A 13-LOX hierarchy for defence in Arabidopsis thaliana

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Thesis

Reference

A 13-LOX hierarchy for defence in Arabidopsis thaliana

CHAUVIN, Adeline

Abstract

13-lipoxygenases (13-LOXs) initiate the first enzymatic reaction in jasmonate synthesis which is essential for the activation of defence against herbivores. Jasmonates are wound- inducible compounds that accumulate in a wide time frame from seconds to hours after tissue damage.

Jasmonate production begins in plastids and, in Arabidopsis thaliana, four 13-LOXs (LOX2, LOX3, LOX4 and LOX6) incorporate molecular oxygen on the C13 of the α-linolenic acid.

Among the four 13-LOXs, LOX2 has been the most widely studied while LOX3, LOX4 and LOX6 remain poorly described. In the present work, we found that each 13-LOX acts differently in response to insect attacks with 1) distinct promoter activities 2) different signalling dynamics and 3) controlling different defences against herbivores.

CHAUVIN, Adeline. A 13-LOX hierarchy for defence in Arabidopsis thaliana. Thèse de doctorat : Univ. Genève, 2014, no. Sc. 4721

URN : urn:nbn:ch:unige-419999

DOI : 10.13097/archive-ouverte/unige:41999

Available at:

http://archive-ouverte.unige.ch/unige:41999

Disclaimer: layout of this document may differ from the published version.

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‘What is a weed? A plant whose virtues have yet not been discovered, and every one of the two hundred thousand, probably yet to be of utility in the arts. As Bacchus of the vine, Ceres of the wheat, - as Arkwright and Whitney were the demi-gods of cotton, so prolific Time will yet bring an inventor to every plant. There is no property in nature, but a mind is born to seek and find it’

Ralph Waldo Emerson, 1863

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Abstract

13-lipoxygenases (13-LOXs) initiate the first enzymatic reaction in jasmonate synthesis which is essential for the activation of defence against herbivores. Jasmonates are wound- inducible compounds that accumulate in a wide time frame from seconds to hours after tissue damage. Jasmonate production begins in plastids and, in Arabidopsis thaliana, four 13-LOXs (LOX2, LOX3, LOX4 and LOX6) incorporate molecular oxygen on the C13 of the α-linolenic acid. Among the four 13-LOXs, LOX2 has been the most widely studied while LOX3, LOX4 and LOX6 remain poorly described. In the present work, we found that each 13-LOX acts differently in response to insect attacks with 1) distinct promoter activities 2) different signalling dynamics and 3) controlling different defences against herbivores. The LOX2 promoter in rosettes is active in most of the leaf while the LOX3 and LOX4 promoters are mainly active in veins. LOX6 promoter activity and protein accumulation is restrained to the vasculature. The major early wound events are mediated by LOX2 and LOX6 while, at this early stage, LOX3 and LOX4 have low activities. Differently from LOX2 which is more specific for the local wound response, LOX6 participates in early jasmonic acid (JA) and jasmonoyl isoleucine (JA-Ile) accumulation in distal leaves. The LOX2 and LOX6 proteins mediate the induction of the early marker genes JASMONATE-ZIM DOMAIN 10 (JAZ10), LOX3 and LOX4 in a wounded leaf and in distal leaves on wounded plants and, in turn, LOX3 and LOX4 mediate the expression of later wound inducible genes such as VSP2. Thus, the wound response is mediated via a 13-LOX hierarchy. Contrasting with the LOX6 protein localisation, LOX6 mediates the activity of the JAZ10 promoter in interveinal tissue. The activity of the JAZ10 promoter was restored in the jasmonate synthesis mutant allene oxide synthase (aos) JAZ10p:GUSPLUS and oxo-phytodienoate reductase 3 (opr3) JAZ10p:GUSPLUS reporter lines when AOS and OPR3 coding sequences are drived by the

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LOX6 promoter. Our data thus provides strong evidence for cell-to-cell communications via jasmonate movements in the early steps of jasmonate signalling. Together, the specific contribution of each 13-LOX in space and time leads to different types of defence. When the generalist herbivore Spodoptera littoralis attacks Arabidopsis thaliana, LOX2, LOX3 and LOX4 orchestrate active defence that impairs the growth of the herbivore while LOX6 does not. LOX2 is predominantly involved in direct defence and, on its own, is sufficient to confer defence to the wild type (WT). LOX3 and LOX4, on the other hand, are less essential to impair insect weight gain but do contribute partially in this process. Despite a weak role in direct defence, LOX6 has a key role in long-distance signalling and participate in the orchestration of the defence by mediating the protection of young leaves from attack of Spodoptera littoralis.

The present thesis work enabled us to unravel the interplay between the different 13-LOXs giving detailed insights into their hierarchy and their roles in plant defence. This opens new questions and provides new tools with which to obtain a comprehensive view of the mechanisms involved in the wound response to herbivores.

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Résumé

Les 13-lipoxygenases (13-LOXs) initient la première réaction enzymatique de la voie de biosynthèse des jasmonates, une étape essentielle pour la défense contre les herbivores. Les jasmonates sont des composés induits en réponse à la blessure des feuilles qui s’accumulent sur un laps de temps qui peut aller de quelques secondes à plusieurs heures après qu’un dommage aux tissus ait eu lieu. Chez Arabidopsis thaliana, une telle voie de biosynthèse prend place au sein de plastes, où les quatre 13-LOXs (LOX2, LOX3, LOX4 et LOX6) incorporent un oxygène moléculaire sur le C13 de l’acide α-linolénique. Parmi ces quatre 13- LOXs, LOX2 a fait l’objet de diverses études, alors que LOX3, LOX4 et LOX6 restent peu connues. Au cours de ce travail, nous avons pu démontrer que chacune des 13-LOXs agit différemment en réponse à l’attaque d’un herbivore par 1) des activités distinctes de promoteurs 2) des dynamiques de signalisation différentes ainsi que par 3) le contrôle de différentes formes de défense contre les herbivores. Dans les feuilles adultes, le promoteur LOX2 est actif majoritairement dans la feuille entière alors que les promoteurs LOX3 et LOX4 sont principalement actifs dans les veines. L’activité du promoteur LOX6 ainsi que l’accumulation de la protéine sont restreintes au tissu vasculaire. Les évènements majeurs de la réponse précoce à la blessure sont sous le contrôle de LOX2 et LOX6, alors que LOX3 et LOX4 ont une faible activité. Contrairement à LOX2, qui est plus spécifique à la réponse locale à la blessure, LOX6 participe à l’accumulation précoce d’acide jasmonique (JA) ainsi que du jasmonoyl-isoleucine (JA-Ile) dans les feuilles distales d’une plante blessée. Les protéines LOX2 et LOX6 contribuent à l’induction de gènes-marqueurs précoces tels que JASMONATE-ZIM DOMAIN 10 (JAZ10), LOX3 et LOX4 dans la feuille locale blessée ainsi que dans la feuille voisine. LOX3 et LOX4 à leur tour contribuent à l’induction de gènes appartenant à la réponse tardive à la blessure tels que VSP2. Ainsi, la réponse à la blessure est

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médiée par une hiérarchie au sein des 13-LOXs. Contrairement à la localisation de sa protéine, LOX6 contribue à l’activité du promoteur JAZ10 dans les tissus situés entre les veines. L’activités du promoteur JAZ10 a été restauré dans les lignées rapportrices qui sont affectées dans la voie des jasmonates, allene oxide synthase (aos) JAZ10p:GUSPLUS et oxo- phytodienoate reductase 3 (opr3) JAZ10p:GUSPLUS, lorsque les séquences codantes de AOS et de OPR3 étaient dirigées par le promoteur LOX6. Ainsi, nos résultats plaident en faveur d’une communication de cellule à cellule par des mouvements de jasmonates, lors de la phase précoce de la réponse à la blessure. Ainsi, la contribution spécifique de chaque 13-LOX au niveau spatial et temporel mène à différents types de défense. Lorsque l’herbivore généraliste Spodoptera littoralis attaque Arabidopsis thaliana, LOX2, LOX3 and LOX4 orchestre une défense active qui altère la croissance de l’herbivore, alors que LOX6 n’est pas impliquée dans ce facteur de la défense. LOX2 est majoritairement impliquée dans la défense directe et son activité est suffisante pour permettre la défense du phénotype sauvage (WT). Bien que LOX3 et LOX4, altèrent moins nettement la croissance de l’insecte, elles contribuent au processus. Malgré un faible impact en défense directe, LOX6 a un rôle clé dans la signalisation à longue-distance et ainsi promeut la protection des jeunes feuilles au centre de la rosette contre l’attaque de Spodoptera littoralis.

Le présent travail de thèse a permis de comprendre quelles interactions existent entre les différentes 13-LOXs, leur hiérarchie et de leurs rôles pour la défense des plantes. Cette approche ouvre de nouvelles questions tout en procurant de nouveaux outils pour comprendre de façon exhaustive et détaillées les mécanismes impliqués dans la réponse à la blessure aux herbivores.

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Remerciements

Je tiens à remercier les Professeurs Wolfender et Farmer d’avoir bien voulu m’accueillir au sein de leur laboratoire, ce qui a rendu possible l’élaboration de ma thèse de doctorat. Je les remercie de m’avoir accordé leur confiance et de m’avoir apporté leur soutien à travers toutes les discussions et tous les entretiens enrichissants que nous avons eus durant près de cinq années.

Je veux leur dire également combien je leur suis reconnaissante de m’avoir offert l’opportunité de travailler sur ce projet de collaboration qui lie la phytochimie à la biologie moléculaire.

Je remercie mes collègues de travail, aussi bien à l’UNIGE qu’à l’UNIL, pour les discussions scientifiques ou personnelles que nous avons partagées. Plus particulièrement, je remercie Florence Mehl et Soura Challal ainsi que Natalie Schregle de m’avoir soutenue lors des moments difficiles.

Je remercie également, Steeve Lassueur, Blaise Tissot, Aurore Chetlat, Raphael Roux, Caroline Mathon ainsi que Olga Stepushchenko pour leur bonne humeur permanente ainsi que leurs petites blagues.

Je remercie Ivan Acosta, le Post-Doc le plus fantastique et le plus passionné que je connaisse.

Je remercie Willam Deakin, de m’avoir transmis sa passion pour les sciences et le goût du travail en équipe, mais également de m’avoir appris comment travailler dans un laboratoire de recherche. Tous ses enseignements, généreusement dispensés tout au long de mon Master, m’ont été d’une grande utilité et ont représenté une aide précieuse sur laquelle j’ai pu m’appuyer pour mener à terme l’élaboration de ma thèse.

Si je pouvais faire de l’Arabette des Dames une personne, je la remercierais, ainsi que ses quatre 13-LOXs pour tout le bonheur dont elle m’a comblée, et pour toutes les merveilleuses surprises qu’elle m’a réservées.

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Je remercie ma famille et mes amis, pour leur soutien et leur amicale présence et plus particulièrement Liliane et Daniel pour leurs bons petits plats et leur accueil chaleureux, ainsi que leur gentillesse naturelle.

Et enfin, je remercie tout particulièrement Jean-Baptiste, pour sa présence sécurisante et son soutien indéfectible dans les périodes difficiles et les moments de doutes, ainsi que pour sa complicité totale dans les bons moments. Il a su, quand il le fallait, m’apporter le réconfort et l’énergie nécessaire pour mener mon travail à son terme.

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Abbreviations

α-LeA: α-linolenic acid AOC : allene oxide cyclase AOS : allene oxide synthase ANOVA: analysis of variance

COI1: CORONATINE INSENSITVE 1 GLR: Glutamate Receptor-like

GLV: green leaf volatiles GSH: glutathione

HPL :hydroperoxy lyase

HPLC : high performance liquid chromatography 13-HPOT: 13-hydroperoxy octadecatrienoic acid JA: jasmonic-acid

JA-AA: JA-amino acid JA-Ile: jasmonoyl-isoleucine

JAR1: JASMONATE RESISTANT 1 JAZ: JASMONATE-ZIM DOMAIN JRG: JA-regulated gene

LA: linoleic acid

LC: liquid chromatography LOX: lipoxygenase

MRM: multiple reaction monitoring MS : mass spectrometry

NASC: European Arabidopsis Stock Center

OPC-8:0: 3-oxo-2-pentenyl cyclopentane-1-octanoic acid OPDA : oxophytodienoic-acid

OPR: OPDA REDUCTASE ORGs: OPDA-regulated gene RES: reactive electrophiles species ROS: reactive oxygen species t-test: student test

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VSP2: VEGETATIVE STORAGE PROTEIN2 WAK: wound associated kinase receptor

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Contributions

Original research papers

Chauvin A, Caldelari D, Wolfender J-L, Farmer EE. 2013. Four 13-lipoxygenases contribute to rapid jasmonate synthesis in wounded Arabidopsis thaliana leaves: a role for lipoxygenase 6 in responses to long-distance wound signals. New Phytologist 197(2): 566- 575.

Mousavi SAR, Chauvin A, Pascaud F, Kellenberger S, Farmer E E. 2013. GLUTAMATE RECEPTOR-LIKE genes mediate leaf-to-leaf wound signalling. Nature 500(7463): p. 422- 426.

Chauvin A, Wolfender J-L, Farmer EE. Paired hierarchical organization of jasmonate- producing lipoxygenase in Arabidopsis. Submitted.

Review

Chauvin A, Acosta IF, Farmer EE, Wolfender J-L. Function of plastidic 13- LIPOXYGENASES in plant defenses. To be submitted.

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Oral communications

Adeline Chauvin, Daniela Caldelari, Jean-Luc Wolfender and Edward E. Farmer. 2013.

LIPOXYGENASE 6: driving long distance defense signalling in Arabidopsis thaliana. PhD day UNIGE, Hermance.

Long distance defense signalling in Arabidopsis thaliana: a key role for LOX6. 22th Edition of the Swiss Plant Molecular and Cell biology, Meiringen.

Posters

Rapid quantitative analysis of jasmonic acid in plant extracts by LC-MS/MS. Swiss chemical Society (SCS) Fall meeting, Zurich.

Identifying enzymes necessary for jasmonate production less than 40s after wounding. 8th Tri-National Arabidopsis Meeting (TNAM), Lausanne.

Long distance defense signalling in Arabidopsis thaliana: a key role for LOX6. National Centre of Competence in Research (NCCR) Plant survival Meeting, Neuchâtel.

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Introduction

Function of plastidic 13-LIPOXYGENASES in plant defenses

To be submitted

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Function of plastidic 13-LIPOXYGENASES in plant defenses

Adeline Chauvin^1,2*, Ivan Acosta^2*, Anna Kostikova³, Edward E. Farmer^1,* and Jean-Luc Wolfender²

1. Department of Plant Molecular Biology, University of Lausanne, Biophore, 1015 Lausanne, Switzerland.

2. School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland

3. Anna Kostikova, Department of Ecology and Evolution, University of Lausanne, UNIL- Sorge, 1015, Lausanne, Switzerland

* this authors contributed equally to this work

Author Contribution:

AC and IF wrote the manuscript AK provide run the tree analysis JLW and EEF provide comment

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Introduction

Lipoxygenases (LOXs) catalyse the formation of hydroperoxides from unsaturated fatty-acids in a wide range of organisms including prokaryotic bacteria, protists, fungi, plants and animals (Brash, 1999; Ivanov et al., 2010). LOX-mediated peroxidation is the first step of several metabolic pathways that generate lipid signaling molecules such as leukotrienes and jasmonates, which respectively mediate inflammatory responses in vertebrates (Feussner &

Wasternack, 2002; Porta & Rocha-Sosa, 2002; Duroudier et al., 2009; Moreno, 2009) and defense and development in plants (Feussner & Wasternack, 2002; Porta & Rocha-Sosa, 2002; Duroudier et al., 2009; Moreno, 2009). LOX proteins contain two well conserved domains, the C-terminal catalytic domain composed of α-helices, and the N-terminal PLAT/LH2 domain forming a β-barrel believed to mediate membrane or substrate attachment to the protein depending on divalent cation availability (Brash, 1999; Schneider et al., 2007) (Oldham et al., 2005; Walther et al., 2011) (Figure 1).

In flowering plants the predominant LOX substrates are linoleic acid (LA) and α-linolenic acid (α-LeA). Two main classes of plant LOXs exist according to the positional specificity of LA oxygenation, which occurs at carbon 9 of the hydrocarbon backbone in the 9-LOX types, and at carbon 13 in the 13-LOX types (Feussner & Wasternack, 2002). Additionally, 9-LOXs have mainly been found in the cytosol, while 13-LOXs from the vast majority of plants carry a chloroplast transit peptide (cTP) at the N-terminus. In Arabidopsis, 9-LOXs regulate lateral root development and stomatal closure, modulate defense under bacterial infection, and promote infestation by a phloem-feeding aphid (Vellosillo et al., 2007; Nalam et al., 2012;

Vicente et al., 2012; Montillet et al., 2013).

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Fig. 1: Lipoxygenases have two conserved domains. (A) The structure of the human 5-LOX is adapted from (Gilbert et al., 2011). The PLAT/LH2 domain is formed of β-barrels and the lipoxygenase catalytic domain is predominantly α-helices. The N-terminal domain (PLAT/LH2) binds to the substrate while the C-terminal domain (lipoxygenase) incorporates molecular oxygen on to the unsaturated fatty-acids backbone. (B) LOX2, LOX3, LOX4 and LOX6 from Arabidopsis thaliana also have chloroplast transit peptides (cTPs). The different gradings (from black to white) are representative of the percentage identities (% ID) among

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the four AtLOXs. LOX2, LOX4 and LOX6 % identity at the amino acid level was performed with LOX3 as reference (using MUSCLE). % ID of cTP (C), PLAT/LH2 (L2) and lipoxygenase (L) is displayed by (C/L2/L). The LOX2 elF4E-binding motif is displayed with dashed boxes. Conserved residue are displayed in red for the iron binding (H: histidine, N:

asparagine and I: isoleucine). Calcium biding domains (G17, T18, D19, N44, D45, E47, D79 and D80) are displayed by green lines.

Among other functions, 13-LOXs are important for male fertility and defense against pathogens and herbivores (Acosta et al., 2009; Glauser et al., 2009; Caldelari et al., 2011;

Christensen et al., 2013).

13-LOXs carrying a putative cTP incorporate molecular oxygen in α-LeA to produce 13- hydroperoxy octadecatrienoic acid (13-HPOT, Figure 2). Further processing of this substance can occur through at least two enzymes, a hydroperoxy lyase (HPL) or an allene oxide synthase (AOS), which define two main branches of the 13-LOX metabolic pathway (Figure 2) (Zimmerman & Vick, 1970; Hamberg, 1988; Gardner, 1991; Ziegler et al., 2000). The AOS branch leads to the formation of jasmonates (Figure 2), which are major inducers of multiple lines of defense in response to microbial or herbivore attack. These include proteins and other compounds that directly impair the performance of the attacker (direct defences) (Yan et al., 2013) and volatile terpenes that attract herbivore predators or parasitoids (indirect defence) (Dicke et al., 1999). AOS catalyses the dehydration of 13-HPOT into a very unstable allene oxide that undergoes cyclization through an allene oxide cyclase (AOC) to form the cyclopentenone (+)-12-oxo-phytodienoic acid (OPDA). OPDA is exported from plastids by an unknown mechanism and then imported into peroxisomes in part through the ABC transporter COMATOSE (Theodoulou et al., 2005). In the peroxisome, an OPDA reductase

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(OPR) catalyses the reduction of OPDA’s cyclopentenone ring to form 3-oxo-2-pentenyl cyclopentane-1-octanoic acid (OPC-8:0). Shortening of the fatty acid chain by three rounds of β-oxidation gives rise to (+)-7-iso-jasmonic acid (JA). The JA-amino synthetase

JASMONATE RESISTANT 1 (JAR1) can conjugate JA to several amino acids (Staswick &

Tiryaki, 2004) but the transconjugate jasmonoyl-isoleucine (JA-Ile) is the single most bioactive jasmonate known so far (Suza & Staswick, 2008; Fonseca et al., 2009). JA-Ile acts as a molecular glue in a receptor complex formed by a JAZ transcriptional repressor and the F-box protein CORONATINE INSENSITVE 1 (COI1) (Sheard et al., 2010). This results in the ubiquitylation and subsequent degradation of JAZ repressors (Chini et al., 2007; Thines et al., 2007; Glauser et al., 2009), which allows the transcriptional activators MYC2, MYC3 and MYC4 to promote the expression of defence-related genes such as VSP2 (Fernandez-Calvo, 2011).

In the HPL pathway, the HPL enzyme cleaves 13-HPOT into volatile (Z)-3-hexenal and traumatin (Figure 2). (Z)-3-hexenal and its derivatives are called green leaf volatiles (GLV) and capable of inducing defence gene expression (Bate & Rothstein, 1998) and attracting insect parasitoids and predators (Kessler & Baldwin, 2001; Shiojiri et al., 2006). Traumatin and its derivatives mediate wound healing (Bonner & English, 1937; Bonner & English, 1938) althought this has not been shown independantly (Farmer et al., 1994).

Here we present the current knowledge of the functional diversification of plastidic 13-LOXs in the synthesis of AOS- and/or HPL-derived lipid signals during plant defence and development. In addition to drawing from genetic and biochemical data gathered from several plant species in the last few years, we identify questions that remain open and provide an overview of how this family of diverse but related enzymes orchestrate plant defence.

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Figure 2: 13-lipoxygenases initiate the first step of direct and indirect defense responses against insects and pathogens.

In plastids, 13-LOXs catalyse the oxygenation α-linolenic acid, this enzymatic reaction forms 13-hydroperoxy-octadecatrienoic acid (13-HPOT). 13-HPOT is a substrate for two competitive branches mediated by allene oxide synthase (AOS) or 13-hydroperoxide lyase (13-HPL). AOS and allene oxide cyclase (AOC) generate oxo-phytodienoic acid (OPDA), a RES that has two different roles in the AOS branch. Firstly, in a complex mechanism OPDA recruits cyclophilin 20-3 and glutathione (GSH) for the regulation of genes called OPDA- regulated gene (ORGs) that are transcribed by TGA transcription factors. Secondly, in peroxisome, OPDA is reduced and then oxidized to form jasmonic-acid (JA). In the cytosol, JAR1 conjugates JA and isoleucine to form the bioactive hormone JA-Ile. JA-Ile is involved in JA-regulated gene (JRG) expression mediated via MYC family transcription factors. The 13-HPL protein cleaves 13-HPOT and form (Z)-3-hexenal and 9-OH-traumatin. These are involved in tritrophic interaction and possibly cell division.

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Evolution of 13-LOXs

Phylogenetic analysis of plastidic 13-LOXs indicate that they diverged in two main clades before the dicot-monocot split (Figure 3) and that each clade underwent further distinct duplication patterns. While clade 2 simply diverged into monocot and dicot subclades, clade 1 duplicated into subclades 1a and 1b before the divergence of monocots and dicots and subclade 1b was lost in the monocots. In both clade 1 and 2, cereal (monocot) 13-LOXs have undergone one or two additional duplication rounds. The increase in the number of plastidic 13-LOXs within flowering plants opens the possibility of functional specialization.

Interestingly, each clade is enriched in LOXs that have been shown to mainly feed either the AOS pathway (clade 1) or the HPL pathway (clade 2, Figure 3) but this distinction is not absolute as discussed below.

Clade 1a 13-LOXs

Several 13-LOXs from this clade provide substrate for the AOS pathway since genetic analysis indicates that they are directly involved in the synthesis of JA and in JA-activated processes such as defence in response to herbivory or flower development (Halitschke &

Baldwin, 2003; Wang et al., 2008; Acosta et al., 2009; Caldelari et al., 2011; Christensen et al., 2013; Yan et al., 2013). The clade includes AtLOX3 and AtLOX4, which resulted from a recent duplication specific to Arabidopsis and are still close enough to act redundantly; black nightshade SnLOX3, tomato TomLOXD, wild tobacco NaLOX3 and maize ZmLOX8.

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Fig. 3: Phylogenetic analysis of 13-LOXs in flowering plants.

The sequence alignment was run using Muscle with default settings and MrBayes used for reconstructing species phylogenies. A model jumping approach was used in order to sample across fixed amino acid rate matrices. Two independent MCMC analyses were run for 5 000 000 generations with 4 independent chains. MCMC chains in Tracer were examined in order to ensure convergence of MCMC chains. The consensus topology was obtained after disregarding a burning fraction of 500 000 generations in TreeAnnotator. The green box surrounds the 13-LOXs from clade 1, while the pink box surrounds the 13-LOXs from clade 2. Underlined 13-LOXs in green or pink have been described to be involved respectively in the AOS or HPL pathway. 13-LOXs underlined with pink and green are 13-LOXs that have a direct or indirect dual role for the AOS and the HPL pathway. See table 1 for other latin name abbreviations

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Table 1: 13-LOXs from monocots and dicots. 13-LOXs for which sequence or function has been reported mainly in recent publications are listed according to their group. Note that for some 13-LOXs alternative names are present in the literature.

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The genes encoding these enzymes are expressed at relatively low levels in unchallenged seedlings and mature leaves. However, they are rapidly induced by leaf wounding with peaks between 30 min and 1 h after stimulation (Vellosillo et al., 2007; Allmann et al., 2010;

Grebner et al., 2013; Yan et al., 2013; Shen et al., 2014). This is a feature shared with other members of the JA synthesis pathway, including AOS, AOC and OPR3 (Ziegler et al., 2001;

Stenzel, I. et al., 2003; Stenzel, Irene et al., 2003; Koo et al., 2009; Christensen et al., 2013).

Addition of insect oral secretions to wounds boosts NaLOX3 up-regulation by two-fold (Allmann et al., 2010). Depending on the plant species and the strength of the stress, the time frame for LOX gene induction can vary and extend to between 2 and 8 hours (Allmann et al., 2010; Vandoorn et al., 2011; Christensen et al., 2013; Yan et al., 2013).

JA accumulation mediated by these 13-LOXs and activated by mechanical damage induces a wide panel of chemical defences such as proteinase inhibitors, leucine amino-peptidases and terpenes (Halitschke & Baldwin, 2003; Kessler et al., 2004; Wang et al., 2008; Allmann et al., 2010; Mariutto et al., 2011; Christensen et al., 2013; Yan et al., 2013). Mutant or knock- down analysis of clade 1 13-LOXs has been instrumental in uncovering their important role in defence. For example, the tomato mutant spr8 is defective in TomLoxD and does not accumulate proteinase inhibitors after mechanical wounding or insect attack (Yan et al., 2013). This and similar mutants or knockdowns in other species are strongly susceptible to attack by herbivores and necrotrophic fungi (Halitschke & Baldwin, 2003; Kessler et al., 2004; Diezel et al., 2011; Vandoorn et al., 2011; Yan et al., 2013). Interestingly, a Na-lox3 knockdown line of wild tobacco grown in the field becomes an appetizing prey for new herbivore communities that do not normally feed on this plant (Kessler et al., 2004; Wang et al., 2008).

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Mutants or underexpressors of clade 1a 13-LOXs are not completely impaired in JA accumulation 30 min to 1 hour after wounding. In Na-lox3 antisense lines and the Tom-loxD / spr8 mutant some JA is still made after wounding but it reaches ~2 and ~4 times less than wild-type, respectively (Halitschke & Baldwin, 2003; Kessler et al., 2004; Wang et al., 2008;

Allmann et al., 2010; Mariutto et al., 2011; Christensen et al., 2013; Yan et al., 2013). Yet, such reduction is sufficient to render these plants sensitive to herbivory. In contrast, the lox3 lox4 double mutant of Arabidopsis accumulates wild-type levels of JA 30 min after wounding (Grebner et al., 2013). Although further analysis is still required, it is possible that losing AtLOX3 and AtLOX4 function in Arabidopsis will have a less obvious impact in defence against herbivores than found for 13-LOXs from this clade in other species. Alternatively, AtLOX3 and AtLOX4 could participate in JA biosynthesis at later time points if their induction by wounding follows a different kinetics, as indicated by a proteomic study where the AtLOX3 protein was found to accumulate 6 h after wounding (Gfeller et al., 2011).

In summary, although most clade 1a 13-LOXs have been shown to be necessary for the bulk of JA synthesis after wounding, they do not seem to be the only contributors. As discussed in other sections below, it is likely that 13-LOXs from the other clades also participate in JA synthesis or attempt to compensate if clade 1a 13-LOXs are not functional.

Functions of clade 1a 13-LOXs in reproductive development

In addition to its function in plant defence, JA produced through clade 1 13-LOXs is important for at least two aspects of flower development. First, AtLOX3 and AtLOX4 redundantly promote the last stages of Arabidopsis stamen development, including filament elongation and anther dehiscence (Caldelari et al., 2011). Second, ZmLOX8 / tasselseed1, a single maize LOX gene out of three present in this clade, has specialized in male sex

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determination of maize tassels (Acosta et al., 2009). Since a COI1 knockdown line in tobacco and an AOC mutant of rice also display defects in male fertility (Riemann et al., 2003; Li et al., 2004; Paschold et al., 2007), it is expected that clade 1a 13-LOXs from those species are also necessary for normal fertility but this remains to be found. Another developmental function for JA in the organization of floral organs in rice spikelets has recently been described (Cai et al., 2014). Therefore, it is possible that rice OsLOX5 and/or OsLOX6 are required for this process.

Clade 1b 13-LOXs

Arabidopsis LOX6 is the sole member of this clade whose function has been investigated. In contrast with 13-LOXs in the other clades, AtLOX6 is not wound inducible (Grebner et al., 2013) and its expression in leaves is restricted to a few cells surrounding the xylem vessels (Chauvin et al., 2013). Crushing leaf tissue causes a very fast accumulation of JA within 30 sec in regions proximal to the wound and 90 sec in distal connected leaves. Analysis of lox6 mutants demonstrated that AtLOX6 is required for this very first JA bursts in regions away from crushed leaf tissue (Chauvin et al., 2013; Glauser et al., 2009). Additionally, unchallenged leaf and root tissue from wild-type plants contain significant amounts of OPDA, which are completely absent in lox6 mutants (Grebner et al., 2013). This suggests a mechanism for fast JA production involving pre-formed OPDA pools that are basally maintained by AtLOX6 function and quickly processed into JA upon perception of wound signals. These signals may include recently described electrical activities that are generated through two Glutamate Receptor-like (GLR) channels (Mousavi et al., 2013) to activate early JA responses in tissues distal to wounds (Chauvin et al., 2013). Then, it is possible that GLR-

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mediated ion fluxes activate the metabolism of pre-formed OPDA pools for distal, rapid synthesis of JA after wounding.

30 min after leaf wounding, JA signaling in the lox6 mutant is almost completely normal in the tissue proximal to the wound while it remains impaired in leaves farthest away (Chauvin et al., 2013; Grebner et al., 2013). Strikingly, a similar dependence on AtLOX6 alone for JA accumulation at later time points was found in wounded root tissue (Glauser et al., 2009;

Grebner et al., 2013). It seems that locally produced JA after root wounding is also initiated from pre-formed OPDA pools made through AtLOX6 (Glauser et al., 2009; Grebner et al., 2013) .

Since LOX6 was dispensable for late JA accumulation in Arabidopsis leaf tissue proximal to wounds, it was not surprising that lox6 mutants resisted herbivory as well as wild-type plants.

Furthermore, the triple mutant lox2 lox3 lox4 where only LOX6 is functional was as sensitive to herbivory as a mutant lacking all four 13-LOXs where insect weight gain was similar.

However, insects attacked apical regions of the quadruple mutant but not the triple mutant (Chauvin et al., 2013). These findings indicate that LOX6 is not sufficient nor responsible for maximum JA accumulation which is required to mount full defence responses. However, LOX6 was found to mediate two other aspects of defence in Arabidopsis. First, lox6 mutants were unable to enhance defence responses when plants were ‘primed’ by wounding before the herbivory challenge (Chauvin et al., 2013; Grebner et al., 2013). Second, even if herbivore performance was equally good in the triple mutant lox2 lox3 lox4 and the quadruple mutant lox2 lox3 lox4 lox6, functional LOX6 in the triple mutant allowed the survival of young leaves and shoot apical meristems (Chauvin et al., 2013; Grebner et al., 2013).

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In brief, AtLOX6 is necessary for JA accumulation in tissues away from wounds and for JA- mediated protection of young shoot tissue against herbivores. One prediction of these findings is that AtLOX6 functions are a common feature of clade 1b 13-LOXs but this remains to be tested. Furthermore, since this clade is exclusively present in dicots, it will be worth investigating if similar functions have independently evolved in the monocots by specialization of 13-LOXs in other clades.

Clade 2 13-LOXs

Genetic evidence supports a major role for several members of this clade in providing substrate for the HPL pathway, including TomLOXC, ZmLOX10 and NaLOX2. Mutants or knockdowns of these 13-LOXs suppress GLV formation in response to wounding. (Allmann et al., 2010; Christensen et al., 2013; Shen et al., 2014). The transcripts encoding these enzymes are relatively abundant in unchallenged leaves (Nemchenko et al., 2006; Allmann et al., 2010; Christensen et al., 2013; Shen et al., 2014) , a feature that may facilitate the rapid release of GLVs. They are also induced by wounding but transcription peaks later than clade 1a 13-LOXs (Nemchenko et al., 2006; Allmann et al., 2010; Christensen et al., 2013; Shen et al., 2014). Genes encoding HPL enzymes follow a similar kinetics and their basal expression is also abundant (Howe et al., 2000; Halitschke et al., 2004; Allmann et al., 2010; Liu et al., 2012).

Although the absolute requirement of several clade 2 13-LOXs in GLV synthesis is a unifying feature, the possible direct or indirect participation of some members in JA synthesis varies

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widely between species. At one end of the spectrum, wild tobacco NaLOX2 or potato StLOX2 have no influence on JA formation (León et al., 2002; Allmann et al., 2010;

Christensen et al., 2013; Shen et al., 2014). At the other end, rice (OsLOX7 / OsHI-LOX) participates exclusively in JA production under herbivory instead of GLV synthesis (Zhou et al., 2009). It is possible that the duplicate monocot subclade containing OsLOX7 has specialized on JA production during the diversification of clade 2 13-LOXs in monocots.

Finally, Arabidopsis AtLOX2 and maize ZmLOX10 are found in between these two extremes as described next.

Most research on Arabidopsis LOX2 has been performed in the Columbia (Col) accession, where the HPL pathway is not functional and GLVs are hardly produced because of a natural mutation in the HPL gene (Duan et al., 2005). Other Arabidopsis accession do synthesize GLVs and traumatin derivatives (Kessler & Baldwin, 2001; Shiojiri et al., 2006; Nakashima et al., 2013), most likely via AtLOX2 but this has not yet been tested. In the Col ecotype AtLOX2 feeds the AOS pathway to contribute a large portion of wound-inducible JA in leaves and defences against herbivory (Glauser et al., 2009; Koo et al., 2009; Birtic et al., 2011). In contrast with tomato and potato where herbivory defence is mainly dependent on clade 1a 13-LOXs, Arabidopsis relies strongly in clade 2 AtLOX2 for defence although full herbivore performance is only reached in a mutant lacking all three AtLOX2, AtLOX3 and AtLOX4 (Schommer et al., 2008; Glauser et al., 2009; Chauvin et al., 2013; Grebner et al., 2013).

While it is possible that most plant LOXs use free unsaturated fatty acids as substrates, 13- LOX-mediated peroxidation of membrane-bound substrates occurs exclusively in two Arabidopsis species (A. thaliana and A. arenosa). These products remain esterified while

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processed by the AOS pathway and the resulting galactolipids containing esterified OPDA are termed arabidopsides (Boettcher et al., 2007; Stelmach et al., 2001). These are found in large quantities in several A. thaliana ecotypes and at least in the Col ecotype their synthesis is fully dependent on AtLOX2 (Glauser et al., 2009; Nakashima et al., 2013). Arabidopsides serve not only as major JA sources but also as direct defence compounds that inhibit the growth of fungus, bacteria and possibly herbivores (Andersson et al., 2006; Kourtchenko et al., 2007; Glauser et al., 2008).

Similar to Arabidopsis AtLOX2, maize ZmLOX10 is required for wound-induced JA accumulation. In consequence, Zm-lox10 mutants are impaired in resistance to herbivory.

ZmLOX10 function in JA synthesis is in part indirect through its role in GLV formation since GLVs strongly induce JA accumulation in maize and external GLV application partially restores JA synthesis upon wounding in the Zm-lox10 mutant (Engelberth et al., 2004;

Christensen et al., 2013). Additionally, it is still formally possible that ZmLOX10 directly feeds the AOS pathway for JA synthesis, similar to Arabidopsis AtLOX2. Further research is required to test the idea that the products of ZmLOX10 and AtLOX2 activity are able to feed both the AOS and HPL pathways.

HPL-generated lipids have important roles in plant defence such as the effective attraction of predators or parasitoid wasps that are natural herbivore enemies (Kessler & Baldwin, 2001;

Shiojiri et al., 2006). However, although these type of defence can be enhanced or reduced by increasing or decreasing HPL levels (Kessler & Baldwin, 2001; Shiojiri et al., 2006), down regulating the HPL pathway does not have a major negative impact on overall resistance to herbivores or pathogens (Kessler et al., 2004; Chehab et al., 2008). The Zm-lox10 mutant is impaired in parasitoid attraction but this is only in part due to the absence of GLVs since

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other volatile terpenes activated by JA also contribute to attraction(Christensen et al., 2013).

Moreover, as stated above, the reduced herbivory resistance of Zm-lox10 is ultimately attributable to the lack of JA production. In our view, the activity of jasmonate pathway is sufficient to resist herbivory in the laboratory if the HPL pathway is eliminated.

Concluding remarks

Flowering plants have organized defence responses to herbivory around plastidic 13-LOXs that generate several lipid-derived signals through the AOS or HPL pathways. While it seems clear that clade 1 13-LOXs specifically feed the AOS pathway for JA biosynthesis, clade 2 members may work in either one or both pathways. A wealth of evidence supports that JA synthesized through plastidic 13-LOXs is an indispensable signal for resistance to herbivory.

Maximum JA accumulation under attack is required to mount a full defence response. This may explain why the ancient divergence of plastidic 13-LOXs in two clades, which predates the monocot-dicot split, has not sufficed to completely entrust JA synthesis for defence to one clade or subclade over another (the Solanaceae being a possible exception). Even after such a long evolutionary path, 13-LOXs from both clades in Arabidopsis and maize still work together to directly or indirectly guarantee maximum JA synthesis during herbivory.

Additionally, each clade seems to have acquired particular temporal expression patterns that may contribute to that purpose. Thus, clade 2 13-LOXs are abundantly present in unchallenged leaves and may stimulate early JA accumulation (possibly in collaboration with clade 1b 13-LOXs such as AtLOX6 in the dicots). These first JA molecules probably activate the expression of clade 1a 13-LOXs, which are required for maximum JA production. At later

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times, JA also enhances the expression of clade 2 13-LOXs maybe as a priming mechanism.

The genes encoding 13-LOXs in Arabidopsis are also expressed in different tissues within the leaf (Chauvin et al., 2013), suggesting that non-overlapping spatial distributions may have enhanced the dependence on all 13-LOXs for full JA synthesis upon wounding in this species.

Differences in 13-LOX spatial localization may also facilitate channelling into the AOS or HPL pathways by expressing 13-LOXs that feed one or another in different organelles (Christensen et al., 2013) or even different sub-organelle compartments (Froehlich et al., 2001; Farmaki et al., 2007).

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Molecular biology and biochemistry approaches to study the jasmonate pathway

The jasmonate pathway belong to four critical steps 1) initiation 2) biosynthesis 3) perception and 4) JA gene expression (Figure 1). The initiation is thought to be controlled by a specific enzyme subfamilly, the 13-lipoxygenases (13-LOXs), followed the biosynthesis step mainly where JA and its biocative form JA-Ile play a key role (Feussner & Wasternack, 2002; Suza

& Staswick, 2008; Fonseca et al., 2009). The perception step is depending on whether the COI1 receptor detects JA-Ile, thus leading to the last step : jasmonate gene expression (Chini et al., 2007; Thines et al., 2007). Along this thesis project we focused a specific attention on the influence of the initiation steps of the biosynthesis of jasmonates and the jasmonate gene expression. We used a set of 13-lox single, double, triple and quadruple mutants ; JA and JA- Ile quantitative analysis and finally JAZ10 or VSP2 marker genes to understand which mechanisms in Arabidopsis leaves modulate the response to the wound and whether the four 13-LOXs are necessary for such a defence mechanism.

13-lox mutants

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A set of 13-lox single mutants was crossed in order to get combinations of double, triple and quadruple mutants (Caldelari et al., 2011). All these combinations provide powerful tools to understand the role of each 13-LOXs and underlines possible redundancy among these proteins or even compensatory mechanisms led by the 13-LOXs themselves. As shown for exemple in the case of male fertility, the importance of LOX3 and LOX4 could be only evidenced by the use of the double mutant lox3 lox4 (Figure 2).

Figure 1 : The four key steps of the jasmonate pathway. (A-B) JA is a derivative from lipid oxidation via lipoxygenase (LOX), allene oxide synthase (AOS), allene oxide cyclase (AOC) (plastids) and OPDA reductase 3 (OPR3) (peroxisome). Once in the cytosol JA is conjugated to isoleucine (Ile) via the jasmonate resistant 1 (JAR1) enzyme. (A) In the nucleus, under steady state, jasmonate-ZIM domain (JAZ) repressor binds to MYC2 transcription factor and jasmonate gene expression is blocked. (B) Once the plant is wounded JA-Ile migrates to the nucleus and promotes JAZ-COI1 interaction leading to the degradation of the JAZ repressors. Once MYC2 is free from the JAZ repressor, jasmonate-regulated genes such as JAZ10 and VSP2 are expressed. Pd : plasid, Px : peroxisome, C : cytosol and N : nucleus.

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Figure 2 : Redundancy among 13-LOXs in Arabidopsis flower development. LOX3 and LOX4 are redundant and important for anther maturation and normal dehiscence. Image from (Caldelari et al., 2011).

JA and JA-Ile

JA is a key component in the jasmonate pathway, being substrate for multiple JA dervatives such as JA-Ile (Suza & Staswick, 2008; Fonseca et al., 2009). JA-Ile is the bioactive form of JA and is necessary for the JAZ-COI1 complex to occur (Chini et al., 2007; Thines et al., 2007), thus making JA-Ile essential for the perception step and necessary for jasmonate gene expression (Figure 1).

Early and late jasmonate-responsive genes

JAZ10 and VSP2 are robust marker genes respectively for the early and the late wound

response (Chung et al., 2008; Dombrechta et al., 2007). In a wounded leaf, less than 15 minutes are necessary for JAZ10 gene to be induced (Glauser et al., 2009). JAZ10 gene expression reaches a peak at 1h after the wound and is then down-regulated. Although JAZ10

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gene is used as a marker for the early wound response, the JAZ10 protein belongs to the JAZ repressor family. JAZ are important repressors of the jasmonate signalling and sequester the MYC2 transcription factor, repressing thereby jasmonate gene expression (Figure 1).

In contrast to the JAZ10 protein, the VSP2 protein has been described to influence coleopteran and dipteran mortality and reducing their growth, however such a study on lepidopteran such as Spodoptera littoralis used in this thesis remains to be done (Liu et al., 2005). Despite evidence for the VSP2 protein to deter lepidopteran, the VSP2 gene gives an essential information on defence kinetic complementary to that of JAZ10 gene. Under herbivory attacks at least 6h are necessary for the full induction of VSP2 gene (Dombrechta et al., 2007; Verhage et al., 2011).

Spodoptera littoralis

Spodoptera littoralis, the Egyptian cotton worm, is widely used for herbivory bioassays (Chauvin et al., 2013; Glauser et al., 2009; Schweizer et al., 2013; Mérey et al., 2013). This generalist herbivore underlined the importance of the jasmonate pathway for defence against insect aggressions. Indeed, this insect grew almost four times faster on the jasmonate deficient line aos (allene oxide cyclase) and almost twice faster on a lox2-1 mutant than on a WT plant (Figure 3) (Glauser et al., 2009).

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Figure 3 : Jasmonates reduce Spodoptera littoralis voracity. Insects gain more weight and eat more on the jasmonate deficient line aos than on the WT.

WT! aos!

A 13-LOX hierarchy for defence in Arabidopsis thaliana

Thesis

Reference

A 13-LOX hierarchy for defence in Arabidopsis thaliana

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A*13<LOX*Hierarchy*For*Defence*in*Arabidopsis!thaliana

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Abstract

Résumé

Remerciements

Abbreviations

Contents

Contributions

Introduction

Function of plastidic 13-LIPOXYGENASES in plant defenses

Function of plastidic 13-LIPOXYGENASES in plant defenses

Introduction

Concluding remarks

References

Molecular biology and biochemistry approaches to study the jasmonate pathway

**A13<LOXHierarchyForDefenceinArabidopsis!thaliana**

AdelineChauvin