Sami Kazaz
email: sami.kazaz@inrae.fr

Romane Miray
email: romane.miray@inrae.fr

Sébastien Baud
email: sebastien.baud@inrae.fr

Acyl-Acyl Carrier Protein Desaturases and Plant Biotic Interactions

Keywords: acyl-acyl carrier protein desaturase, fatty acid, monounsaturated, plant, biotic interactions

Interactions between land plants and other organisms such as pathogens, pollinators, or symbionts usually involve a variety of specialized effectors participating in complex cross-talks between organisms. Fatty acids and their lipid derivatives play important roles in these biological interactions. While the transcriptional regulation of genes encoding acyl-acyl carrier protein (ACP) desaturases appears to be largely responsive to biotic stress, the different monounsaturated fatty acids produced by these enzymes were shown to take active part in plant biotic interactions and were assigned with specific functions intrinsically linked to the position of the carbon-carbon double bond within their acyl chain. For example, oleic acid, an omega-9 monounsaturated fatty acid produced by ∆ 9 -stearoyl-ACP desaturases, participates in signal transduction pathways affecting plant immunity against pathogen infection. Myristoleic acid, an omega-5 monounsaturated fatty acid produced by ∆ 9 -myristoyl-ACP desaturases, serves as a precursor for the biosynthesis of omega-5 anacardic acids that are active biocides against pests. Finally, different types of monounsaturated fatty acids synthesized in the labellum of orchids are used for the production of a variety of alkenes participating in the chemistry of sexual deception, hence favoring plant pollination by hymenopterans.

Introduction

In plant cells, fatty acids produced in the plastids are used for the biosynthesis of a variety of lipid compounds. Acyl lipids derived from fatty acids are basic structural components of the cellular membranes [START_REF] Mamode Cassim | Plant lipids: Key players of plasma membrane organization and function[END_REF]. Triacylglycerols represent an important carbon and energy store in many oleaginous species [START_REF] Baud | Seeds as oil factories[END_REF][START_REF] Miray | Molecular control of oil metabolism in the endosperm of seeds[END_REF]. Cuticular lipids such as cutin, suberin, and cuticular waxes constitute lipophilic cell-wall associated barriers [START_REF] Kunst | Plant cuticles shine: Advances in wax biosynthesis and export[END_REF]. Aside from this, some fatty acids, acyl lipids, and their derivatives participate in signal transduction pathways that influence plant development and responses to environmental cues [START_REF] Nandi | Arabidopsis sfd mutants affect plastidic lipid composition and suppress dwarfing, cell death, and the enhanced disease resistance phenotypes resulting from the deficiency of a fatty acid desaturase[END_REF][START_REF] Wasternack | Jasmonates: An update on biosynthesis, signal transduction and action in plant stress response, growth and development[END_REF]. These various categories of lipids play fundamental roles in biotic interactions. First, cuticular lipid layers, together with cell walls, can serve as structural obstruction and limit the entry of pathogenic microorganisms. Changes in cuticular permeability have thus been associated with altered plant responses to microbes [START_REF] Bessire | A permeable cuticle in Arabidopsis leads to a strong resistance to Botrytis cinerea[END_REF][START_REF] Xia | The glabra1 mutation affects cuticle formation and plant responses to microbes[END_REF][START_REF] Xia | Acyl CoA binding proteins are required for cuticle formation and plant responses to microbes[END_REF]. Early in plant-pathogen interactions, plants recognize specific molecular signatures in microbial cells called pathogenassociated molecular patterns (PAMPs) via cell surface-localized pattern recognition receptors. This recognition results in activation of PAMP-triggered immunity (PTI), a form of basal resistance minimizing or preventing microbial colonization [START_REF] Singh | Transport of chemical signals in systemic acquired resistance[END_REF]. Before and concurrent with the onset of PTI, changes in membrane lipid composition and adjustment of membrane fluidity largely mediated by desaturases appear to be critical for the function of integral membrane proteins that participate in this response [START_REF] Upchurch | Fatty acid unsaturation, mobilization, and regulation in the response of plants to stress[END_REF][START_REF] Walley | Fatty acids and early detection of pathogens[END_REF]. Virulent pathogens can overcome PTI by deploying virulence effectors that interfere with components of the PTI transduction pathway. In response, plants have another induced defense response called effector-triggered immunity (ETI), which is induced upon recognition of pathogenencoded avirulence factors by the products of plant resistance (R) genes. ETI leads to the activation of a signal transduction pathway resulting in the hypersensitive response (HR) and systemic acquired resistance (SAR), two important components of a plant's defense arsenal against pathogens [START_REF] Lim | Fatty acid-and lipid-mediated signaling in plant defense[END_REF]. The HR is a rapid response characterized by the production of reactive oxygen species (ROS) referred to as the oxidative burst, which prevents further spread of the pathogen, and by localized programmed cell death [START_REF] Yaeno | Role of chloroplast trienoic fatty acids in plant disease defense responses[END_REF]. Concurrent with the HR, a systemic signal inducing long-lasting SAR in uninfected plant tissues is released. ETI shares many signaling components with PTI, but results in much stronger and more durable resistance. Enzymatic and non-enzymatic genesis of bioactive lipid mediators such as oxylipins, jasmonic acid, or azelaic acid, among others, play a key role in the deployment of plant immune responses leading to genome-wide massive transcriptional reprogramming [START_REF] Singh | Transport of chemical signals in systemic acquired resistance[END_REF]. As for storage lipids, the fatty acid composition of oilseeds is regarded as a component of pathogen susceptibility and seed colonization in some species. For example, colonization of Glycine max (soybean) seeds by Cercospora kikuchii has been found to be correlated with the ω-9 18:1 (oleic acid):18:2 (linoleic acid) ratio and mid-oleic genotypes are more extensively colonized by the fungal pathogen in the field [START_REF] Xue | Relationships between oleic and linoleic acid content and seed colonization by Cercospora kikuchii and Diaporthe phaseolorum[END_REF].

Biosynthesis of fatty acids and their derivatives therefore play a pivotal role in plant biotic interactions, and the importance of unsaturated fatty acids is increasingly documented [START_REF] Walley | Fatty acids and early detection of pathogens[END_REF]. This review aims to summarize how plant monounsaturated fatty acids and their biosynthesis by acyl-acyl carrier protein (ACP) desaturases (AADs) contribute to plant defenses against pathogens and also participate in various plant-insect interactions. Stimulating discoveries in this research field have improved the basics of our understanding of plant-pathogen interactions. They have also impacted the improvement of agronomic varieties with respect to stress resistance. For example, ω-7 16:1 (palmitoleic acid) has been shown to directly inhibit the growth of the fungus Verticillium dahlia. Transgenic Solanum Melongena (eggplant) lines expressing the yeast ∆ 9 desaturase-coding gene ELO1 and consequently accumulating increased levels of ω-7 16:1 display improved resistance to the fungus [START_REF] Xing | Modification of fatty acids in eggplant affects its resistance to Verticillium dahlia[END_REF]. Similarly, expression of the yeast ELO1 gene in Solanum esculentum (tomato) improves resistance to Erysiphe polygoni (powdery mildew) [START_REF] Wang | Changes of fatty acids and fatty acid-derived flavor compounds by expressing the yeast ∆-9 desaturase gene in tomato enhances its resistance to powdery mildew[END_REF].

Synthesis of Monounsaturated Fatty Acids by Acyl-Acyl Carrier Protein (ACP) Desaturases

Plant de novo fatty acid synthesis occurs in plastids [START_REF] Harwood | Recent advances in the biosynthesis of plant fatty acids[END_REF][START_REF] Troncoso-Ponce | New insights on the organization and regulation of the fatty acid biosynthetic network in the model higher plant Arabidopsis thaliana[END_REF]. The first committed step for this pathway consists in the ATP-dependent formation of malonyl-CoA from acetyl-CoA and bicarbonate through the action of acetyl-CoA carboxylase [START_REF] Turnham | Changes in the activity of acetyl-CoA carboxylase during rape-seed formation[END_REF]. The malonyl group of malonyl-CoA is then transferred to a protein cofactor named ACP by a malonyl-CoA:ACP S-malonyltransferase. The malonyl-ACP thus formed provides two-carbon units for each cycle of the fatty acid biosynthetic process catalyzed by a type II fatty acid synthase, which uses acetyl-CoA as a starting unit and malonyl-ACP as the elongator [START_REF] Brown | Tandem affinity purification tagging of fatty acid biosynthetic enzymes in Synechocystis sp PCC6803 and Arabidopsis thaliana[END_REF]. The fatty acid synthase complex associates four monofunctional enzymes: a 3-ketoacyl-ACP synthase catalyzing a condensation reaction, a 3-ketoacyl-ACP reductase catalyzing a first reduction step, a hydroxyacyl-ACP dehydratase catalyzing a dehydration reaction, and an enoyl-ACP reductase catalyzing a second reduction step yielding saturated acyl chains [START_REF] Shimakata | Purification and characterization of β-ketoacyl-[acyl-carrier-protein] reductase, β-hydroxyacyl-[acylcarrier-protein] dehydrase, and enoyl-[acyl-carrier-protein] reductase from Spinacia oleracea leaves[END_REF][START_REF] Jaworski | A cerulenin insensitive short chain 3-ketoacyl-acyl carrier protein synthase in Spinacia oleracea leaves[END_REF][START_REF] Ohlrogge | Lipid biosynthesis[END_REF]. A fraction of these saturated acyl chains is hydrolyzed by acyl-ACP thioesterases and directly used for the elaboration of different classes of lipids inside and outside the plastids [START_REF] Salas | Characterization of substrate specificity of plant FatA and FatB acyl-ACP thioesterases[END_REF]. However, stromal AADs efficiently introduce carbon-carbon double bonds (also called unsaturations) within these saturated acyl chains to form cis-monounsaturated fatty acyl chains before their hydrolysis by thioesterases [START_REF] He | Plant unsaturated fatty acids: Biosynthesis and regulation[END_REF].

∆ 9 -Stearoyl-ACP desaturases represent the predominant AAD isoforms in most land plants. They efficiently desaturate 18:0 (stearic acid) to form ω-9 18:1, a major monounsaturated fatty acid of membrane and storage lipids [START_REF] Kachroo | The Arabidopsis stearoyl-acyl carrier proteindesaturase family and the contribution of leaf isoforms to oleic acid synthesis[END_REF]. Once esterified to a glycerol backbone to form membrane lipids, oleic acid can be further desaturated by membranebound fatty acid desaturases (FADs), yielding polyunsaturated fatty acids like 18:2 and 18:3 (alpha-linolenic acid). While archetypal ∆ 9 -stearoyl-ACP desaturases can be found in every land plant, plant genomes usually code multiple AAD isoforms. Some of these isoforms can exhibit different substrate specificities or regioselectivities associated with the biosynthesis of unusual monounsaturated fatty acids. ∆ 9 -palmitoyl-ACP desaturases identified in several plant species prefer 16:0 (palmitic acid) instead of 18:0 as a substrate, resulting in the production of ω-7 16:1 [START_REF] Cahoon | A determinant of substrate specificity predicted from the acyl-acyl carrier protein desaturase of developing cat's claw seed[END_REF][START_REF] Bryant | ACYL-ACYL CARRIER PROTEIN DESATURASE2 and 3 are responsible for making omega-7 fatty acids in the Arabidopsis aleurone[END_REF][START_REF] Troncoso-Ponce | Transcriptional activation of two delta-9 palmitoyl-ACP desaturase genes by MYB115 and MYB118 is critical for biosynthesis of omega-7 monounsaturated fatty acids in the endosperm of Arabidopsis seeds[END_REF]. The ∆ 4 -and ∆ 6 -palmitoyl-ACP desaturases respectively identified in Coriandrum sativum (coriander) and Thunbergia alata (black-eyed Susan vine) also use 16:0 as a preferential substrate, but differ in their regiospecificity, leading to the production of ω-12 16:1 (∆ 4 hexadecanoic acid) and ω-10 16:1 (∆ 6 hexadecenoic acid) [START_REF] Cahoon | ∆6 Hexadecenoic acid is synthesized by the activity of a soluble ∆6 palmitoyl-acyl carrier protein desaturase in Thunbergia alata endosperm[END_REF][START_REF] Cahoon | Metabolic evidence for the involvement of a ∆4-palmitoyl-acyl carrier protein desaturase in petroselinic acid synthesis in coriander endosperm and transgenic tobacco cells[END_REF].

AADs perform dioxygen-dependent dehydrogenation reactions resulting in the introduction of double bonds into fatty acyl chains [START_REF] Thompson | Primary structures of the precursor and mature forms of stearoyl-acyl carrier protein desaturase from safflower embryos and requirement of ferredoxin for enzyme activity[END_REF][START_REF] Shanklin | Stearoyl-acyl-carrier-protein desaturase from higher plants is structurally unrelated to the animal and fungal homologs[END_REF][START_REF] Shanklin | Emerging models for understanding functional diversification of diiron-containing enzymes[END_REF]. These reactions are initiated by the energy-demanding abstraction of a hydrogen from a methylene group with the use of an active-site diiron cluster recruiting and activating molecular oxygen [START_REF] Fox | Stearoyl-acyl carrier protein ∆9 desaturase from Ricinus communis is a diiron-oxo protein[END_REF]. The reducing equivalents needed for the reaction in the form of NADPH are transferred from ferredoxin reductase to ferredoxin, and then to AAD [START_REF] Fox | Reactions of the diiron enzyme stearoyl-acyl carrier protein desaturase[END_REF]. Crystal structures of Ricinus communis (castor) ∆ 9 -stearoyl-ACP desaturase [START_REF] Lindqvist | Crystal structure of delta9 stearoyl-acyl carrier protein desaturase from castor seed and its relationship to other di-iron proteins[END_REF] and Hedera helix (English ivy) ∆ 4 -palmitoyl-ACP desaturase [START_REF] Guy | The crystal structure of the ivy ∆4-16:0-ACP desaturase reveals structural details of the oxidized active site and potential determinants of regioselectivity[END_REF] showed the AADs to be homodimeric proteins. Each monomer is folded into a compact single domain. The diiron active site is buried within a conserved four-helix bundle at the core of the monomer. This active site is positioned alongside a deep, narrow hydrophobic cavity [START_REF] Shanklin | Emerging models for understanding functional diversification of diiron-containing enzymes[END_REF]. The methyl end of a saturated fatty acyl chain bound to ACP can enter this hydrophobic cavity and, once this substrate is accommodated, a cis double bond is created between two adjacent carbon atoms of the acyl chain facing the active site, concomitantly with the reduction of a dioxygen molecule into water [START_REF] Shanklin | Desaturation and related modifications of fatty acids[END_REF]. The boomeranglike shape of the hydrophobic channel imparts an eclipsed substrate conformation, leading to the formation of a cis double bond and explains the mechanism of stereoselectivity [START_REF] Behrouzian | Exploring the hydroxylation-dehydrogenation connection: Novel catalytic activity of castor stearoyl-ACP ∆ 9 desaturase[END_REF]. The structural basis for the different chain-length and double bond positional specificities of AADs were identified through the characterization of desaturases exhibiting different functional properties despite sharing high amino acid sequence similarity [START_REF] Cahoon | Characterization of a structurally and functionally diverged acyl-acyl carrier protein desaturase from milkweed seed[END_REF]. In this respect, approaches of site-directed mutagenesis aimed at converting the activity of one type of AAD to that of another by replacing specific residues have been proven to be very informative [START_REF] Cahoon | Redesign of soluble fatty acid desaturases from plants for altered substrate specificity and double bond position[END_REF]. First, the conversion of a ∆ 9 -stearoyl-ACP desaturase into a ∆ 9 -palmitoyl-ACP desaturase was made possible by substituting amino acid residues lining the bottom part of the substrate pocket. A group of eight residues was shown to set constraints on the chain lengths of fatty acid substrates, thus determining the substrate specificity of the enzymes [START_REF] Cahoon | A determinant of substrate specificity predicted from the acyl-acyl carrier protein desaturase of developing cat's claw seed[END_REF][START_REF] Troncoso-Ponce | Transcriptional activation of two delta-9 palmitoyl-ACP desaturase genes by MYB115 and MYB118 is critical for biosynthesis of omega-7 monounsaturated fatty acids in the endosperm of Arabidopsis seeds[END_REF][START_REF] Cahoon | Redesign of soluble fatty acid desaturases from plants for altered substrate specificity and double bond position[END_REF]. Then, determination of crystal structures of AADs in complex with acyl-ACPs identified residues at the entrance of the substrate-binding cavity that are determinants for the binding modes of ACP with respect to the desaturase, predisposing the potential insertion depth of the acyl chain and thereby influencing regioselectivity [START_REF] Guy | Remote control of regioselectivity in acyl-acyl carrier protein-desaturase[END_REF].

Transcriptional Responses of AAD Genes to Biotic Stress in Arabidopsis Leaves

Twenty years ago, identification by forward-genetic approaches of a first AAD-coding gene in A. thaliana [START_REF] Kachroo | A fatty acid desaturase modulates the activation of defense signaling pathways in plants[END_REF] coincided with the release of the complete genome sequence of the model species [START_REF] Genome | Analysis of the genome sequence of the flowering plant Arabidopsis thaliana[END_REF]. It appeared that FATTY ACID BIOSYNTHESIS2 (FAB2)/SUPPRESSOR OF SALICYLIC ACID INSENSITIVE2 (SSI2) was part of larger multigene family comprising seven members (Figure 1A) [START_REF] Kachroo | The Arabidopsis stearoyl-acyl carrier proteindesaturase family and the contribution of leaf isoforms to oleic acid synthesis[END_REF]. FAB2, AAD1, AAD5, and AAD6 encode ∆ 9 -stearoyl-ACP desaturases [START_REF] Kachroo | The Arabidopsis stearoyl-acyl carrier proteindesaturase family and the contribution of leaf isoforms to oleic acid synthesis[END_REF][START_REF] Cao | Heterologous expression of stearoyl-acyl carrier protein desaturase (S-ACP-DES) from Arabidopsis thaliana in Escherichia coli[END_REF][START_REF] Klinkenberg | Two fatty acid desaturases, STEAROYL-ACYL CARRIER PROTEIN ∆9-DESATURASE6 and FATTY ACID DESATURASE3, are involved in drought and hypoxia stress signaling in Arabidopsis crown galls[END_REF][START_REF] Kazaz | Differential activation of partially redundant ∆9 stearoyl-ACP desaturase genes is critical for omega-9 monounsaturated fatty acid biosynthesis during seed development in Arabidopsis[END_REF] while AAD2 and AAD3 are divergent ∆ 9 -palmitoyl-ACP desaturase isoforms [START_REF] Bryant | ACYL-ACYL CARRIER PROTEIN DESATURASE2 and 3 are responsible for making omega-7 fatty acids in the Arabidopsis aleurone[END_REF][START_REF] Troncoso-Ponce | Transcriptional activation of two delta-9 palmitoyl-ACP desaturase genes by MYB115 and MYB118 is critical for biosynthesis of omega-7 monounsaturated fatty acids in the endosperm of Arabidopsis seeds[END_REF]. To date, the enzymatic function of AAD4 remains unclear [START_REF] Kachroo | The Arabidopsis stearoyl-acyl carrier proteindesaturase family and the contribution of leaf isoforms to oleic acid synthesis[END_REF]. In vegetative organs, FAB2 exhibits the highest expression levels (Figure 1B) [START_REF] Kazaz | Differential activation of partially redundant ∆9 stearoyl-ACP desaturase genes is critical for omega-9 monounsaturated fatty acid biosynthesis during seed development in Arabidopsis[END_REF] and the corresponding enzyme is usually regarded as the main ∆ 9 -stearoyl-ACP desaturase isoform in this species. After the release of the A. thaliana genome sequence, the exploration of microarray data has quickly become a vital aspect of post-genomic research in the field. Large-scale transcriptome analyses indicate that metabolic pathways are frequently influenced by the presence of pathogens [START_REF] Jiang | Large-scale transcriptome analysis reveals arabidopsis metabolic pathways are frequently influenced by different pathogens[END_REF]. The Arabidopsis eFP Browser, which displays microarray data from Affymetrix [START_REF] Winter | Electronic Fluorescent Pictograph" browser for exploring and analyzing large-scale biological data sets[END_REF], allows viewing of the expression levels of approximately 22,000 genes in the leaves of plants exposed to various pathogens such as Phytophthora infestans and oomycete-derived elicitors, Botrytis cinerea, Golovinomyces orontii, Pseudomonas syringae, and bacterial-derived elicitors, among others (Figure 1C). The exploration of the AAD expression profiles among the biotic stress series suggests that expression of several AAD genes respond to stress. Interestingly, while genes encoding ∆ 9 -stearoyl-ACP desaturases isoforms appear to be essentially repressed, an opposite trend was observed for ∆ 9 -palmitoyl-ACP desaturase-coding genes. When performing a large-scale transcriptional data analysis based on expression samples from Gene Expression Omnibus [START_REF] Barrett | NCBI GEO: Archive for functional genomics data sets-Update[END_REF] and ArrayExpress [START_REF] Brazma | ArrayExpress-A public repository for microarray gene expression data at the EBI[END_REF], Jiang and colleagues [START_REF] Jiang | Large-scale transcriptome analysis reveals arabidopsis metabolic pathways are frequently influenced by different pathogens[END_REF] also showed that FAB2 was significantly repressed in A. thaliana leaves exposed to B. cinerea, Blumeria graminis [START_REF] Jensen | Transcriptional regulation by an NAC (NAM-ATAF1,2-CUC2) transcription factor attenuates ABA signalling for efficient basal defence towards Blumeria graminis f. sp. hordei in Arabidopsis[END_REF], Cabbage leaf curl virus (CaLCuV) [START_REF] Ascencio-Ibáñez | Global analysis of Arabidopsis gene expression uncovers a complex array of changes impacting pathogen response and cell cycle during geminivirus infection[END_REF], G. orontii, P. syringae, and Sclerotinia sclerotinium. More recently, Mine and colleagues [START_REF] Mine | The defense phytohormone signaling network enables rapid, high-amplitude transcriptional reprogramming during effector-triggered immunity[END_REF] conducted an RNA-sequencing analysis of plants treated with ETI-triggering avirulent strain of P. syringae to gain insights into the gene regulatory networks controlling transcriptional reprogramming during ETI. A coexpression network analysis was implemented, which allowed clustering coexpressed genes into modules. This approach revealed that FAB2 belongs to a module that gathers genes exhibiting strong and statistically significant transcriptional decrease in early hours after infection, whereas AAD3 belongs to a module enriched for genes associated with immunity-related GO terms with strong transcriptional induction at early hours. Interestingly, using wildtype plants and mutants deficient in components of the signaling network upon challenge with virulent or ETI-triggering avirulent strains of P. syringae, the authors then showed that susceptible plants exhibit almost identical transcriptional responses to resistant plants, although with several hours delay.

The necrotrophic pathogen B. cinerea is endemic throughout the world and causes severe pre-and post-harvest losses in many crops [START_REF] Zhang | Plastic transcriptomes stabilize immunity to pathogen diversity: The jasmonic acid and salicylic acid networks within the Arabidopsis/Botrytis Pathosystem[END_REF]. Using the A. thaliana-B. cinerea pathosystem to investigate how the host's defense system functions against genetic variation in the pathogen, Zhang and colleagues [START_REF] Zhang | Plastic transcriptomes stabilize immunity to pathogen diversity: The jasmonic acid and salicylic acid networks within the Arabidopsis/Botrytis Pathosystem[END_REF] measured defense-related phenotypes and transcriptomic responses in A. thaliana challenged with 96 diverse B. cinerea isolates. This study failed to identify any singular gene or subset of genes highly correlated with resistance and lesion areas, supporting the perspective that host resistance to B. Cinerea is highly polygenic. It should be noted, however, that a negative correlation between FAB2 expression and lesion size was observed [START_REF] Zhang | Plastic transcriptomes stabilize immunity to pathogen diversity: The jasmonic acid and salicylic acid networks within the Arabidopsis/Botrytis Pathosystem[END_REF]. To further characterize this pathosystem and its genetic interactions dominated by complex small-effect loci displaying a high degree of interaction between the host and pathogen, a cotranscriptome study with simultaneous analysis of the host and pathogen's transcripts was recently performed through single-sample RNA-sequencing. In addition, a GWA analysis of both host and pathogen transcriptomes was implemented to identify loci in the B. Cinerea genome that may affect the transcriptomes of either or both organisms [START_REF] Zhang | Plastic transcriptomes stabilize immunity to pathogen diversity: The jasmonic acid and salicylic acid networks within the Arabidopsis/Botrytis Pathosystem[END_REF][START_REF] Zhang | Plant-necrotroph co-transcriptome networks illuminate a metabolic battlefield[END_REF][START_REF] Soltis | Pathogen genetic control of transcriptome variation in the Arabidopsis thaliana-Botrytis cinerea pathosystem[END_REF]. This analysis revealed mostly smalleffect polymorphisms dispersed throughout the pathogen genome, with several B. cinerea trans-eQTL hotspot loci associated with specific host or pathogen transcript coexpression modules and variation in lesion size. These loci exhibit regulatory potential in controlling the plant-pathogen interaction via modulation of gene expression to influence the lesions outcome. Several of these hotspots have an overrepresentation of photosynthesisrelated functions within their targets, in good agreement with the previously characterized downregulation of photosynthesis transcripts described as a hallmark of plant immune processes [START_REF] Bilgin | Biotic stress globally downregulates photosynthesis genes[END_REF]. Interestingly, FAB2 was identified among the genes targeted by the B. cinerea trans-eQTL hotspot 10_2268522 whose nearest gene, Bcin10g05900, encodes a winged helix-turn-helix transcription factor [START_REF] Soltis | Pathogen genetic control of transcriptome variation in the Arabidopsis thaliana-Botrytis cinerea pathosystem[END_REF]. Unfortunately, the molecular mechanisms underpinning the transcriptional control of AAD genes in response to pathogen attacks remain totally unknown. The only transcription factors known to regulate the expression of gene members of the AAD family, WRINKLED1 and MYB115-MYB118, participate in the developmental control of seed metabolism [START_REF] Troncoso-Ponce | Transcriptional activation of two delta-9 palmitoyl-ACP desaturase genes by MYB115 and MYB118 is critical for biosynthesis of omega-7 monounsaturated fatty acids in the endosperm of Arabidopsis seeds[END_REF][START_REF] Kazaz | Differential activation of partially redundant ∆9 stearoyl-ACP desaturase genes is critical for omega-9 monounsaturated fatty acid biosynthesis during seed development in Arabidopsis[END_REF]. So far, these trans-acting factors have never been associated with stress signaling, suggesting the existence of alternative regulatory mechanisms affecting the expression of AAD genes in response to biotic stress.

exhibiting strong and statistically significant transcriptional decrease in early hours after infection, whereas AAD3 belongs to a module enriched for genes associated with immunity-related GO terms with strong transcriptional induction at early hours. Interestingly, using wild-type plants and mutants deficient in components of the signaling network upon challenge with virulent or ETI-triggering avirulent strains of P. syringae, the authors then showed that susceptible plants exhibit almost identical transcriptional responses to resistant plants, although with several hours delay. Expression levels were calculated using the data displayed on the Arabidopsis eFP Browser [START_REF]Arabidopsis eFP Browser[END_REF]. (C) Variations in AAD transcript levels in leaves of A. thaliana in response to various pathogens and elicitors and expressed in fold changes with respect to control. Bold lines between AAD2 and AAD4 reflect the impossibility of distinguishing between AAD2 and AAD4 transcripts using the Affymetrix technology due to the very similar sequences of these transcripts. Flg62, bacterial derived elicitor; GST-NPP1, oomycete-derived elicitor; HrpZ, bacterial derived elicitor.

∆ 9 -Stearoyl-ACP Desaturases and Plant-Pathogen Interactions

In several subtropical fruits, preformed biologically active phytochemicals have been associated with fruit resistance to pathogen attack [START_REF] Rodríguez-Sánchez | Isolation and structure elucidation of avocado seed (Persea americana) lipid derivatives that inhibit Clostridium sporogenes endospore germination[END_REF]. In Persea americana (avocado) fruits, for example, Colletotrichum gloeosporioides initiates its attack by conidium germination and appressorium formation, followed by the penetration of infection hyphae into epidermal cells [START_REF] Yakoby | pH regulation of pectate lyase secretion modulates the attack of Collectotrichum gloeosporioides on avocado fruits[END_REF]. It has been proposed that the presence of preformed antifungal compounds such as (Z,Z)-1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene and (Z,Z,E)-1-acetoxy-2hydroxy-4-oxo-heneicosa-5,12,15-triene in the fruit peel regulates fruit resistance [START_REF] Domergue | Antifungal compounds from idioblast cells isolated from avocado fruits[END_REF]. If the biosynthetic pathway for these compounds has never been fully elucidated, production of ω-9 18:1, followed by its desaturation, is thought to yield the starting compound 18:2, which is further elongated by conventional two-carbon extension (Figure 2). Interestingly, expression of an avocado gene encoding a ∆ 9 -stearoyl-ACP desaturase was enhanced by different stresses including treatment with ethylene and inoculation by C. gloeosporioides. Furthermore, this transcriptional activation was associated with increased 18:2 levels, higher incorporation of 14C-linoleate into the antifungal dienes, and improved resistance to C. gloeosporioides [START_REF] Madi | Stress on avocado regulates ∆9-stearoyl ACP desaturase expression, fatty acid composition, antifungal diene level and resistance to Collectrichum gloeosporioides attack[END_REF], suggesting that ∆ 9 -stearoyl-ACP desaturase activity not only participates in the pathway leading to antifungal diene formation, but also regulates this pathway. compound 18:2, which is further elongated by conventional two-carbon extension (Figure 2). Interestingly, expression of an avocado gene encoding a Δ 9 -stearoyl-ACP desaturase was enhanced by different stresses including treatment with ethylene and inoculation by C. gloeosporioides. Furthermore, this transcriptional activation was associated with increased 18:2 levels, higher incorporation of 14C-linoleate into the antifungal dienes, and improved resistance to C. gloeosporioides [START_REF] Madi | Stress on avocado regulates ∆9-stearoyl ACP desaturase expression, fatty acid composition, antifungal diene level and resistance to Collectrichum gloeosporioides attack[END_REF], suggesting that Δ 9 -stearoyl-ACP desaturase activity not only participates in the pathway leading to antifungal diene formation, but also regulates this pathway. In A. thaliana, crown galls induced by Agrobacterium tumefaciens encounter hypoxia, drought stress, and ROS during their development [START_REF] Deeken | An integrated view of gene expression and solute profiles of Arabidopsis tumors: A genome-wide approach[END_REF]. Tolerance of plant cells for that type of stress was previously shown to be associated with their capacity to maintain or increase fatty acid unsaturation of polar lipids [START_REF] Gigon | Effect of drought stress on lipid metabolism in the leaves of Arabidopsis thaliana (ecotype Columbia)[END_REF] and, accordingly, levels of lipids containing 18:3 are significantly higher in tumors compared with reference stems, despite the limited oxygen availability in crown galls [START_REF] Klinkenberg | Two fatty acid desaturases, STEAROYL-ACYL CARRIER PROTEIN ∆9-DESATURASE6 and FATTY ACID DESATURASE3, are involved in drought and hypoxia stress signaling in Arabidopsis crown galls[END_REF]. Two genes encoding the Δ 9 -stearoyl-ACP desaturase AAD6 and the fatty acid desaturase FAD3, respectively, were shown to be strongly expressed in tumors and proposed to participate in increasing the levels of unsaturated fatty acids. Transcriptional activation of AAD6 is thought to be driven by hypoxia conditions associated with crown gall development [START_REF] Klinkenberg | Two fatty acid desaturases, STEAROYL-ACYL CARRIER PROTEIN ∆9-DESATURASE6 and FATTY ACID DESATURASE3, are involved in drought and hypoxia stress signaling in Arabidopsis crown galls[END_REF]. However, beyond the correlation observed between AAD6 expression and increased levels of unsaturated fatty acids in tumors, functional evidence for a role of this desaturase in stress tolerance is still lacking.

While the above examples illustrate how ω-9 18:1 produced by Δ 9 -stearoyl-ACP desaturases can serve as a precursor for the biosynthesis of biochemical compounds playing key roles in plant-pathogen interactions, the involvement of ω-9 18:1 itself in plant immunity against pathogen infection has been disclosed by a mutant of A. thaliana deficient in FAB2, the major Δ 9 -stearoyl-ACP desaturase isoform in this species [START_REF] Kachroo | A fatty acid desaturase modulates the activation of defense signaling pathways in plants[END_REF] (Figure 3). Plant response to pathogen infection relies on activation of a signal transduction pathway In A. thaliana, crown galls induced by Agrobacterium tumefaciens encounter hypoxia, drought stress, and ROS during their development [START_REF] Deeken | An integrated view of gene expression and solute profiles of Arabidopsis tumors: A genome-wide approach[END_REF]. Tolerance of plant cells for that type of stress was previously shown to be associated with their capacity to maintain or increase fatty acid unsaturation of polar lipids [START_REF] Gigon | Effect of drought stress on lipid metabolism in the leaves of Arabidopsis thaliana (ecotype Columbia)[END_REF] and, accordingly, levels of lipids containing 18:3 are significantly higher in tumors compared with reference stems, despite the limited oxygen availability in crown galls [START_REF] Klinkenberg | Two fatty acid desaturases, STEAROYL-ACYL CARRIER PROTEIN ∆9-DESATURASE6 and FATTY ACID DESATURASE3, are involved in drought and hypoxia stress signaling in Arabidopsis crown galls[END_REF]. Two genes encoding the ∆ 9 -stearoyl-ACP desaturase AAD6 and the fatty acid desaturase FAD3, respectively, were shown to be strongly expressed in tumors and proposed to participate in increasing the levels of unsaturated fatty acids. Transcriptional activation of AAD6 is thought to be driven by hypoxia conditions associated with crown gall development [START_REF] Klinkenberg | Two fatty acid desaturases, STEAROYL-ACYL CARRIER PROTEIN ∆9-DESATURASE6 and FATTY ACID DESATURASE3, are involved in drought and hypoxia stress signaling in Arabidopsis crown galls[END_REF]. However, beyond the correlation observed between AAD6 expression and increased levels of unsaturated fatty acids in tumors, functional evidence for a role of this desaturase in stress tolerance is still lacking.

While the above examples illustrate how ω-9 18:1 produced by ∆ 9 -stearoyl-ACP desaturases can serve as a precursor for the biosynthesis of biochemical compounds playing key roles in plant-pathogen interactions, the involvement of ω-9 18:1 itself in plant immunity against pathogen infection has been disclosed by a mutant of A. thaliana deficient in FAB2, the major ∆ 9 -stearoyl-ACP desaturase isoform in this species [START_REF] Kachroo | A fatty acid desaturase modulates the activation of defense signaling pathways in plants[END_REF] (Figure 3). Plant response to pathogen infection relies on activation of a signal transduction pathway leading to HR and SAR. Accumulation of salicylic acid (SA) and expression of PATHOGENESIS-RELATED (PR) genes, some of which encode proteins exhibiting antimicrobial activities, correlate with the appearance of HR and SAR. The AtNPR1 gene is a component of the SA signal transduction pathway participating in the induction of PR genes. The ssi2 (suppressor of SA insensitivity 2) mutant was identified in a genetic screen for mutants restoring SA signaling in the npr1 genetic background [START_REF] Shah | A recessive mutation in the Arabidopsis SSI2 gene confers SA-and NPR1-independent expression of PR genes and resistance against bacterial and oomycete pathogens[END_REF], and the corresponding mutation appeared to affect the ∆ 9 -stearoyl-ACP desaturase-coding gene FAB2 [START_REF] Kachroo | A fatty acid desaturase modulates the activation of defense signaling pathways in plants[END_REF]. Among the phenotypes of fab2/ssi2 mutants are dwarfing, accumulation of high levels of SA, constitutive activation of defense signaling, and spontaneous cell death. These characteristics confer broad spectrum disease resistance to multiple pathogens such as P. syringae, Peronospora parasitica, Golovinomyces cichoracearum, Turnip crinkle virus (TCV), and Cucumber mosaic virus (CMV) [START_REF] He | Plant unsaturated fatty acids: Biosynthesis and regulation[END_REF][START_REF] Sekine | Enhanced resistance to Cucumber mosaic virus in the Arabidopsis thaliana ssi2 mutant is mediated via an SA-independent mechanism[END_REF][START_REF] Chandra-Shekara | Plastidial fatty acid levels regulate resistance gene-dependent defense signaling in Arabidopsis[END_REF][START_REF] Song | Overexpression of a wheat stearoyl-ACP desaturase (SACPD) gene TaSSI2 in Arabidopsis ssi2 mutant compromise its resistance to powdery mildew[END_REF]. However, A. thaliana fab2/ssi2 mutant plants are also defective in jasmonic acid (JA)-regulated signaling and thereby are hypersusceptible to necrotrophic pathogens like B. cinerea [START_REF] Kachroo | A fatty acid desaturase modulates the activation of defense signaling pathways in plants[END_REF]. This is consistent with the fact that SA-mediated signaling usually participates in defense against biotrophs, while defense to many necrotrophic pathogens requires JA-derived signaling [START_REF] Gao | Low oleic acid-derived repression of jasmonic acid-inducible defense responses requires the WRKY50 and WRKY51 proteins[END_REF]. Importantly, although FAB2/SSI2 catalyzes the initial desaturation step required for JA biosynthesis, the fab2/ssi2 mutation does not alter the levels of the JA precursor 18:3 or the induced endogenous levels of JA and cannot be rescued by exogenous JA [START_REF] Kachroo | A fatty acid desaturase modulates the activation of defense signaling pathways in plants[END_REF]. Loss or knockdown of ∆ 9 -stearoyl-ACP desaturase-coding genes is also associated with similar phenotypes in other plant species. In G. max, for example, silencing of the GmSACPD-A and GmSACPD-B genes confers enhanced resistance to bacterial and oomycete pathogens, namely P. syringae and Phytophthora sojae [START_REF] Kachroo | An oleic acid-mediated pathway induces constitutive defense signaling and enhanced resistance to multiple pathogens in soybean[END_REF], while suppression of OsSSI2 in Oryza sativa (rice) enhances resistance to the leaf-blight bacteria Xanthomonas oryzae and blast fungus Magnaporthe grisea [START_REF] Jiang | Suppression of the rice fatty-acid desaturase gene OsSSI2 enhances resistance to blast and leaf blight diseases in rice[END_REF].

The fab2/ssi2 mutation confers high 18:0 level and reduced ω-9 18:1 level. Identification and characterization of fab2/ssi2 suppressor mutants showed that the altered defense phenotype observed in fab2/ssi2 mutant plants is due to the reduction in ω-9 18:1 level in the chloroplast (Figure 3). As a matter of fact, mutations restoring levels of plastid ω-9 18:1 in the fab2/ssi2 background while maintaining high levels of 18:0 also restore wild-type phenotypes [START_REF] Nandi | Arabidopsis sfd mutants affect plastidic lipid composition and suppress dwarfing, cell death, and the enhanced disease resistance phenotypes resulting from the deficiency of a fatty acid desaturase[END_REF]. For example, a loss-of-function mutation affecting the ACT1 gene, which encodes a glycerol-3-phosphate acyltransferase catalyzing the acylation of glycerol-3-phosphate with ω-9 18:1 [START_REF] Kunst | Altered regulation of lipid biosynthesis in a mutant of Arabidopsis deficient in chloroplast glycerol-3-phosphate acyltransferase activity[END_REF], restores wild-type levels of ω-9 18:1 and completely reverses SA-and JA-mediated phenotypes in fab2/ssi2 [START_REF] Kachroo | Plastidial fatty acid signaling modulates salicylic acid-and jasmonic acid-mediated defense pathways in the Arabidopsis ssi2 mutant[END_REF]. A similar phenotypic reversion was obtained with a loss-of-function mutation of ACP4 gene, which inhibits the ACT1-catalyzed reaction because ω-9 18:1-ACP4 is the preferred substrate for ACT1 [START_REF] Xia | An intact cuticle in distal tissues is essential for the induction of systemic acquired resistance in plants[END_REF]. A mutation affecting the glycerol-3-phosphate dehydrogenase-coding gene GLY1/SFD1, which disrupts the generation of glycerol-3-phosphate from dihydroxyacetone phosphate and thereby the ACT1-catalyzed reaction, also restores a wild-type phenotype [START_REF] Nandi | Arabidopsis sfd mutants affect plastidic lipid composition and suppress dwarfing, cell death, and the enhanced disease resistance phenotypes resulting from the deficiency of a fatty acid desaturase[END_REF][START_REF] Lorenc-Kukula | Biochemical and molecular-genetic characterization of SFD1's involvement in lipid metabolism and defense signaling[END_REF]. Conversely, exogenous application of glycerol on wild-type plants increases endogenous levels of glycerol-3-phosphate and activates the prokaryotic pathway of glycerolipid biosynthesis, resulting in a reduction in plastid ω-9 18:1 level and fab2/ssi2-like phenotypes [START_REF] Kachroo | Oleci acid levels regulated by glycerolipid metabolism modulate defense gene expression in Arabidopsis[END_REF]. Since the ACT1-catalyzed reaction is rate limiting, the quenching of ω-9 18:1 is even more efficient in glycerol-treated ACT1 over-expressing lines.

It was later proposed that plastid ω-9 18:1 regulates defense signaling via suppressing NO production [START_REF] Mandal | Oleic acid-dependent modulation of NITRIC OXIDE ASSOCIATED1 protein levels regulates nitric oxide-mediated defense signaling in Arabidopsis[END_REF]. In A. thaliana, at least two routes for pathogen-responsive NO production have been described, one via the reduction of nitrate by the nitrate reductases NIA1 and NIA2, and the other via an unknown mechanism involving NITRIC OXIDE ASSOCIATED1 (NAO1), a protein bearing GTPase activity. In chloroplasts, ω-9 18:1 physically interacts with NAO1, hence repressing the GTPase activity of NAO1, in turn leading to its degradation in a protease-dependent manner. As a consequence, decreased ω-9 18:1 content stabilizes NAO1 and stimulates NO production. Furthermore, expression of NIA1 and NIA2 appears to be upregulated under low ω-9 18:1 condition, contributing to NO accumulation and further launching of downstream signaling, resulting in the reprogrammation of the immune response, with SA-and JA-signaling pathways being affected. Notably, the altered defense signaling in fab2/ssi2 mutant plants can be partially restored by a mutation in NAO1 and completely restored by double mutations in NAO1 and in either of the two NIA genes. Since the fab2/ssi2 nao1 double mutant retains fab2/ssi2-like levels of ω-9 18:1 while exhibiting partial phenotypic reversion, NOA1 was proposed to function downstream of ω-9 18:1 and to provide a direct mechanistic link between membrane characteristics and transcriptional regulation of plant defense responses [START_REF] Walley | Fatty acids and early detection of pathogens[END_REF][START_REF] Mandal | Oleic acid-dependent modulation of NITRIC OXIDE ASSOCIATED1 protein levels regulates nitric oxide-mediated defense signaling in Arabidopsis[END_REF]. Interestingly, ω-9 18:1 also inhibits NO synthase activity in humans [START_REF] Davda | Oleic acid inhibits endothelial nitric oxide synthase by a protein kinase C-independent mechanism[END_REF]. For the sake of clarity, export of fatty acids from the plastids and the eukaryotic lipid metabolic pathway have been omitted. Production of oleic acid by the ∆ 9 -stearoyl-ACP desaturase FAB2/SSI2 is highlighted in green. Blue-colored circles denote enzymatic steps whose blockage (by mutation of corresponding genes) complements the fab2/ssi2 mutant phenotype: darker shades of blue denote full phenotypic reversion while lighter shades of blue denote partial phenotypic reversion. Lipid-derived signaling molecules (JA and AzA) and NO, whose biosynthesis is regulated by oleic acid, are presented on a white background. AAD, acyl-acyl carrier protein desaturase; ACP, acyl carrier protein; AzA, azelaic acid; CDP-DAG, cytidinediphosphate-diacylglycerol; DAG, diacylglycerol; CDP-DAGS, cytidinediphosphate-diacylglycerol synthase; DHAP, dihydroxyacetone phosphate; DGDG, digalactosyldiacylglycerol; DGDGS, digalactosyldiacylglycerol synthase; FAD, fatty acid desaturase; G3P, glycerol-3-phosphate; G3PDH, glycerol-3-phosphate dehydrogenase; GK, glycerol kinase; GPAT, glycerol-3-phosphate acyltransferase; JA, jasmonic acid; KAS, fatty acid synthase complex comprising 3-ketoacyl-ACP Figure 3. Lipid metabolism in the chloroplasts of Arabidopsis thaliana. The scheme depicts the different pathways involved in the biosynthesis of fatty acids and the elaboration of membrane lipids in the chloroplasts of A. thaliana. For the sake of clarity, export of fatty acids from the plastids and the eukaryotic lipid metabolic pathway have been omitted. Production of oleic acid by the ∆ 9 -stearoyl-ACP desaturase FAB2/SSI2 is highlighted in green. Blue-colored circles denote enzymatic steps whose blockage (by mutation of corresponding genes) complements the fab2/ssi2 mutant phenotype: darker shades of blue denote full phenotypic reversion while lighter shades of blue denote partial phenotypic reversion. Lipid-derived signaling molecules (JA and AzA) and NO, whose biosynthesis is regulated by oleic acid, are presented on a white background. AAD, acyl-acyl carrier protein desaturase; ACP, acyl carrier protein; AzA, azelaic acid; CDP-DAG, cytidinediphosphatediacylglycerol; DAG, diacylglycerol; CDP-DAGS, cytidinediphosphate-diacylglycerol synthase; DHAP, dihydroxyacetone phosphate; DGDG, digalactosyldiacylglycerol; DGDGS, digalactosyldiacylglycerol synthase; FAD, fatty acid desaturase; G3P, glycerol-3-phosphate; G3PDH, glycerol-3-phosphate dehydrogenase; GK, glycerol kinase; GPAT, glycerol-3-phosphate acyltransferase; JA, jasmonic acid; KAS, fatty acid synthase complex comprising 3-ketoacyl-ACP synthase; LIP, lipase; LPA, lysophosphatidic acid; LPAAT, 1-acylglycerol-3-phosphate acyltransferase; MGDG, monogalactosyldiacylglycerol; MGDGS, monogalactosyldiacylglycerol synthase; NAO1, NITRIC OXID ASSOCIATED1; NO, nitric oxide; PA, phosphatidic acid; PG, phosphatidylglycerol; PGP, phosphatidylglycerol-phosphate; PGPP, phosphatidylglycerol-phosphate phosphatase; PGPS, phosphatidylglycerol-phosphate synthase; PP, phosphatidate phosphatase; SQD2, UDP-sulfoquinovose:diacylglycerol sulfoquinovosyltransferase; SQDG, sulfoquinovosyldiacylglycerol. Picture credit: [START_REF] Nguyen | Wikimedia Commons. File Licensed under the Creative Commons Attribution 2.5 Generic License[END_REF].

Cells 2021, 10, 674 9 of 19 However, many unknowns remain concerning the molecular mechanisms underpinning this defense response. First, it is not clear how pathogen attacks alter ω-9 18:1 level in plastids of wild-type plants, if at all. Then, the complex and intricated signaling pathways lying downstream of ω-9 18:1/NOA1 have not been fully elucidated despite the identification of several actors participating in this regulatory network including the signaling component ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) [START_REF] Venugopal | Enhanced disease susceptibility 1 and salicylic acid act redundantly to regulate resistance gene-mediated signaling[END_REF] and the WORKY transcription factors WRKY50 and WRKY51 [START_REF] Gao | Low oleic acid-derived repression of jasmonic acid-inducible defense responses requires the WRKY50 and WRKY51 proteins[END_REF]. EDS1 functions redundantly with SA to modulate resistance to biotrophic pathogens under low ω-9 18:1 condition, but do not participate in the repression of JA signaling, whereas WRKY50 and WRKY51 are both required for SA accumulation and the suppression of JA-responsive induction in a fab2/ssi2 background. Finally, it remains unclear why partial phenotypic reversion was observed in the fab2/ssi2 fad6 double mutant [START_REF] Nandi | Arabidopsis sfd mutants affect plastidic lipid composition and suppress dwarfing, cell death, and the enhanced disease resistance phenotypes resulting from the deficiency of a fatty acid desaturase[END_REF][START_REF] Kachroo | Plastidial fatty acid signaling modulates salicylic acid-and jasmonic acid-mediated defense pathways in the Arabidopsis ssi2 mutant[END_REF] and in the fab2/ssi2 fad7 fad8 triple mutant [START_REF] Kachroo | Role of salicylic acid and fatty acid desaturation pathways in ssi2-mediated signaling[END_REF] while the fab2/ssi2 fad5 double mutant exhibited a slightly aggravated phenotype [START_REF] Kachroo | Role of salicylic acid and fatty acid desaturation pathways in ssi2-mediated signaling[END_REF]. These contrasting observations suggest the existence of a complex interplay between the low ω-9 18:1-triggered signaling cascade and other lipid-derived signals originating from plastid galactolipids. Recent advances in the characterization of SAR have, for instance, revealed that NO and ROS, which serve as inducers of SAR in a concentration-dependent manner [START_REF] Wang | Free radicals mediate systemic acquired resistance[END_REF], triggered the generation of azelaic acid, a signaling molecule derived from the hydrolysis of unsaturated C18 fatty acids on galactolipids [START_REF] Singh | Transport of chemical signals in systemic acquired resistance[END_REF] and behaving as a chemical inducer of SAR together with SA and glycerol-3-phosphate [START_REF] Lim | Plasmodesmata localizing proteins regulate transport and signaling during systemic acquired immunity in plants[END_REF].

Synthesis of ω-Anacardic Acids and Resistance to Pests in Geranium

Fatty acid derivatives consisting of a polar aromatic ring and a hydrophobic alkyl chain constitute a class of specialized metabolites known as phenolic lipids. Among them, anacardic acids (2-hydroxy-6-alkylbenzoic acids) feature a salicylic acid system substituted with saturated or unsaturated alkyl chains that have 15-17 carbons at the 6-position. They are produced by a relatively limited number of plant species, mostly in the Anacardiaceae family, as in Anacardium occidentale (cashew) [START_REF] Osman | A highly sensitive GC-MS method for simultaneous determination of anacardic acids in cashew (Anacardium occidentale) nut shell oil in the presence of other phenolic lipid derivatives[END_REF] and Pistacia vera (pistachio) [START_REF] Yalpani | The phenolics acids of Pistachia vera[END_REF]. These compounds are also synthesized by some species of the Ginkgoaceae (Ginkgo biloba) [START_REF] Li | Efficient purification of ginkgolic acids from Ginkgo biloba leaves by selective adsorption on Fe 3 O 4 magnetic nanoparticles[END_REF], Geraniaceae (Pelargonium x horturum) [START_REF] Gerhold | Analysis of trichome exudate from mite-resistant geraniums[END_REF], Araceae (Philodendron scandens) [START_REF] Reffstrup | Philodendron scandens Koch Et Sello subsp. oxycardium (Schott) Bunting, a new source of allergenic alkyl resorcinols[END_REF], Schoepfiaceae (Schoepfia californica) [START_REF] Chen | Inhibitors of DNA polymerase β from Schoepfia californica[END_REF], Violaceae (Viola Websteri) [START_REF] Lee | Bioassay-guided isolation of antiplasmodial anacardic acids derivatives from the whole plants of Viola websteri Hemsl[END_REF], and Myristicaceae families (Knema elegans and knema hookeriana) [START_REF] Spencer | Alkyl and phenylalkyl anacardic acids from Knema elegans seed oil[END_REF][START_REF] Gény | Anacradic acids from Knema kookeriana as modlulators of Bcl-xl/Bak and Mcl-1/bid interactions[END_REF]. Anacardic acids have received great attention by the community of chemicobiology researchers and pharmaceutical companies due to their potent biological activity against Alzheimer's disease [START_REF] Lemes | Cardanol-derived AChE inhibitors: Towards the development of dual binding derivatives for Alzheimer's disease[END_REF] as well as antitumor [START_REF] Huang | Anacardic acid induces cell apoptosis associated with induction of ATF4-dependent endoplasmic reticulum stress[END_REF], anti-inflammatory, and anti-obese activity [START_REF] Hemshekhar | Natural product anacardic acid from cashew nut shells stimulates neutrophil extracellular trap production and bactericidal activity[END_REF]. They also display bactericide [100], fungicide [101,102], insecticide [103,104], anti-parasite [START_REF] Lee | Bioassay-guided isolation of antiplasmodial anacardic acids derivatives from the whole plants of Viola websteri Hemsl[END_REF]105,106], and molluscicide properties [START_REF] Hemshekhar | Natural product anacardic acid from cashew nut shells stimulates neutrophil extracellular trap production and bactericidal activity[END_REF].

Despite their great potential as therapeutic molecules and crop protection products, little is known about the in-planta biosynthesis and functions of anacardic acids. The production of these specialized metabolites seems to take place in different parts of the plant depending on the species considered. In cashew, for example, anacardic acids accumulate at high levels in a viscous pericarp fluid of the nut called 'cashew nut shell liquid'. In contrast, zonal geranium secretes anacardic acids from glandular trichomes, specialized tissues comprised of a basal stalk topped by secretary cells accumulating essential oils. Moreover, geranium is likely one of the only plants that produce anacardic acids as a single class of phenolic lipids rather than as a complex mixture of related phenolic lipid components. The biosynthesis of anacardic acids was shown to happen through the addition of six carbons to C16 and C18 fatty acid precursors [107]. Elongation of starter fatty acyl-CoA esters is probably catalyzed by type III polyketide synthase through sequential condensation of three acetate units from malonyl-CoA [108]. The polyketide intermediate thus obtained is ultimately folded to the ring system specific to anacardic acids by aldol condensation.

So far, a physiological function of anacardic acids has been determined uniquely in zonal geranium, which has evolved a multifaceted defensive mechanism against small pests like spider mites and aphids based on the secretion of anacardic acids by glandular trichomes. Two modes of actions of these specialized metabolites have been put forward [109].

The first consists in a physical entrapment of the pests: the sticky secretion impedes pest movement, thereby reducing potential feeding and oviposition behavior. The second relies on a toxic effect of anacardic acids that increases mortality and reduces fecundity of the pests. If the exact mechanisms of action underpinning this toxic effect have not been determined with certainty, anacardic acids have been ascribed an increasing number of inhibitory activities against enzymes such as acetylcholinesterase [110], prostaglandin endoperoxide synthase [111], lipoxygenase [112], tyrosinase [113], or histone acetyltransferases [114], highlighting some interesting research areas for future investigations. Tyrosinase is indeed an important molting enzyme [113], whereas prostaglandin synthase and lipoxygenase can influence insect fecundity and maturation of eggs [111]. Conversely, in some primitive insect species lacking prostaglandins, products of the lipoxygenase can play a prostaglandin role. It is also possible that anacardic acids disrupt pest feeding by making the tissue non-nutritive or non-palatable.

Importantly, the effective resistance of zonal geranium to pests not only depends on the secretion of anacardic acids, but also on the chemical structure of these compounds (Figure 4). Specifically, pest resistance is mediated by the alkyl group desaturation [109]. Pest-resistant and pest-susceptible genotypes of geranium have been thoroughly characterized. The exudate from resistant plants contains approximately 90% of ω-5 monounsaturated C22 and C24 anacardic acids whereas in the susceptible genotype, the saturated 22:0 and 24:0 anacardic acids predominate, accounting for over 80% of the exudate composition [111]. These contrasted compositions result in distinct physical properties of trichome secretions. The exudate of the pest-resistant genotype is fluid under normal growth conditions, composing an efficient sticky trap, whereas the highly saturated exudate of the pest-susceptible genotype is a solid material that fails to impede pest movement. However, exudate fluidity also influences the effectiveness of the application of the potential toxin: the secretions from the resistant genotype are more effectively applied to the pest, yielding a higher level of mortality when the solid exudate of the susceptible genotype does not adhere to the exoskeleton of the pest. The biosynthesis of ω-5 C22 and C24 anacardic acids requires a Δ 9 -myristoyl-ACP desaturase [116]. The gene encoding this desaturase is only expressed in trichomes of the pest-resistant genotype, correlating with the production of ω-5 anacardic acids and the pest-resistant phenotype. The product of the myristoyl-ACP desaturase activity, ω-5 14:1, is thought to be further elongated by the fatty acid synthase system, yielding ω-5 16:1 and ω-5 18:1 that are further metabolized through the polyketide synthase pathway. So far, the determinant of substrate specificity of the myristoyl-ACP desaturase has never been elucidated. Likewise, the identity of the transcription factor(s) regulating the transcriptional activation of the Δ 9 -myristoyl-ACP desaturase-coding gene in trichomes remains The biosynthesis of ω-5 C22 and C24 anacardic acids requires a ∆ 9 -myristoyl-ACP desaturase [116]. The gene encoding this desaturase is only expressed in trichomes of the pest-resistant genotype, correlating with the production of ω-5 anacardic acids and the pest-resistant phenotype. The product of the myristoyl-ACP desaturase activity, ω-5 14:1, is thought to be further elongated by the fatty acid synthase system, yielding ω-5 16:1 and ω-5 18:1 that are further metabolized through the polyketide synthase pathway. So far, the determinant of substrate specificity of the myristoyl-ACP desaturase has never been elucidated. Likewise, the identity of the transcription factor(s) regulating the transcriptional activation of the ∆ 9 -myristoyl-ACP desaturase-coding gene in trichomes remains unknown. If the single dominant locus identified in geranium for conditioning ω-5 anacardic acid synthesis and subsequent pest resistance most likely corresponds to the desaturase-coding gene [117], the recalcitrance of geranium to transformation has so far prevented the confirmation of this hypothesis. The alternative hypothesis of the dominant factor encoding a transcriptional activator cannot be completely ruled out though [116].

Although anacardic acids do not have a known physiological role in most plant species producing such specialized metabolites, the wide range of bioactivity displayed by these phytochemicals suggests that they may be involved in pathogen resistance in other plants, with a mode of action distinct from the sticky trap of zonal geranium. Accumulation of anacardic acids in leaves and fruits might be related to the consumption of these tissues by pests. As a matter of fact, anacardic acids consumed as part of a diet negatively affect numerous facets of Leptinotarsa decemlineata (Colorado potato beetle) larval growth and development [103]. Anacardic acids therefore represent a valuable resource for integrated management in the population control of disease vectors and agricultural pests, applied as an ecofriendly chemical spray or through bioengineering production in crops. Their biological activities encourage further investigations to look for the mode of actions of these phytochemicals and for the genes encoding transcriptional regulators and biosynthetic enzymes of the anacardic acid pathway.

Synthesis of Alkenes and Attraction of Pollinators in Orchids

Orchids occur in a great variety of geographical areas and their high level of pollinator specialization is regarded as one key to their success [118]. They have evolved various cues to attract pollinators and increase efficiency in pollen delivery, which is ensured by hymenopterans like solitary bees [119]. Plant-pollinator associations are not always mutualistic and parasitism has frequently evolved on both sides [120]. Whereas two thirds of orchids reward their visitors, typically with nectar, the remaining third is enabled to produce that type of exudate and has consequently evolved alternative mechanisms based on deception. Food deception, where orchids advertise floral cues resembling those from rewarding plants, targets generalist pollinators while sexual deception, where flowers mimic the sexual signals of pollinators, attracts highly specialized pollinators [118]. The conspicuous, insect-like flowers of sexually deceptive orchids mimic both morphological characteristics and the female sex pheromones of insect pollinators, which attempt courtship or copulatory behavior with the flower and thereby remove or deliver the orchids' pollen packets, termed pollinia [119]. Research has shown that semiochemicals were of paramount importance for pollinator attraction [121].

To elucidate the chemistry of sexual deception, pollinator perception of semiochemicals can be investigated using calcium imaging of antennal lobe activity in the pollinator brain during exposure to floral scents [122,123]. More commonly, semiochemicals can be individually tested for their ability to stimulate the pollinator's antennal chemoreceptors using gas chromatography coupled with electroantennography [124]. A broad range of compounds pivotal for pollination has thus been discovered. Among those, known or predicted fatty acid derived semiochemicals include alkanes, alkenes, carboxylic acids, aldehydes, carboxylic esters, and chiloglottones. In many species of the Mediterranean orchid genus Ophrys, a blend of very long-chain alkanes and alkenes differing in carbon chain length and the position of unsaturation is the main basis for olfactory mimicry [120,125]. Bioassays have emphasized the importance of the alkene unsaturation position for controlling pollinator preference. For example, O. sphegodes mostly produces 9-and 12-alkenes, whereas O. exaltata produces high levels of 7-alkenes. The pollinator of O. sphegodes, Andrena nigroaenea, is attracted to 9-and 12-alkenes, and the addition of 7-alkenes to the odor blend reduces its attractiveness. Conversely, Colletes cunicularius, the pollinator of O. exaltata, is attracted to 7-alkenes, and the addition of 9-and 12-alkenes reduces this attraction [126].

All currently identified Ophrys semiochemicals can be formed biosynthetically from fatty acid precursors. The first stage in alkane production is the de novo synthesis of C16 and C18 fatty acids in the plastid. These acyl chains are then elongated into very long-chain fatty acyl-CoAs (C20-C34) in the endoplasmic reticulum [124]. After this elongation sequence, the enzymatic complex formed by ECERIFERUM1 and ECERIFERUM3 efficiently converts very long-chain acyl-CoAs into odd-numbered very long-chain alkanes via a two-step reaction of aldehyde formation followed by decarbonylation [127,128]. The semiochemicals may then be exported just as the other components of the cuticular layer of the epidermal cells of the flower. Alkenes should follow the same biosynthetic pathway as alkanes, with the noticeable exception of an additional desaturation step potentially catalyzed by AADs prior to the elongation step [124]. In light of the essential role played by the alkene unsaturation position for governing pollinator preference, research efforts have been dedicated to identify AADs participating in alkene production in Ophrys. AAD multigene families were therefore thoroughly characterized in closely related sympatric Ophrys species reproductively isolated from each other due to the production of different types of alkenes and the subsequent attraction of different pollinators [126,129]. These studies have led to the discovery of specialized AAD isoforms preferentially expressed in one or the other of these species and displaying distinct enzymatic activities that could be associated with the production of different categories of alkenes (Figure 5). For example, the expression level of SAD2 is much higher in O. sphegodes flowers than in O. exaltata flowers [126], and in vitro assays have established that O. sphegodes SAD2 exhibits both ∆ 9 -stearoyl-ACP desaturase and ∆ 4 -palmitoyl-ACP desaturase activities [129]. As such, it is very likely that SAD2 produces ω-9 18:1 and ω-12 16:1 intermediates required to build the 9-and 12-alkenes responsible for A. nigroaenea attraction. Conversely, SAD5 is predominant in flowers of O. exaltata, where this specialized ∆ 9 -palmitoyl-ACP desaturase synthesizes ω-7 16:1 used for the production of 7-alkenes attracting C. cunicularius.

Remarkably, protein structural modeling and reconstruction of inferred ancestral state proteins have shown how the 16:0 specificity of the specialized ∆ 9 -palmitoyl-ACP desaturase SAD5 is linked to just three amino acid residues lining the bottom part of its substrate-binding pocket [132]. Reconstructed ancestral state proteins at these three amino acid sites exhibit a marked preference for 18:0 substrates similar to that of the archetype ∆ 9 -stearoyl-ACP desaturase. The substrate-binding pocket of the archetype desaturase is deep enough to accommodate 18:0 substrates. In contrast, changed amino acids obstruct the lower end of the substrate pocket in SAD5, leaving just enough space to accommodate a shorter 16:0 substrate with carbons 9 and 10 of the acyl chain facing the active site of the enzyme, leading to the production of ω-7 monounsaturated fatty acids. The replacement of a hydrophobic residue by a bulkier and still hydrophobic residue (as in the L145F substitution observed in SAD5) appears to be a key determinant of modified substrate specificity for all the specialized ∆ 9 -palmitoyl-ACP desaturases characterized thus far [START_REF] Cahoon | A determinant of substrate specificity predicted from the acyl-acyl carrier protein desaturase of developing cat's claw seed[END_REF][START_REF] Troncoso-Ponce | Transcriptional activation of two delta-9 palmitoyl-ACP desaturase genes by MYB115 and MYB118 is critical for biosynthesis of omega-7 monounsaturated fatty acids in the endosperm of Arabidopsis seeds[END_REF]. The evolutionary trajectory of SAD5 is proposed to have arisen by gene duplication and diversification from an ancestral 'housekeeping' ∆ 9 -stearoyl-ACP desaturase. Modeling approaches illustrate how variations in AAD paralogs associated with floral scent variations can drive evolutionary divergence between orchid species, with subsequent reproductive isolation allowing for rapid sympatric speciation by pollinator shift [125]. AAD genes responsible for contrasted alkene production between species therefore appear as potential 'speciation genes', supporting the model where orchids are prime candidates for rapid pollinator-driven 'genic' speciation [124]. In this model, changes in a limited subset of genes with large effects do make speciation more likely than changes in many genes with small effects [119].

Δ 9 -stearoyl-ACP desaturase and Δ 4 -palmitoyl-ACP desaturase activities [129]. As such, it is very likely that SAD2 produces ω-9 18:1 and ω-12 16:1 intermediates required to build the 9-and 12-alkenes responsible for A. nigroaenea attraction. Conversely, SAD5 is predominant in flowers of O. exaltata, where this specialized Δ 9 -palmitoyl-ACP desaturase synthesizes ω-7 16:1 used for the production of 7-alkenes attracting C. cunicularius. Remarkably, protein structural modeling and reconstruction of inferred ancestral state proteins have shown how the 16:0 specificity of the specialized Δ 9 -palmitoyl-ACP desaturase SAD5 is linked to just three amino acid residues lining the bottom part of its substrate-binding pocket [132]. Reconstructed ancestral state proteins at these three amino acid sites exhibit a marked preference for 18:0 substrates similar to that of the archetype Δ 9 -stearoyl-ACP desaturase. The substrate-binding pocket of the archetype desaturase is deep enough to accommodate 18:0 substrates. In contrast, changed amino acids obstruct the lower end of the substrate pocket in SAD5, leaving just enough space to accommodate a shorter 16:0 substrate with carbons 9 and 10 of the acyl chain facing the active site of the enzyme, leading to the production of ω-7 monounsaturated fatty acids. The replacement of a hydrophobic residue by a bulkier and still hydrophobic residue (as in the L145F substitution observed in SAD5) appears to be a key determinant of modified substrate specificity for all the specialized Δ 9 -palmitoyl-ACP desaturases characterized thus far [START_REF] Cahoon | A determinant of substrate specificity predicted from the acyl-acyl carrier protein desaturase of developing cat's claw seed[END_REF][START_REF] Troncoso-Ponce | Transcriptional activation of two delta-9 palmitoyl-ACP desaturase genes by MYB115 and MYB118 is critical for biosynthesis of omega-7 monounsaturated fatty acids in the endosperm of Arabidopsis seeds[END_REF]. The evolutionary trajectory of SAD5 is proposed to have arisen by gene duplication and diversification from an ancestral 'housekeeping' Δ 9 -stearoyl-ACP desaturase. Modeling approaches illustrate how variations in AAD paralogs associated with floral scent variations can drive evolutionary divergence between orchid species, with subsequent reproductive isolation allowing for rapid sympatric speciation by pollinator shift [125]. AAD genes responsible for contrasted alkene production between species therefore appear as potential 'speciation genes', supporting the model where orchids are prime candidates for rapid pollinator-driven 'genic' speciation [124]. In this model, changes in a limited subset of genes with large effects do make speciation more likely than changes in many genes with small effects [119]. While the question of the molecular bases of substrate specificity has been successfully addressed for one AAD isoform in orchids, opening interesting research perspectives in the field of pollinator-driven speciation, the equally important regulatory mechanisms that differentially activate expression of the AAD genes remain completely unknown. The AAD transcripts encoding desaturases involved in alkene production are specifically accumulated in the epidermal cell layer of the labellum [132,133]. Aside from this tissuespecific accumulation pattern [134], AAD transcripts exhibit contrasted accumulation levels from one species to another [126]. Preliminary analyses relying on expression studies in hybrids suggest that both cisand trans-acting factors regulate these gene expression differences. Importantly, these factors also constitute potential speciation elements that cannot be neglected.

Interestingly, recent reports have provided examples of sexual deception in the Iridaceae [135] and Asteraceae [136], suggesting that this pollination strategy may be more common than is currently known [121]. Considering both the important proportion of sex pheromones secreted by female insects to attract conspecific mates that derive from fatty acids [137] and the relatively frequent occurrence of unsaturations within the acyl chains of such compounds [138,139], it is tempting to speculate that more AADs involved in the production of semiochemicals aimed at favoring plant pollination are yet to be discovered in the plant kingdom.

Conclusions

The regio-and stereospecific unsaturation of saturated acyl chains by different types of AADs in plants provides a large pool of monounsaturated fatty acids used for the biosynthesis of different kinds of lipids and derivatives. Past research efforts have shown that a number of these molecules participate in regulatory processes and signal transduction pathways essential for plant biotic interactions. As research initiatives aimed at exploring the diversity of plant specialized metabolites become more numerous while benefiting from increasingly sophisticated and affordable-omics technologies, it is very likely that many molecules derived from unsaturated fatty acids and bearing unusual chemical functionalities will soon be discovered to participate in responses to biotic stress. As a striking example of these approaches, a pathogen-responsive gene cluster responsible for the biosynthesis of falcarindiol was recently identified in Solanum lycopersicum (tomato). This prototypical acetylenic lipid is derived from 18:2 and is present in edible plants like Daucus carota (carrot), S. lycopersicum, and Apium graveolens (celery) [140]. This dietary metabolite bearing conjugated acetylenic functionality, oxidation, and an unusual terminal vinyl group inhibits the growth of fungi as well as human cancer cell lines [141]. Future work investigating the biosynthesis, mode of action, and effectiveness of these types of compounds will be essential to better understand how plants interact with pollinators and pathogens. An improved knowledge of the underpinning biological mechanisms could be particularly beneficial for improving yields of agronomically important species and for preserving plant biodiversity in a context of climate change and modified ecological balance.

Figure 1 .

 1 Figure 1. Transcriptional regulation of AAD genes in the leaves of Arabidopsis thaliana in response to biotic stress. (A) Phylogram, with branch lengths in arbitrary units, using the alignment of the seven A. thaliana AAD sequences (with gaps). Enzymatic activities of the AAD isoforms are indicated below the phylogram (blue squares). The activity of AAD4 remains unknown. (B) Relative AAD transcript levels in control leaves of A. thaliana expressed as a percentage of FAB2 transcript levels.Expression levels were calculated using the data displayed on the Arabidopsis eFP Browser[START_REF]Arabidopsis eFP Browser[END_REF]. (C) Variations in AAD transcript levels in leaves of A. thaliana in response to various pathogens and elicitors and expressed in fold changes with respect to control. Bold lines between AAD2 and AAD4 reflect the impossibility of distinguishing between AAD2 and AAD4 transcripts using the Affymetrix technology due to the very similar sequences of these transcripts. Flg62, bacterial derived elicitor; GST-NPP1, oomycete-derived elicitor; HrpZ, bacterial derived elicitor.

Figure 2 .

 2 Figure 2. Biosynthesis of antifungal dienes in Persea americana. ∆ 9 -18:0 AAD, ∆ 9 -stearoyl-acyl carrier protein desaturase; FAD2, fatty acid desaturase 2. Picture credit: [66].

Figure 2 .

 2 Figure 2. Biosynthesis of antifungal dienes in Persea americana. ∆ 9 -18:0 AAD, ∆ 9 -stearoyl-acyl carrier protein desaturase; FAD2, fatty acid desaturase 2. Picture credit: [66].

Figure 3 .

 3 Figure 3. Lipid metabolism in the chloroplasts of Arabidopsis thaliana. The scheme depicts the different pathways involved in the biosynthesis of fatty acids and the elaboration of membrane lipids in the chloroplasts of A. thaliana. For the sake of clarity, export of fatty acids from the plastids and the eukaryotic lipid metabolic pathway have been omitted. Production of oleic acid by the ∆ 9 -stearoyl-ACP desaturase FAB2/SSI2 is highlighted in green. Blue-colored circles denote enzymatic steps whose blockage (by mutation of corresponding genes) complements the fab2/ssi2 mutant phenotype: darker shades of blue denote full phenotypic reversion while lighter shades of blue denote partial phenotypic reversion. Lipid-derived signaling molecules (JA and AzA) and NO, whose biosynthesis is regulated by oleic acid, are presented on a white background. AAD, acyl-acyl carrier protein desaturase; ACP, acyl carrier protein; AzA, azelaic acid; CDP-DAG, cytidinediphosphate-diacylglycerol; DAG, diacylglycerol; CDP-DAGS, cytidinediphosphate-diacylglycerol synthase; DHAP, dihydroxyacetone phosphate; DGDG, digalactosyldiacylglycerol; DGDGS, digalactosyldiacylglycerol synthase; FAD, fatty acid desaturase; G3P, glycerol-3-phosphate; G3PDH, glycerol-3-phosphate dehydrogenase; GK, glycerol kinase; GPAT, glycerol-3-phosphate acyltransferase; JA, jasmonic acid; KAS, fatty acid synthase complex comprising 3-ketoacyl-ACP

Cells 2021 ,

 2021 10, x 11 of 20 yielding a higher level of mortality when the solid exudate of the susceptible genotype does not adhere to the exoskeleton of the pest.

Figure 4 .

 4 Figure 4. Biosynthesis of anacardic acids in Pelargonium x hortorum. The simplified scheme depicts the different pathways involved in the biosynthesis of fatty acids and omega-anacardic acids (AnAc) in trichomes of Pelargonium x hortorum. Production of myristoleic acid by ∆ 9 -myristoyl-ACP desaturases in resistant genotypes is highlighted in orange. AAD, acyl-acyl carrier protein desaturase; KAS, fatty acid synthase complex comprising 3-ketoacyl-ACP synthase. Picture credit: [115].

Figure 4 .

 4 Figure 4. Biosynthesis of anacardic acids in Pelargonium x hortorum. The simplified scheme depicts the different pathways involved in the biosynthesis of fatty acids and omega-anacardic acids (AnAc) in trichomes of Pelargonium x hortorum. Production of myristoleic acid by ∆ 9 -myristoyl-ACP desaturases in resistant genotypes is highlighted in orange. AAD, acylacyl carrier protein desaturase; KAS, fatty acid synthase complex comprising 3-ketoacyl-ACP synthase. Picture credit: [115].

Figure 5 .

 5 Figure 5. Biosynthesis of alkenes in Ophrys sphegodes and Ophrys exaltata. The simplified scheme depicts the different pathways involved in the biosynthesis of fatty acids and alkenes in the epidermal cell layer of the labellum in Ophrys sphegodes and Ophrys exaltata. Different types of acyl-acyl carrier protein desaturases with contrasted substrate specificities and regiospecificities produce different categories of monounsaturated fatty acids in the Ophrys species considered. As a consequence, different types of alkenes are synthesized and used as semiochemicals that attract different pollinators. AAD, acyl-acyl carrier protein desaturase; KAS, fatty acid synthase complex comprising 3-ketoacyl-ACP synthase. Picture credit: [130,131].

Figure 5 .

 5 Figure 5. Biosynthesis of alkenes in Ophrys sphegodes and Ophrys exaltata. The simplified scheme depicts the different pathways involved in the biosynthesis of fatty acids and alkenes in the epidermal cell layer of the labellum in Ophrys sphegodes and Ophrys exaltata. Different types of acyl-acyl carrier protein desaturases with contrasted substrate specificities and regiospecificities produce different categories of monounsaturated fatty acids in the Ophrys species considered. As a consequence, different types of alkenes are synthesized and used as semiochemicals that attract different pollinators. AAD, acyl-acyl carrier protein desaturase; KAS, fatty acid synthase complex comprising 3-ketoacyl-ACP synthase. Picture credit: [130,131].

Acknowledgments: We are grateful to A. To, M. Miquel, L. Lepiniec, B. Dubreucq, and M. Corso for their helpful discussions. We apologize to those researchers whose work we were not able to cite in this review.

Funding:

The Institut Jean-Pierre Bourgin benefits from the support of Saclay Plant Sciences-SPS (ANR-17-EUR-0007).

Conflicts of Interest:

The authors declare no conflict of interest.


Chad Plumet

Achmet Said

Tang Vendeuvre

Brigitte Renoux

Jonathan Clarhaut

S Ébastien Papot
email: sebastien.papot@univ-poitiers.fr

Cell-cell interactions via non-covalent click chemistry †

Metabolic glycoengineering with unnatural sugars became a valuable tool for introducing recognition markers on the cell membranes via bioorthogonal chemistry. By using this strategy, we functionalized the surface of tumor and T cells using complementary artificial markers based on both b-cyclodextrins (b-CDs) and adamantyl trimers, respectively. Once tied on cell surfaces, the artificial markers induced cell-cell adhesion through non-covalent click chemistry. These unnatural interactions between A459 lung tumor cells and Jurkat T cells triggered the activation of natural killer (NK) cells thanks to the increased production of interleukin-2 (IL-2) in the vicinity of cancer cells, leading ultimately to their cytolysis. The ready-to-use surface markers designed in this study can be easily inserted on the membrane of a wide range of cells previously submitted to metabolic glycoengineering, thereby offering a simple way to investigate and manipulate intercellular interactions.

Results and discussion

 Based

Introduction

In nature, intercellular recognition is typically achieved by means of complementary adhesion molecules present on the surface of each cell partner. During the last decade, metabolic glycoengineering, in which unnatural monosaccharides bearing bioorthogonal functional groups are metabolically incorporated into cell-surface glycans, [START_REF] Kayser | This eld was rst pioneered[END_REF][2][3] emerged as a powerful strategy for manipulating cellular interactions. 4 As early as 2009, Gartner and Bertozzi were the rst to use this approach for the construction of 3-dimensional micro-tissues, by conjugating single stranded DNA nucleotides at the membrane of azidolabelled Jurkat T cells. 5 Cells bearing complementary DNA sequences led to the formation of aggregates with well-dened interconnectivities. More recently, Iwasaki and co-workers used the thiol-ene reaction to introduce aptamers on the surface of methacryloyl-functionalized macrophages, enabling them to recognize human T lymphoblasts in vitro. 6 The manipulation of cell-cell interactions was also investigated by forming covalent bonds between cells through bioorthogonal ligation reactions. [7][8][9][10] Within this framework, the strain-promoted azidealkyne cycloaddition (SPAAC) reaction was employed by Ma and Cai to bind tumor cells and T cells. 9 Intercellular covalent bonding triggered an increased cytotoxicity of T cells for tumor cells, highlighting the potential of this approach for the development of novel cancer immunotherapy. In 2016, Qu and co-workers reported an elegant strategy enabling the manipulation of cellular adhesion via photo-responsive host-guest recognition. 11 By using a photoisomerizable azobenzene dimer as a reversible cell binder, they were able to control the assembly of tumor cells, previously functionalized on the surface by b-cyclodextrins (b-CD) via copper(I)-catalysed azidealkyne cycloaddition (CuAAC). Herein, we present the development of complementary cell surface markers allowing unnatural cell-cell adhesion through non-covalent click chemistry (Fig. 1). 12 We designed b-CD and adamantyl trimers (Tri-b-CD and Tri-Adam, respectively) bearing a dibenzocyclooctyne (DBCO) that can be easily attached on the surface of azido-labelled cells by the SPAAC reaction. When installed on the membrane of cells that do not recognize naturally, these articial recognition markers promoted cell-cell adhesion through host-guest interactions.

As proof of principle, we coated tumor cells and T lymphocytes with Tri-b-CD and Tri-Adam respectively, and we demonstrated that their forced interaction activated natural killer (NK) cells to kill tumor cells. This strategy, that combines metabolic glycan labelling with both covalent and non-covalent click chemistry, provides a simple way to manipulate cell-cell interactions, hence facilitating the study of resulting biological processes. on a gallic acid core, the latter one enables indeed the attachment of both a DBCO and three copies of the recognition unit in only a few synthetic steps (Scheme 1). Thus, the adamantyl moieties were rst introduced on the primary amines of 1 by nucleophilic substitution using the N-hydroxysuccinimide ester 2. Cleavage of the tert-butoxycarbonyl protecting group (Boc) followed by coupling with the activated ester of DBCO 3 afforded Tri-Adam (56% yield over three steps). On the other hand, Tri-b-CD was prepared via a four-step strategy from compound 1. The reaction between 1 and the N-hydroxysuccinimide ester 4 allowed the introduction of three terminal alkynes on the molecular platform. Aer removal of the Boc protecting group, the platform was then functionalized with three equivalents of azido-b-CD 5 through the CuAAC reaction. Finally, the DBCO moiety was coupled with the remaining primary amine using the precursor 3, thereby providing Tri-b-CD with an overall yield of 41%.

We next investigated the ability of the complementary host/ guest pair Tri-b-CD/Tri-Adam to trigger cell-cell adhesion. For this purpose, A549 human cancer cells and human Jurkat T lymphocytes were rst treated for three days with tetraacetylated N-azidoacetyl-D-mannosamine (Ac 4 ManNAz) in order to install azides within cell surface glycoconjugates (see the ESI †). A549 and Jurkat cells were then incubated for thirty minutes with Tri-b-CD and Tri-Adam, respectively, for binding the arti-cial surface markers on the cell membrane via the SPAAC reaction. Such a procedure of cell surface engineering did not affect the viability of cells for at least forty eight hours postfunctionalization. Furthermore, it is worth mentioning that with the trimeric structure of our articial markers, each click reaction permits the introduction of three recognition units (host/guest), hence multiplying the potential interactions between complementary cells.

Once modied as described above, A549 (green) and Jurkat (red) cells were incubated together in order to analyze the effect of the articial markers Tri-b-CD and Tri-Adam on cell recognition (Fig. 2). Thus, Jurkat T cells were seeded on A549 adherent cells (5 : 1 Jurkat : A549 ratio) for ten minutes, then the supernatant was removed. The adherent cells were next washed and xed prior to monitoring intercellular interactions by 3D confocal microscopy. As shown in Fig. 2a, Jurkat T cells remained bound to A549 cells when both cell lines were previously submitted to metabolic labelling with Ac 4 ManNAz followed by the bioorthogonal introduction of either Tri-Adam or Tri-b-CD on the surface glycans. In contrast, when Jurkat cells were not modied beforehand by the full procedure of cell engineering, they did not adhere to A549 cells, as testied by the absence of red uorescence. 3D imaging analysis conrmed the establishment of contacts between modied cells and showed that each A549 tumor cell interacted with several Jurkat cells (Fig. 2b).

The interactions between both modied A549 and Jurkat T cells were also investigated by electron microscopy (Fig. 3). These experiments conrmed the adhesion of Jurkat T cells on the surface of A549 tumor cells (Fig. 3c). Interestingly, the images suggested that interactions between the two cell types induced the remodeling of the actin cytoskeleton with the formation of laments 14 (Fig. 3d).

Overall, these results demonstrate that the functionalization of the cell surface with complementary articial recognition markers, based on host-guest pairs (e.g. cyclodextrin-adamantyl), induces cell-cell adhesion via non-covalent click chemistry. Since such a bioorthogonal conjugation enables tying of cells that do not recognize naturally, it provides a valuable opportunity for studying and manipulating intercellular interactions. Under these circumstances, we decided to pursue our investigations by exploring the potential of our articial surface markers within the framework of the destruction of cancer cells (Fig. 4a).

Indeed, Jurkat T cells readily secrete cytokines such as interleukin-2 (IL-2) when stimulated by lectins. 15 Therefore, we postulated that the accumulation of Jurkat T cells at the surface of A549 cells could generate an IL-2 local concentration which may be sufficient to activate natural killer (NK) cells (step 1). Once activated by IL-2, NK cells could then initiate the lysis of cancer cells through the exocytosis of perforin (step 2), 16 leading ultimately to tumor cell death (step 3). To study this hypothesis, Tri-Adam-modied Jurkat T cells were seeded on A549 adherent cells previously functionalized with Tri-b-CD. Aer ten minutes of incubation, the culture medium was washed with PBS in order to remove non-adherent cells. Remaining cells were treated for forty eight hours with phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA) to stimulate IL-2 production by Jurkat T cells. 17 The supernatant was then harvested and the amount of secreted IL-2 was analyzed by human IL-2 enzyme-linked immunosorbent assay (ELISA).

As shown in Fig. 4b, the IL-2 production was similar under almost all conditions (Fig. 4b, entries 1-3), except when both modied Jurkat and A549 cells were incubated together (Fig. 4b, entry 4). In the latter case, the IL-2 concentration was approximately 4-fold higher than that measured in all the control experiments. These results demonstrated a direct correlation between cell recognition promoted by non-covalent click chemistry and the increased IL-2 concentration. Indeed, the presence of Tri-b-CD and Tri-Adam on the cell surface prevented Jurkat T cells from being washed away during the experimental protocol, hence enabling the release of a larger IL-2 amount in the vicinity of A549 adherent cells.

We next investigated whether such an IL-2 production can trigger the lysis of tumor cells by NK cells. Toward this end, A549 and NK cells were incubated for four hours with the supernatants collected from previous experiments and the resulting cytotoxicity for cancer cells was monitored using a calcein-release assay (Fig. 4c, for the full experimental procedure see the ESI †). In this study, the basal toxicity of NK cells for A549 cells was used as the control experiment (Fig. 4c, entry 1). Thus, when unmodied Jurkat and A549 cells were incubated together, no supplementary toxicity was observed for tumor cells (Fig. 4c, entry 2). In contrast, the incubation of both glycoengineered Jurkat and A549 cells triggered a signicant cytotoxic effect (Fig. 4c, entry 3) that was correlated with the increased concentration of IL-2 secreted by T cells (Fig. 4b, entry 4).

To prove that the killing of tumor cells was the consequence of NK cell activation by the cytokine, we conducted a positive control experiment in which A549 and NK cells were incubated in the presence of recombinant IL-2 (Fig. 4c, entry 4). Under these conditions, strong toxicity toward tumor cells was recorded, highlighting the role of IL-2 in the process of A549 cell destruction by NK cells. Taken altogether, these results showed that the interaction between A549 and Jurkat cells, induced by the presence on their surface of the complementary cell recognition markers Tri-b-CD and Tri-Adam, was necessary to launch the observed cytotoxic activity. This study also indicated that non-covalent click chemistry can be a powerful tool for modulating the behavior of cellular networks.

Conclusions

In summary, we designed complementary articial recognition markers that can be easily introduced on the surface of glycoengineered cells via the SPAAC reaction. These articial markers led to unnatural intercellular interactions via non-covalent click chemistry. We demonstrated that such cell-cell contacts offered the possibility to manipulate cell networks with potential medicinal applications. Since metabolic glycan labelling using azido-sugars has been applied to remodel the membranes of a wide range of cells, the Tri-b-CD and Tri-Adam recognition markers can be useful for associating cells that cannot recognize each other naturally. Therefore, these readyto-use articial markers should greatly facilitate the study of intercellular interactions, providing a better comprehension of biological mechanisms associated with cell adhesion, as well as new opportunities for the development of cell-based therapies.

Fig. 1

 1 Fig. 1 Principle of cell surface engineering with complementary artificial recognition markers based on host-guest pairs. Step 1: metabolic glycan labelling with Ac 4 ManNAz resulting in azide tag incorporation at cell surfaces. Step 2: introduction of the complementary artificial markers Tri-b-CD and Tri-Adam on the cell membranes via the bioorthogonal SPAAC ligation reaction. Step 3: cell-cell adhesion through non-covalent click chemistry.

Fig. 2

 2 Fig. 2 Confocal microscopy imaging of cellular recognition. (a) A549 adherent tumor cells (green) and Jurkat T cells (red) (1 : 5 ratio) were incubated together for 10 minutes and washed with PBS prior to imaging; (b) 3D imaging of cell-cell interactions.

Fig. 3

 3 Fig. 3 Electron microscopy imaging of cellular recognition. (a) Imaging of A549 cells bearing Tri-b-CD artificial surface markers; (b) imaging of Jurkat T cells functionalized with Tri-Adam markers; (c) and (d) imaging of Jurkat T cell on the surface of A549 cells at two different magnifications, showing the appearance of filaments (arrows).

Fig. 4

 4 Fig. 4 (a) Potential cell interaction network engendered by an unnatural cell recognition leading to the destruction of tumor cells; (b) Concentration of IL-2 secreted by Jurkat T cells when incubated with A549 cells in the presence of PHA and PMA (GI: glycoengineered cells, WT: wild-type cells); (c) NK cells-mediated cytotoxicity for A549 cells directed by cell-cell interactions.

© 2021 The Author(s). Published by the Royal Society of Chemistry

| Chem. Sci., 2021, 12, 9017-9021 © 2021 The Author(s). Published by the Royal Society of Chemistry

Acknowledgements

The authors thank La Ligue contre le Cancer (Comités Vienne, Charente, Charente-Maritime and Deux-Sèvres), the Région Nouvelle Aquitaine and the program PAUSE for nancial support of this study. The authors also thank Jérémie Rousseau for his precious help in the design of gures and artwork

Notes and references

Author contributions

C. P. synthesised, puried and characterised the articial markers. J. C. designed and conducted in vitro biological experiments. A. S. M. and B. R. supervised the synthesis work and analysed the data. T. V. analysed biological data and provide advices for clinical transfer. S. P. designed the study and wrote the manuscript. All the authors regularly discussed the results of the study and jointly decided on the directions to be followed.

Conflicts of interest

There are no conicts to declare.