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