Delphine Marion

Céline Masclaux-Daubresse

Françoise Simon-Plas

Suayib Üstün

Juliette Jouhet

Keywords: Autophagy, Membranes, Lipids, Plants, Stress

Dernière partie à écrire mais sans doute la plus importante. Ça fait déjà quatre ans que j'ai intégré ce laboratoire, le temps passe vite. Tout au long de ma thèse, ainsi que pendant mon stage, je me suis senti comme en famille, et en étant si loin de mon pays, croyez-moi que ça a été très important pour moi. Que ce soit sur le plan scientifique ainsi qu'humain, les membres de ce laboratoire vous êtes incroyables et j'en garderai toujours un cher souvenir. Sans vous, cette épreuve aurait été bien plus difficile, et surtout beaucoup moins amusante. J'espère je n'oublie personne mais si c'est le cas, j'en suis désolé. Tout d'abord je tiens à remercier Amélie Bernard, pour ton encadrement, pour la confiance et les libertés que tu m'as accordé, ainsi que pour la rigueur scientifique que tu m'as inculquée. Je te remercie aussi pour ta bonne humeur, ta bonne volonté, le fait que tu m'as toujours motivé à aller plus loin, de faire de mon mieux ; ça m'a vraiment formé. Tu es une grande source d'inspiration, jeune maman, superbe chercheuse, toujours à l'écoute et disponible pour aider les autres, une vraie superwoman ! Je tiens aussi à remercier Fred qui a été un super encadrant de master ainsi qu'une grande source de bonnes vibes (« OH YEAH ») tout le long de cette thèse. Tes conseils et tes vastes connaissances en lipides m'ont beaucoup aidé à rédiger cette thèse. Merci à toi Jérôme pour toutes les blagues, les rires, mais surtout pour tes remarques qui m'ont souvent remis dans le droit chemin quand je me laissais un peu aller. Merci à Steph pour ta bonne humeur et aussi de m'avoir appris plein de choses à mon arrivé au labo. Merci aux nouveaux recrues de l'équipe autophagie Julie, Josselin et Clément, votre arrivée dans le groupe a d'avantage dynamisé l'équipe, great discoveries are on the way ! Vous m'avez aussi beaucoup aidé pour l'écriture de ma thèse, et je vous en remercie fortement. Merci à toi Amazigh pour toutes ces intéressantes discussions que nous avons eues tout au long de ces années. Tu es devenu un bon ami et on reste en contact. Merci aussi à Jessi, Julie,

Titre : Identification et caractérisation de la contribution des lipides dans l autophagie chez les plantes

Résumé :

Les plantes, étant des organismes sessiles, sont fréquemment confrontées à une grande variété de stress environnementaux. Ces conditions peuvent conduire à l'accumulation d'agrégats de protéines ou au disfonctionnement de multiples organites intracellulaires. Pour faire face à ces conditions, les plantes ont mis au point des mécanismes d'adaptation sophistiqués qui permettent le recyclage des composants intracellulaires. Ces mécanismes sont essentiels pour les remodelages métaboliques nécessaires à un recyclage efficace des nutriments ainsi qu'à l'élimination des composants nocifs pour la cellule comme des organites endommagés. L'un de ces mécanismes est l'autophagie, une voie de dégradation intracellulaire qui utilise des vésicules à double membranes qui encapsulent des portions du cytoplasme et les délivrent à la vacuole où elles sont dégradées. L'autophagie repose sur la formation de ces vésicules spécialisées, appelées autophagosomes (AP). Les AP sont des vésicules uniques dans le système endomembranaire, d'abord parce qu'elles sont constituées d'une double bicouche lipidique, et ensuite parce qu'elles ne bourgeonnent pas à partir d'un compartiment déjà existant. La biogenèse des AP est un processus en plusieurs étapes impliquant une machinerie centrale (protéines ATG) qui intervient dans la formation de novo d'une membrane initiale ; puis, par l'addi ion de lipide , ce e membrane agrandi e de ient ne r c re en forme de co pe a ec de bord for emen inc r l i perme an d englo ir la cargaison autophagique. Une fois la cargaison engloutie, ses bords fusionnent afin de fermer la structure, qui circule ensuite vers la vacuole, où sa membrane externe fusionne avec la membrane de la vacuole ce qui libère la membrane interne et la cargaison à l'intérieur de la vacuole pour leur dégradation. Ainsi, la biogenèse des AP repose sur de nombreux événements de remodelage membranaire, d'abord pour initier la membrane initiale, puis pour maintenir sa forme très incurvée tout en assurant son expansion, et enfin pour sceller les structures matures et promouvoir sa fusion ultérieure à la vacuole. Dans les membranes biologiques, les lipides, grâce à leurs propriétés physico-chimiques, définissent des caractéristiques importantes telles que la fluidité, la courbure ainsi que les champs électrostatiques des membranes. Par conséquent, le rôle crucial des lipides dans l'autophagie a émergé ces dernières années. Chez les plantes, on ne connait encore que très peu de choses sur la composition lipidique des membranes autophagiques et les fonctions des lipides dans la formation des AP restent largement méconnues. Mon travail de thèse a consisté à identifier des acteurs lipidiques et protéiques impliqués dans l'autophagie chez les plantes dans le but de caractériser leurs fonctions dans le processus. En effectuant un criblage d'inhibiteurs enzymatiques nous avons analysé l'impact de l'inhibition de la synthèse de différentes espèces de lipides sur l'autophagie. En utilisant cette approche, nous avons identifié différents candidats lipidiques importants pour l'autophagie des plantes. Notamment, nous avons identifié le phosphatydil-inositol-4-phosphate (PI4P) comme étant critique pour la formation des APs. En l'absence de PI4P, la formation des AP est stoppée à un stade très précoce, ce qui entraîne un blocage total dans le processus. De plus, nous avons obtenu des informations précieuses pour mieux comprendre la formation des APs chez les plantes. En particulier, nos résultats suggèrent que la membrane plasmique (PM) semble jouer un rôle important dans la formation de ces structures. Dans leur ensemble, nos résultats ont confirmé notre hypothèse initiale: les lipides ne sont pas seulement des éléments inertes qui constituent les membranes autophagiques; ils semblent plutôt jouer des rôles distincts et avoir des fonctions spécifiques dans le processus. Enfin, cette thèse a mis en évidence comment les lipides sont des acteurs clés du processus autophagique et par conséquent des adaptations des plantes aux conditions environnementales défavorables.

Mots clés : Autophagie, Membranes, Lipides, Plantes, Stress

Title : UNRAVELLING THE CONTRIBUTION OF LIPIDS IN PLANT AUTOPHAGY

Abstract :

Plants, being sessile organisms, are frequently confronted to a plethora of environmental stresses and harsh conditions. Enduring these conditions can lead to the accumulation of protein aggregates or organelles that become dysfunctional. To withstand these conditions,

plants have evolved sophisticated adaptation mechanisms for the recycling of intracellular components. These mechanisms are essential for the metabolic transitions required for efficient nutrient use, as well as proper disposal of protein aggregates or damaged organelles.

One of these mechanisms is autophagy, an intracellular degradation pathway that employs specialized double membrane vesicles that encapsulate cytosolic material and delivers it to the vacuole for degradation. Autophagy relies on the formation of these specialized vesicles, called autophagosomes (APs). APs are unique vesicles in the endomembrane system, first because they are made of a double lipid bilayer, and second because they do not but from a pre-existing compartment. AP biogenesis is a multistep process implicating a core machinery (ATG proteins) that mediate the de novo formation of an initial membrane; then, by the addition of lipids, this membrane expands into a cup-shaped structure with highly curved edges to engulf autophagic cargo. Upon completion, the rims of the structure seal and form a mature AP that traffics to the vacuole, where its outer membrane fuses with the tonoplast releasing the inner membrane and cargo inside the vacuole. Thus, AP biogenesis relies on numerous membrane remodeling events, first to initiate the initial membrane, then to maintain the highly curved shape of the structure while ensuring its expansion, and finally to seal the mature structures and its subsequent fusion to the vacuole. Lipids, thanks to their physicochemical properties define important membrane features such as its, fluidity, curvature and electrostatics. Hence, evidence showing the crucial role of lipids in autophagy has emerged in the recent years. In plants however, little is known about the lipid composition of autophagic membranes and thus, about the functional contribution of lipids in plant autophagy. My PhD thesis consisted on identifying crucial lipids for plant autophagy with an aim to characterize their function in the process. By performing a lipid-related enzymes inhibitor screen in which we assayed the impact of inhibiting the synthesis of specific lipids on autophagy, we identified different lipid candidates important for plant autophagy. Notably, we identified the phosphatydil-inositol-4-phosphate (PI4P) as being critical for the formation of APs. In the absence of PI4P, AP formation is stalled at a very early stage resulting in a block in the process.

Furthermore, we have obtained valuable insights to better understand the AP formation. In plants, particularly, our results suggest that the plasma membrane (PM) plays important roles

III -RESULTS AND DISCUSSION………………………………………………………………….

III-1 Identifying lipids required for autophagy: a lipid inhibitor screen…………………………….

III-2 Phosphoinositol-4-phosphate controls autophagosome formation in Arabidopsis thaliana III-3 Putative implication of a phospholipase A2 in autophagy………………………………...

IV -GENERAL DISCUSSION AND PERSPECTIVES……………………………………………

IV-1 Regarding the identification and functional characterization of lipids required for autophagy in plants cells……………………………………………………………………………… IV-2 PI4P is critical for plant autophagy.……………………………………………………………. The introduction consists in 2 reviews, one about lipids in autophagy, published in 2018 in Journal of experimental Botany, for which I participated in the writing at the very beginning of my thesis; the other one is a manuscript in preparation for a review to be published in the special issue of the journal Cells untitled: 'Membrane Lipids in the Interaction of Plants with Their Abiotic and Biotic Environment'. This review has been written in collaboration with J. Lupette, J. Castets and C. Chambaud, I was notably in charge of the section '1. Lipids are fundamental actors of the autophagy machinery Introduction Macroautophagy, hereafter referred to as autophagy, is a critical process in which a portion of the cell is enwrapped in a double membrane vesicle and delivered to the vacuolar lumen for degradation. Breakdown products are recycled back to the cytoplasm, ensuring the turnover of macromolecules used as building blocks to maintain cell homeostasis and promote plant survival upon various stresses (Masclaux-Daubresse et al., 2017). Key to the autophagy pathway is the formation of a unique and very specialized lipoproteic double membrane vesicle called the autophagosome [START_REF] Yang | New insight into the mechanism and function of autophagy in plant cells[END_REF]. The autophagosome is initiated de novo through a hierarchical multistep process starting with the nucleation of the so-called phagophore (Fig. 1). The phagophore expands, surrounding its cargo, and ultimately closes to form the double membrane autophagosome. Upon completion, the outer membrane of the autophagosome fuses with the lytic vacuole to deliver its cargo into the vacuolar lumen.

V -EXPERIMENTAL

Membrane dynamics, membrane sculpting and membrane remodeling events are critically required at several steps of the autophagy pathway. These are regulated by the interplay between the two structural and functional components of biological membranes: lipids and proteins. Early genetic screens in yeast discovered a group of dedicated AuTophaGy-related (ATG) proteins composing the essential core components of the autophagy machinery (Reggiori and Klionsky, 2013). ATGs were later identied by sequence homologies in other organisms such as mammals and plants, revealing a high conservation of the pathway among eukaryotes [START_REF] Kim | Genes for plant autophagy: functions and interactions[END_REF]Reggiori and Klionsky, 2013). The functions of ATG proteins remain poorly characterized in plants yet they are often divided into four functional groups based on their activities in other organisms. (i) The ATG1 complex, notably composed of the protein kinases ATG1 (ATG1a-c in Arabidopsis) and its regulatory subunits ATG3 (ATG13a,b in Arabidopsis), is involved in the induction of the process (Reggiori and Klionsky, 2013), although this complex may act at a later point in the pathway in Arabidopsis (Suttangkakul et al., 2011;Li et al., 2014). (ii) Upon induction of autophagy, the Vps34 complex is recruited at the phagophore to produce the lipid phosphatidylinositol-3-phosphate (PI3P), essential for the formation of the autophagosome [START_REF] Liu | Autophagy regulates programmed cell death during the plant innate immune response[END_REF].

(iii) Enrichment in PI3P notably recruits and/or stabilizes the membrane binding of a complex made of ATG18 and ATG2. These two proteins participate in the mandatory cycling of ATG9 between a cytosolic reservoir of small vesicles and the phagophore (Reggiori and Klionsky, 2013). (iv) Elongation and closure of the phagophore are mediated by ATG8 proteins, a large family composed of nine members in Arabidopsis. ATG8 is conjugated to the lipid phosphatidylethanolamine (PE) through the action of the protease ATG4 as well as two ubiquitin-like conjugation systems composed of ATG5, ATG12, ATG16, ATG10, ATG3, and ATG7. The lipidation of ATG8 to PE is strictly required for its recruitment to the phagophore and its function in the autophagy pathway (Liu and Bassham, 2012). While extensive research on the molecular mechanism of autophagy has mostly focused on the characterization of ATG proteins, studies on autophagy-related lipids remain scarce and comprehensive analyses of the lipid composition of autophagic compartments have not been reported in any kingdom. At present, only two lipids, PI3P and PE, are documented as being present on the phagophore membrane [START_REF] Yang | New insight into the mechanism and function of autophagy in plant cells[END_REF]. Both are strictly essential for autophagy, which speaks to the importance of lipids for the initiation and progression of the pathway. However, their relative concentration, although functionally critical, is still unknown. Other lipids composing the autophagic membrane remain elusive: several species are suggested to be important for autophagy but whether they are present on the phagophore membrane and/or directly or indirectly function in autophagy is unknown. Similarly, the origin of the phagophore membrane remains elusive; essentially every organelle of the endomembrane system has been implicated (Reggiori and Klionsky, 2013). Yet, to better understand the machinery of autophagy, the interplay between proteins and lipids in the formation and functions of the phagophore must be characterized. The present review focuses on our current understanding of membrane dynamics in the autophagy pathway in plants with emphasis on the lipids and membrane-modifying proteins specifying the initiation, expansion, and maturation of the autophagosome.

Autophagosome biogenesis requires a mosaic of complex and tightly regulated membrane shaping events Nucleation of the phagophore Autophagosomes (APs) are unique endomembrane compartments in their composition, morphology and biogenesis. Contrary to other endomembrane vesicles, autophagosomes are (i) formed of a double membrane and (ii) do not bud from a preexisting compartment. Instead, APs form de novo through a hierarchical multistep process starting with the assembly or nucleation of the initial membrane called the phagophore [START_REF] Yang | New insight into the mechanism and function of autophagy in plant cells[END_REF]. At steady state, most ATG proteins reside in the cytoplasm; upon autophagy induction, they rapidly relocalize to particular punctuate structures within the cell known as phagophore assembly sites (PASs; Reggiori and Klionsky, 2013). PASs consist of lipo-proteic cores where all ATG proteins converge and from which emerges the phagophore. The nature and architecture, or even the occurrence, of PASs are still highly debated and therefore the nucleation of the phagophore remains poorly characterized, especially in plants. In yeast and mammals, the Atg1/ULK1 complex composed of Atg1/ULK1, ATG13, and Atg17/29/31/FIP200 is the rst component of the autophagy machinery that is recruited at the PAS and therefore likely de nes the speci c autophagic identity of the membrane forming de novo (Karanasios et al., 2016). In-depth analyses of the ULK1 complex dynamics in mammals using high resolution microscopy proposed that it is recruited to endoplasmic reticulum (ER)-derived tubulovesicular compartments composed of ER-Golgi intermediate compartment (ERGIC) and ER exit site (ERES) coalescing with ATG9-containing vesicles (Fig. 1; Karanasios et al., 2016). A recent biochemical analysis of early autophagic structures suggests instead that the ULK1 complex translocates directly to speci c ER membrane subdomains enriched in phosphatidylinositol (PI) and then recruits the PI3-kinase (PI3K) complex (Nishimura et al., 2017). The authors propose that PI or PI3P shuttles from these domains to phagophore membrane precursors, possibly a coalescence of multiple membrane sources including ATG9 vesicles. A consensus model in mammals is that ULK1/ATG9-decorated membranes are used as a platform to nucleate the phagophore: they recruit further ATG proteins as well as lipids to assemble and shape the initial autophagic membrane that emerges from the PAS. In particular, the Atg1/ULK1 kinase is thought to act as a signal transducer mediating the activating phosphorylation of other ATG proteins while other components of the Atg1/ULK1 complex play a non-catalytic scaffolding role in the organization of the phagophore [START_REF] Lin | Structure and function of the ULK1 complex in autophagy[END_REF]. The yeast Atg1 binds preferentially to small highly curved vesicles in vitro and Atg1 membrane targeting is mediated by its C-terminal domain (Ragusa et al., 2012). This so-called early autophagy targeting/tethering (EAT) domain was found to promote vesicle tethering in vitro. Consequently it was proposed that Atg1 is involved in the tethering of Atg9 vesicles during the initiation of autophagy (Ragusa et al., 2012;[START_REF] Rao | The Atg1kinase complex tethers Atg9-vesicles to initiate autophagy[END_REF]. In mammals, the phagophore was found to nucleate at membrane contact sites between the ER and the plasma membrane (PM) as well as between the ER and the mitochondria, stressing the importance of exchanges of proteins and/or lipids between these organelles for the initiation of autophagy (Hamasaki et al., 2013;Nascimbeni et al., 2017).

In plants, little is known in terms of the function and localization of ATG1 proteins, yet in-depth analysis of another early marker of the autophagosome formation, ATG5, showed that phagophores form at sites in close proximity with speci c cradle-like ER subdomains (Fig. 1). Similarly to mammals, these subdomains could be a platform for the nucleation and further expansion of the phagophore (Le Bars et al., 2014). Fine ultrastructural EM studies and localization analyses of the autophagy-related proteins ATG5 and SH3P2 show occasional contacts between the phagophore and the ER membrane (Zhuang et al., 2013;Le Bars et al., 2014). Further electron tomography analyses of an Arabidopsis atg9 mutant suggested some direct continuity between the ER and a structure positive for ATG8 and ATG18 (Zhuang et al., 2017). From these latter data, the authors proposed that the phagophore emerges from ER subdomains and that ATG9 or ATG9-brought components are required for dissociation of the elongated phagophore from these domains. However, this hypothesis is mainly proposed from observations based on the atg9 mutant, which still presents residual autophagy (Shin et al., 2014), and remains to be validated by further experimental studies. Still and similarly to what has been proposed for mammalian cells, it is plausible that, in plants, ATG9 vesicles coalesce with other vesicles in the vicinity and/or in contact sites with ER subdomains to shape the initial autophagic structure.

Expansion and maturation of the phagophore

The phagophore is an extremely organized structure made of a cup-shaped double membrane with highly curved edges (Fig. 1). To ensure the formation of proper APs, the initial membrane must expand with respect to its particular architecture and avoid premature fusion whilst ensuring cargo sequestration. This implies that the expansion phase of the phagophore is more than a mere elongation of the membrane. Instead, it must be a highly regulated process that orchestrates multiple events to organize the growth, shape and architecture of the structure.

The length of the phagophore membrane is regulated and depends on the type of autophagy. In Arabidopsis cells confocal microscopy analyses showed initial green uorescent protein (GFP)-ATG8e decorated structures expand from a size of 0.5 µm or less to ring-like structures reminiscent of APs with a diameter of over 1 µm [START_REF] Merkulova | Assessment and optimization of autophagy monitoring methods in Arabidopsis roots indicate direct fusion of autophagosomes with vacuoles[END_REF]. On a given stress most APs are in the same size range indicating that the length of the phagophore membrane is nely regulated prior to its closure [START_REF] Merkulova | Assessment and optimization of autophagy monitoring methods in Arabidopsis roots indicate direct fusion of autophagosomes with vacuoles[END_REF]Le Bars et al., 2014). Furthermore, plant selective autophagy mediates the degradation of cargoes of various sizes up to several micrometers for larger organelles such as mitochondria and chloroplasts (Izumi et al., 2017). Therefore, the mobilization (i.e. delivery or synthesis) of lipids for membrane growth must be particularly controlled; their quantity and availability will determine the size and/or number of APs.

The geometry of lipid molecules and their assembly within the bilayer are also critical aspects of membrane morphology. Therefore, the repartition of incoming lipids and proteins at the phagophore must be tightly regulated in time and space, by promoting either lateral diffusion or segregation of independent molecules, creating speci c microenvironments within the phagophore. The existence of particular subdomains, with speci c protein and lipid composition, within the phagophore would likely promote protein binding or activity, membrane uidity and membrane morphology. The highly curved edges of the phagophore membrane are a good example of such specialized domains: their structure likely relies on a speci c lipid composition and by the implication of membrane shaping proteins to stabilize such a spatial structuration. The particular morphology of the phagophore also suggests a differential composition between the inner and the outer layer of each of its two membranes to promote and stabilize its overall curvature. This could be marshalled by actin laments (Wang et al., 2016) as well as Lipids in the formation of autophagic membranes in plants | Page 3 of 13 Downloaded from https://academic.oup.com/jxb/advance-article-abstract/doi/10.1093/jxb/erx392/4626680 by Universite de Bordeaux user on 20 November 2017 other cytoskeleton elements. The activity and fate of the outer membrane of the APs greatly differ from that of the inner membrane, which further supports that they differ in structural and functional composition. (i) On the one hand, most ATG proteins are recycled from the phagophore at the time of its closure (Reggiori and Klionsky, 2013; Le Bars et al., 2014), which implies that they must preferentially locate to the outer membrane of the phagophore. After fusion with the lytic vacuole, the outer lipid bilayer will fuse with the vacuolar membrane, while the inner bilayer enclosing cargo will be delivered into the vacuolar lumen. This suggests that the outer membrane of the phagophore/AP speci cally collects particular lipoproteic components mediating membrane fusion. SNARE proteins are proposed to participate in this step (Surpin et al., 2003) and the formation of SNARE complexes as well as their activity is greatly in uenced by the lipid composition in their vicinity [START_REF] Mima | Complex lipid requirements for SNARE-and SNARE chaperone-dependent membrane fusion[END_REF]. The fusion of the outer membrane of the AP with the tonoplast likely leads to a massive local input of AP lipids. How this impacts on the structure and functions of the vacuolar membrane and how this process is regulated are not known. This is particularly critical for plants given the importance of the tonoplast in osmoregulation [START_REF] Gao | Dynamics of vacuoles and actin filaments in guard cells and their roles in stomatal movement[END_REF]. In regard to this, it is plausible that a speci c lipid composition of AP outer membrane is required to reduce potential effects on vacuolar homeostasis. (ii) On the other hand ATG8, and possibly other proteins, decorates both the outer and the inner membrane of the phagophore where they play different roles [START_REF] Kirisako | Formation process of autophagosome is traced with Apg8/Aut7p in yeast[END_REF]. ATG8 proteins located on the outer bilayer are thought to be involved in the elongation and closure of the phagophore (Xie et al., 2008), while ATG8 located in the inner membrane are required for the recognition of selective cargoes (Xie et al., 2016). Furthermore, upon fusion of APs with the tonoplast, the inner membrane bilayer will be degraded by lumenal hydrolases and lipases. How vacuolar enzymes discriminate between the vacuolar membrane and the inner membrane of the AP remains puzzling. Again, a speci c lipoproteic composition of the inner bilayer may be a structural or physiological key to ensure these events. In mammals, a recent report proposes that the inner membrane of the AP undergoes ATG3-mediated ssion and that this is required for vacuolar enzymes to access and degrade the autophagic bodies [START_REF] Tsuboyama | The ATG conjugation systems are important for degradation of the inner autophagosomal membrane[END_REF]. Whether such a mechanism is also in place in plant cells awaits further studies.

In sum, multiple lines point to a differential partition of both lipids and proteins within the phagophore. At this point, the molecular mechanisms governing these processes remain completely unknown in plants and largely unexplored in other systems.

Completion of the phagophore and fusion with the vacuole

Once the phagophore has reached the desired size and composition, it closes, completing the autophagosome (Fig. 1). The ATG8 conjugation system could play a role in the fusion of the phagophore edges [START_REF] Kaufmann | Molecular mechanism of autophagic membrane-scaffold assembly and disassembly[END_REF] yet the regulation of this process and the speci c proteins and lipids involved are not characterized in plant cells. As mentioned before, most ATG proteins are recycled from the phagophore at the time of its closure to be reused in the next round of AP biogenesis (Reggiori and Klionsky, 2013). In Arabidopsis, at least a portion of ATG1 and ATG13 present in the inner membrane of the phagophore are autophagy cargoes and are subjected to degradation (Suttangkakul et al., 2011). This is also the fate of ATG8 proteins decorating the inner membrane on the AP (Le Bars et al., 2014). However, ATG8 located at the outer membrane is dissociated by the action of the protease ATG4, which cleaves the bond between ATG8 and PE (Yoshimoto et al., 2004;Woo et al., 2014). Our understanding of the mechanism(s) by which other ATG proteins are excluded from the phagophore remains poor. It is largely assumed that a particular membrane composition is involved in the recruitment and binding of ATGs at the phagophore; a particular lipid pro le may also be key to their dissociation upon closure. For example, the hydrolysis of PI3P is required for the ef cient dissociation of ATG proteins from the phagophore and the completion of the autophagosome in yeast (Cebollero et al., 2012). This further suggests that, similar to ATG proteins, speci c lipids involved in phagophore nucleation/elongation may be recycled upon AP completion.

Once closed, the AP reaches the vacuole by an unknown mechanism possibly involving the cytoskeleton (Liu and Bassham, 2012). Along the way, APs may mature through interactions with other endomembrane compartments, such as endosomes, to collect lipids or proteins required for its fusion with the vacuolar membrane (Fig. 1). This hypothesis is supported by recent studies revealing interactions between autophagy and the endosomal sorting complexes required for transport (ESCRT) machinery in plants (Zhuang et al., 2015; for further details see the review by Erika Isono and co-worker in this special issue). Upon fusion of the outer membrane of the AP with the tonoplast, a new compartment composed of the inner membrane surrounding the cargo, called the autophagic body, is delivered to the vacuolar lumen for degradation (Fig. 1). To date, nothing has been reported on the enzymes mediating the degradation of the autophagic body as well on the mechanism by which molecules are recycled from the vacuole to the cytoplasm in plant cells.

Lipids are critical, yet largely unexplored, components of the autophagosome biogenesis machinery

Autophagy consists of myriad membrane events that must be very tightly orchestrated and regulated to ef ciently proceed to AP formation and subsequent cargo delivery and degradation. The molecular mechanisms underlying such events largely depend on the interplay of proteins and lipids conferring the particular membrane properties and activities on the phagophore. Yet, while ATG proteins have been extensively characterized, particularly in yeast and mammals, the identity and functions of lipids composing the phagophore and AP membranes remain largely unexplored.

Lipids in biological membranes

Biological membranes are composed of proteins and lipids that act synergistically to promote membrane structure and functions. Based on their physicochemical properties, lipids Page 4 of 13 | Gomez et al.

Downloaded from https://academic.oup.com/jxb/advance-article-abstract/doi/10.1093/jxb/erx392/4626680 by Universite de Bordeaux user on 20 November 2017 form the physical boundary de ning the boundaries of the cell itself as well as that of organelles within the cell (van Meer et al., 2008). Yet lipids do more than provide a mere barrier. Their great structural and chemical diversity provides uidity, thickness and plasticity to membranes notably controlling curvature, fusion or ssion events (van Meer et al., 2008). Three main classes of lipids are differentiated based on their structures: glycerolipids, sterols and sphingolipids (Fig. 2A). Among glycerolipids, phospholipids and galactolipids are major building blocks of biological membranes while neutral lipids such as glycerides are packed into lipid droplets or oil bodies used for energy storage [START_REF] Furt | Lipids of the plant plasma membrane[END_REF]. Phospholipids and galactolipids as well as sphingolipids and sterols consist of a polar head and a hydrophobic tail (Fig. 2A). The amphipathic properties of these lipids drive the spontaneous formation of membrane bilayers in aqueous environments: the hydrophobic tails, composed of the acyl chains, self-associate and the hydrophilic heads interact with each other as well as with water (Fig. 2B). In phospholipids and galactolipids the polar head is esteri ed on a glycerol backbone with two acyl chains; sphingolipids are composed of a long-chain base (LCB) amidated by an acyl chain and a polar head esteri ed to the LCB and sterols are isoprenoids formed by a moiety made of four rigid rings that is hydroxylated. Phytosterols can be further processed by the addition of a sugar, which can be subsequently acylated (Fig. 2A; [START_REF] Furt | Lipids of the plant plasma membrane[END_REF]. Polar heads greatly differ among different lipids from the relatively small head of sterols composed of a hydroxyl group to the large polyglycosylated forms of sphingolipids. The nature and size of the polar heads, notably that of phospholipids, greatly in uence the organization of the membrane they compose (Jouhet, 2013). Cylinder-shaped lipids, such as phosphatidylcholine (PC), phosphatidylserine (PS) or PI, show a head and a tail of about the same diameter and form bilayers (Fig. 2B). Inverted cones lipids, such as lysophospholipids or phosphoinositides, are characterized by a bigger head than a tail, which promotes the formation of positive curve when they are inserted in membranes. In contrast, inclusion of conical lipids such as PE, phosphatidic acid (PA) or cardiolipins, with a relatively small size of their head group, imposes negative curvature (Jouhet, 2013). Membrane curvature relies not only on the nature of the lipids but also on their asymmetric distribution between the two lea ets of the bilayer as well as the action of membrane-shaping proteins required to stabilize such curvature and/or to promote lipid asymmetry (Fig. 2B,C; van Meer and Sprong, 2004). Multiple proteins are involved in the formation of membrane curvature; insertion of intrinsic proteins can promote localized curvature and membrane asymmetry while scaffolding proteins, such as BAR-containing proteins, assemble into rigid curved oligomers that bind to membranes and force their deformation [START_REF] Shen | SnapShot: membrane curvature sensors and generators[END_REF]. Membrane curvature is essential for many cellular functions, including autophagy, by promoting the ssion and fusion of membranes (Fig. 2C). Such events are also dependent on membrane thickness and uidity, which are modulated by the structure of lipids, and in particular by the length of their hydrophobic tails as well as their degree of unsaturation [START_REF] Furt | Lipids of the plant plasma membrane[END_REF]. Sphingolipids have rather saturated tails, form taller and narrower cylinders than similarly shaped phospholipids, and pack more tightly in the membrane increasing its degree of order (van Meer et al., 2008). Addition of sterols in sphingolipid-enriched membranes is critical to maintain uidity by inducing an intermediate liquid-ordered phase with both a high mobility and a high conformational order in the lipid acyl chain (van Meer et al., 2008). Such liquid-ordered membrane domains, such as lipid rafts in the plasma membrane of plant cells, specify particular functional subdomains within the membrane and carry out critical activities notably acting as signaling platforms [START_REF] Furt | Lipids of the plant plasma membrane[END_REF]. Lipids are further uniquely interactive with proteins in all cellular processes. Individual lipids, such as phosphoinositides, or the assembly of lipids into favorable microenvironments can promote the binding, stability, segregation or dispersion as well as the activity of particular proteins or protein complexes (van Meer et al., 2008). They further participate in the organization of interfaces and/or contact between the membrane of two organelles (i.e. membrane contact sites) and the transfer of components from one to another. In addition, lipids play critical roles as signaling molecules for the regulation of multiple processes (van Meer et al., 2008).

More than 1000 lipid species are present in the plant cell, which points to the variety of process in which they participate [START_REF] Furt | Lipids of the plant plasma membrane[END_REF]; yet, all lipids are not equally distributed among organelles. For examples, galactolipids are almost exclusively plastidial [START_REF] Dubots | Role of phosphatidic acid in plant galactolipid synthesis[END_REF], cardiolipins are found in the mitochondria [START_REF] Paradies | Functional role of cardiolipin in mitochondrial bioenergetics[END_REF] and, while synthesized in the ER, sterols are rapidly transported through the secretory pathway to the plasma membrane [START_REF] Furt | Lipids of the plant plasma membrane[END_REF]. Sphingolipids are also critical components of the PM as well as the Golgi apparatus where part of their biosynthesis occurs (Melser et al., 2010). Speci c lipid composition likely confers identity and function on a speci c compartment notably by shaping its structure but also by organizing the optimal environment for protein structure, mobility and activity.

Structural lipids in the formation of autophagic compartments

Several classes of lipids have been implicated in the signaling and/or regulation of autophagy, such as sphingolipids in mammals (see Dall 'Armi et al., 2013;Carlsson and Simonsen, 2015). Two main lipids, PI3P and PE, are known to be present on the phagophore and are essential structural components of the autophagosome membrane. Their roles in the formation of autophagosomes as well as the potential involvement of other lipids are detailed below.

PI3P.

As mentioned before, the rst step in phagophore assembly in yeast and mammals is the recruitment of the Atg1 (ULK1) complex and it was proposed that this complex is recruited at speci c ER subdomains enriched in PI (Nishimura et al., 2017). Consistently, in vitro experiments showed that yeast Atg1 preferentially binds to highly curved membranes enriched in PI and/or PI3P through its EAT domain (Ragusa et al., 2012;[START_REF] Rao | The Atg1kinase complex tethers Atg9-vesicles to initiate autophagy[END_REF]. At the PAS, the ATG1 complex, and speci cally ATG13, is required to further recruit ATG14 [START_REF] Jao | A HORMA domain in Atg13 mediates PI 3-kinase recruitment in autophagy[END_REF]. Conserved from yeast to mammals but absent in plants, ATG14 is the autophagy-speci c component of the PI3K complex that targets the synthesis of PI3P at the phagophore [START_REF] Diao | ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes[END_REF]. Although its function is not entirely understood, it is well established that PI3P is a critical lipid for the biogenesis of the autophagosome in every kingdom including plants. Arabidopsis and tobacco depleted for ATG6, a component of the PI3P complex, show phenotypes reminiscent of autophagy-de cient mutants (Liu and Bassham, 2012). Further, the use of wortmanin, an inhibitor of PI3K activity, blocks autophagy at a very early stage [START_REF] Yang | New insight into the mechanism and function of autophagy in plant cells[END_REF]. However, the plant autophagy-speci c PI3K complex is poorly characterized: its composition is unknown and its localization at the phagophore remains speculative. In particular, it is still unclear whether a protein with a function similar to that of ATG14 exists in plants and/or how else the complex is targeted to the phagophore. In fact, while the ATG1 complex is also involved in plant autophagy, its involvement in the recruitment of the PI3K complex remains undocumented and the characterization of the hierarchy of ATG recruitment at the phagophore in the plant cells still awaits experimental studies. PI3P likely plays multiple roles in autophagy, notably by recruiting or stabilizing the membrane binding of several autophagy-related proteins. In yeast and mammals, these include the protein Atg18 in complex with Atg2 (Proikas-Cezanne et al., 2015). In Arabidopsis ATG18 is part of a large family of eight proteins (Atg18a-h) with conserved WD repeats characteristic of binding motifs for phosphoinositides (Xiong et al., 2005); yet, whether any of the isoforms effectively bind PI3P is not known. Arabidopsis ATG18a RNAi lines do not form autophagosomes indicating that the corresponding protein is critical for autophagy although its function is still undetermined (Xiong et al., 2005). Likewise, the function and involvement of other ATG18 members in autophagy remain unresolved.

In Arabidopsis, a recently identi ed protein called SH3P2 shows a dual interaction with PI3K: it promotes the formation of PI3K foci in protoplasts and PI3K activity is required for SH3P2 co-localization with ATG8 (Zhuang et al., 2013). Plant lines knocked down for SH3P2 are defective in autophagy and show reduced levels of autophagosomes and autophagic bodies. SH3P2 interacts with ATG8 yet ATG8 lipidation is greatly induced in the mutant indicating that the protein may act at the elongation and/or closure step (Zhuang et al., 2013). SH3P2 is a BAR domain-containing protein with a role in membrane shaping events and vesicle tubulation (Zhuang et al., 2013;[START_REF] Ahn | SH3 domain-containing protein 2 plays a crucial role at the step of membrane tubulation during cell plate formation[END_REF]. The deletion of SH3P2 is lethal, indicating that, in contrast to ATG proteins, SH3P2 also has functions beyond autophagy and suggesting that its involvement in the process may be indirect. In fact, the protein may be itself degraded by the autophagy pathway (Zhuang et al., 2013).

PI3P is also involved in the maturation of the APs, and in mammals it promotes the recruitment of endosome-resident proteins that mediate AP fusion with the lysosome, the functional equivalent of the plant lytic vacuole [START_REF] Pankiv | FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end-directed vesicle transport[END_REF][START_REF] Olsvik | FYCO1 contains a C-terminally extended, LC3A/B-preferring LC3-interacting region (LIR) motif required for efficient maturation of autophagosomes during basal autophagy[END_REF]. In plants, direct crosstalk also exists between endosome-associated components and autophagy (Zhuang et al., 2015). For example the ESCRT protein FREE1 interacts with both ATG8 and the PI3K complex (Gao et al., 2015). The free mutant accumulates structures reminiscent of APs and shows increased interaction between APs and multivesicular bodies. It was also proposed that the ESCRT machinery participates in the phagophore expansion and/or closure through its interaction with PI3P (Zhuang et al., 2015).

PI3P could also participate in the architecture of the phagophore; with a head larger than a tail, PI3P is an inverted conical lipid that, when clustered, could promote the initial membrane deformation/curvature of the phagophore. Additionally, PI3P may participate in the shaping of the phagophore through its interaction with the actin capping protein of muscle Z-line (CapZ), which modulates the morphology of autophagic structures in human cells [START_REF] Mi | CapZ regulates autophagosomal membrane shaping by promoting actin assembly inside the isolation membrane[END_REF]. Nevertheless, the involvement of PI3P in the structure of the autophagic compartments is not characterized, notably because the quantitative and qualitative lipid composition of the phagophore and APs has not been established yet. In yeast, PI3P is concentrated in the internal surface of the phagophore [START_REF] Obara | Transport of phosphatidylinositol 3-phosphate into the vacuole via autophagic membranes in Saccharomyces cerevisiae[END_REF], while in mammals it is enriched at the phagophore rim [START_REF] Zhong | Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1-phosphatidylinositol-3-kinase complex[END_REF]. Further, while enrichment of PI3P is a prerequisite for the formation of the phagophore, PI3P hydrolysis is also required for the dissociation of Atg proteins before closure of the phagophore in yeast (Cebollero et al., 2012). Yet, PI3P is also involved in the maturation of the APs as well as during their fusion with the lysosomes in mammals [START_REF] Olsvik | FYCO1 contains a C-terminally extended, LC3A/B-preferring LC3-interacting region (LIR) motif required for efficient maturation of autophagosomes during basal autophagy[END_REF]. Therefore, concentration and local repartition of PI3P vary in the autophagic structures and are likely critical for autophagy. However, how PI3P molecules are assembled and organized within plant autophagic structures and how levels are regulated by both synthesis and hydrolysis by phosphatases is not known.

Other phosphoinositides are involved in autophagy. In mammals, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) is required for the binding of ATG14 and the assembly of the autophagy-speci c PI3K complex at the phagophore [START_REF] Tan | PtdIns(4,5)P 2 signaling regulates ATG14 and autophagy[END_REF]. Phosphatidylinositol 3,5-bisphosphate (PI(3,5) P 2 ) is implicated in the maturation and turnover of APs in mammals and ies (Dall 'Armi et al., 2013). In yeast, Pik1 produces PI4P and is required for the progression of autophagy. Pik1 is an essential protein that functions in the vesicle sorting at the trans-Golgi network and could participate in ATG9 traf cking to the PAS (Wang et al., 2012). Further studies are needed to better characterize the role of PI3P and other phosphoinositides in plant cells.

PE.

Across kingdoms, ATG8/LC3 (one of the ATG8 equivalents in mammals) is a central protein in the autophagy machinery. ATG8 is conjugated to the lipid PE through a multi-step ubiquitin-like conjugation pathway that requires the activity of seven independent ATG proteins, ATG3,4,5,7,10,12,and 16, which are conserved from yeast to mammals and plants [START_REF] Avin-Wittenberg | Variations on a theme: plant autophagy in comparison to yeast and mammals[END_REF]. In Arabidopsis, ATG8 is part of a large family of nine isoforms with two structurally truncated and putatively 'unconjugatable' ATG8 (isoforms h and i). ATG8 isoforms show differential patterns of expression and potentially different interacting partners, suggesting that they may have specialized functions [START_REF] Avin-Wittenberg | Variations on a theme: plant autophagy in comparison to yeast and mammals[END_REF]. Mutants in the conjugation of ATG8, such as atg5 or atg7, show a complete block in autophagosome formation [START_REF] Kim | Genes for plant autophagy: functions and interactions[END_REF] presumably at the elongation step although ultrastructural EM analyses are missing to determine the nature of any autophagic structures present in these mutants. In yeast, the level of Atg8 correlates with the size of the autophagosomes suggesting that Atg8 is key to the elongation of the phagophore. Therefore, PE plays a critical role in autophagy by recruiting Atg8 to the growing membrane. In Arabidopsis, acyl-CoA-binding protein 3 (ACBP3) is preferentially expressed in senescing conditions and strongly binds PE and PC. ACBP3 may regulate autophagy by competing with ATG8 over the pool of PE upon senescence. In fact, overexpression of ACPB3 decreases the number of ATG8e-labelled structures suggesting that autophagosome formation is impaired [START_REF] Xiao | Overexpression of Arabidopsis acyl-CoA binding protein ACBP3 promotes starvation-induced and age-dependent leaf senescence[END_REF].

ATG3 is involved in the last step of ATG8 conjugation with PE. In mammals, ATG3 is recruited to the phagophore through its amphipathic helix, which preferentially inserts into membrane with lipid-packing defects (Nath et al., 2014). It is speculated that ATG3 is recruited at the highly curved edges of the phagophore where it would localize the lipidation of LC3/ATG8. PE is a conical lipid with a small head-group and a relatively wide unsaturated acyl chain, and therefore high PE concentrations could lead to local lipid packing defects promoting or sustaining high curvature at the edges of the phagophore [START_REF] Shatz | Complex relations between phospholipids, autophagy, and neutral lipids[END_REF]. The Arabidopsis genome contains a homologue to ATG3 that has not been characterized yet [START_REF] Avin-Wittenberg | Variations on a theme: plant autophagy in comparison to yeast and mammals[END_REF]. Likewise, the precise localization of plant ATG8 members within the phagophore membrane still awaits further study.

While PE plays a conserved critical role in autophagy, the concentration of this lipid in the phagophore remains undetermined and the possibility of a local gradient of PE within the phagophore has not been tested. This, however, is of particular importance for the understanding of autophagy and notably for the characterization of ATG8's functions. In fact, ATG8 was predicted to have a fusogenic capacity; however, the particular study relied on the use of liposomes with articially high levels of PE [START_REF] Nakatogawa | Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion[END_REF]. In contrast, ATG8 did not show fusogenic activity using liposomes with levels of PE similar to those found in endomembranes [START_REF] Nair | SNARE proteins are required for macroautophagy[END_REF]. Therefore, it is critical to determine the lipid composition of the phagophore to fully understand its dynamics and mechanism of formation. An interesting hypothesis comes from Nguyen and coworker who raised the idea that ATG8/LC3-PE is only fusion-active at speci c highly curved membranes, such as phagophore extremities and/or membrane donor vesicles (Nguyen et al., 2017).

The complex of ATG5-ATG12 is also required for the lipidation of ATG8 to PE at the phagophore [START_REF] Yang | New insight into the mechanism and function of autophagy in plant cells[END_REF]. In-depth microscopy analysis found the ATG5 protein speci cally at the phagophore aperture in Arabidopsis cells (Le Bars et al., 2014). This suggests that the lipidation of ATG8 is highly localized within the membrane and/or that, as suggested in other organisms (Romanov et al., 2012), ATG5 could be involved in the closure of the phagophore in plants. However, at this time the mechanism by which ATG5-ATG12 is recruited at the phagophore and how it is partitioned within the membrane is not known in plants. In yeast and mammals, the Atg5-Atg12 conjugate forms a complex with Atg16 (ATG16L1 in mammals). ATG16L1 is recruited to the phagophore by the PI3P binding protein WIPI2, the homologue of ATG18 (Dooley et al., 2014). The Arabidopsis genome shows a homologue to ATG16 that has not been functionally characterized to date [START_REF] Avin-Wittenberg | Variations on a theme: plant autophagy in comparison to yeast and mammals[END_REF]. In vitro experiments showed that the recruitment of the yeast Atg12-Atg5/Atg16 complex to membranes relies on Atg5's ability to itself bind membranes (Romanov et al., 2012). Preventing the binding of Atg5 to membrane blocks autophagy yet it does not prevent its localization at PASs, which is mediated by the interaction of Atg12 with Atg8 [START_REF] Kaufmann | Molecular mechanism of autophagic membrane-scaffold assembly and disassembly[END_REF].

PA.

In mammals, phospholipase D1 (PLD1), which synthesizes the lipid PA, localizes at the phagophore in a PI3P-dependent manner, and its absence greatly impacts the progression of autophagy (Dall [START_REF] Armi | The Phospholipase D1 Pathway Modulates Macroautophagy[END_REF]. Further, the recently characterized protein HS1BP3 regulates autophagy by modulating the content of ATG16-positive autophagosome precursor membranes through PLD1 activity and localization (Holland et al., 2016). Therefore, it is speculated that local synthesis and a gradient of PA are prerequisites for AP formation. Whether PA is also involved in plant autophagy is unknown at this point. PA, like PE, is a cone-shaped lipid and therefore could be implicated in the deformation of the membrane to promote sharp negative curvature. In line with PE's properties, a local enrichment in PA could facilitate the insertion of lipid packing defectsensitive proteins such as ATG3 to promote the lipidation of ATG8 (Nath et al., 2014). Besides its possible structural involvement, PLD1-derived PA is an important signaling molecule for the regulation of the mammalian target of rapamycin complex (mTORC1) activity. First, PA competes with the mTORC1 inhibitor rapamycin by directly binding onto mTORC1's FKBP12 rapamycin binding domain with a higher af nity [START_REF] Fang | Phosphatidic acid-mediated mitogenic activation of mTOR signaling[END_REF]. Second, PA binding stimulates mTORC1 activity [START_REF] Yoon | Phosphatidic acid activates mammalian target of rapamycin complex 1 (mTORC1) kinase by displacing FK506 binding protein 38 (FKBP38) and exerting an allosteric effect[END_REF]. Third, PA promotes the removal of the mTORC1 inhibitor DEPTOR [START_REF] Yoon | Rapid mitogenic regulation of the mTORC1 inhibitor, DEPTOR, by phosphatidic acid[END_REF]. TOR is a conserved master regulator of nutrient availability and a repressor of autophagy among eukaryotes; upon nutrient starvation, TOR inhibition is a prerequisite for the induction of autophagy (Liu and Bassham, 2012;[START_REF] Feng | How to control self-digestion: transcriptional, post-transcriptional, and post-translational regulation of autophagy[END_REF]. Therefore, PLD1 derived PA plays opposite roles in autophagy by both promoting the formation of the autophagosomes and repressing the induction of the pathway. One possible explanation is that differentially localized PLD1 pools are differentially regulated and/or provide PA with different roles. At the phagophore, PA would be incorporated in the membrane playing a proautophagy role, while at the vacuole PA could be required to nely tune TOR and autophagy activity. The Arabidopsis genome contains 12 PLDs annotated as PLDα (1)(2)(3), β (1,2), γ (1-3), δ, ε and ζ(1,2) based on sequence similarities and enzymatic properties (Hong et al., 2016). PLDs differ in expression patterns and subcellular localization. Their activity increases rapidly in response to various environmental stresses and plants defective in individual PLDs show alterations in diverse biological processes suggesting that they cover a broad range of functions. Mammalian PLD1 and PLD2 prefer PC over PE as substrate; in Arabidopsis, most PLDs utilize PC, PE and phosphatidylglycerol (PG) albeit with different preferences. PLDα1 and PLDα3 prefer PC to PE whereas PLDδ and PLDγ1 hydrolyse PE at a higher rate (Hong et al., 2016). Although a role for PLDs and PA has not been reported in plant autophagy, PLDε is an important factor in nitrogen signaling in Arabidopsis as well as in Brassica napus. Overexpression of PLDε increases PA levels and enhances root growth under severe nitrogen deprivation in both plants [START_REF] Hong | Phospholipase Dε and phosphatidic acid enhance Arabidopsis nitrogen signaling and growth[END_REF][START_REF] Lu | Phospholipase Dε enhances Braasca napus growth and seed production in response to nitrogen availability[END_REF]. Conversely, the Arabidopsis pldε knock-out mutant shows a decrease in biomass production in conditions of low nitrogen, a phenotype somewhat reminiscent of mutants with defects in the autophagy pathway [START_REF] Hong | Phospholipase Dε and phosphatidic acid enhance Arabidopsis nitrogen signaling and growth[END_REF]. These data suggest that PLDε could be involved in autophagy, but a direct link has yet to be proven. Of note, while PLDε shows higher activity towards PC in vitro, the content of PC is not affected in the pldε mutant. Instead, 10 levels of PE decrease concomitant to an increase in PA when PLDε is overexpressed suggesting that the enzyme may prefer PE to PC as a substrate [START_REF] Hong | Phospholipase Dε and phosphatidic acid enhance Arabidopsis nitrogen signaling and growth[END_REF]. This provides another potential interplay of PLDε with autophagy given the critical importance of PE for the progression of autophagosome formation.

Glycerides. In the cell, neutral lipids such as glycerides are stocked in cytosolic structures called lipid droplets (LDs; [START_REF] Walther | Lipid droplets and cellular lipid metabolism[END_REF]. In mammals, a selective type of autophagy called macrolipophagy mediates the speci c degradation of LDs [START_REF] Weidberg | Lipophagy: selective catabolism designed for lipids[END_REF]. In rice anthers but not in Arabidopsis, autophagy mediates the degradation of speci c lipids that is essential for pollen development and plant fertility [START_REF] Kurusu | OsATG7 is required for autophagy-dependent lipid metabolism in rice postmeiotic anther development[END_REF]. Whether lipophagy exists in other plants remains undocumented. In mammals, oleatedriven formation of lipid droplets induces autophagy and provokes transient interaction of LDs with LC3 structures. In these conditions, autophagy is positively regulated by phospholipase domain-containing protein 5 (PNPLA5), which shows co-localization with ATG16L1 on LDs [START_REF] Dupont | Neutral lipid stores and lipase PNPLA5 contribute to autophagosome biogenesis[END_REF]. PNPLA5 is a neutral lipase that cleaves triacylglycerols (TAGs) to yield diacylglycerol (DAG), and AP biogenesis on LDs depends on the ef cient conversion and remodeling of PC from DAG. This pathway seems to be speci c for mammalian lipophagy, although LD lipids have been proposed to participate in autophagy in yeast [START_REF] Shpilka | Lipid droplets and their component triglycerides and steryl esters regulate autophagosome biogenesis[END_REF][START_REF] Velázquez | Lipid droplet-mediated ER homeostasis regulates autophagy and cell survival during starvation[END_REF]. In this organism, the inhibition of LD formation was accompanied with a block in the biogenesis of APs [START_REF] Shpilka | Lipid droplets and their component triglycerides and steryl esters regulate autophagosome biogenesis[END_REF]. Yet, this block only occurs upon nitrogen starvation and not when autophagy is induced by other conditions [START_REF] Velázquez | Lipid droplet-mediated ER homeostasis regulates autophagy and cell survival during starvation[END_REF]. In fact, the synthesis of LD-associated lipids highly increases in conditions of nutrient scarcity. When LD synthesis is blocked, neutral lipids massively accumulate and cause proliferation of unstructured ER membranes [START_REF] Velázquez | Lipid droplet-mediated ER homeostasis regulates autophagy and cell survival during starvation[END_REF]. This impairs overall ER functions and endomembrane traf cking and could indirectly block autophagy. Yet it cannot be ruled out that some neutral lipids directly participate in the formation of APs although this hypothesis may prove dif cult to address. Among neutral lipids DAG may play a particular role in the formation of the phagophore as (i) conversion of PA to DAG is required for autophagy in a cancer cell line [START_REF] Brohée | Lipin-1 regulates cancer cell phenotype and is a potential target to potentiate rapamycin treatment[END_REF], (ii) DAG may be involved in the shaping of the phagophore edges (Nath et al., 2014), and (iii) a DAG-dependent signaling pathway regulates antibacterial autophagy in mammals [START_REF] Shahnazari | A diacylglycerol-dependent signaling pathway contributes to regulation of antibacterial autophagy[END_REF].

A lipid's journey to the phagophore Where do lipids come from-multiple membrane sources?

Over the years, extensive microscopy studies of autophagyrelated proteins led to the proposal of multiple membrane sources for nucleation and/or expansion of the phagophore in mammals and yeast. In fact, essentially every compartment of the endomembrane system has been implicated: the ER, ERES, ERGIC, COPI, and COPII vesicles, the PM, endosomes, mitochondria, ER-mitochondria or ER-PM contact sites, as well as Golgi-derived vesicles (Fig. 3; Reggiori and Klionsky, 2013;[START_REF] Davis | Sec24 phosphorylation regulates autophagosome abundance during nutrient deprivation[END_REF]Karanasios et al., 2016). Several possibilities can explain these apparent discrepancies. (i) Autophagy is induced by various stresses that lead to various cell signaling cascades (Yang and Bassham,Fig. 3. Hypothetical sources of membrane/lipid delivery for the autophagy pathway in plant cells. Vesicular trafficking including ATG9 reservoirs (ATG9) (in red), ER-derived (in black) or post-Golgi vesicles (in dark red) may be involved for the nucleation/expansion of the phagophore. Lipids may further be delivered through membrane contact sites (MCS, represented in orange) including potential interaction between the ER and the phagophore as well as ER junctions with the mitochondria, the PM or the chloroplast. Additionally, lipid synthesis and/or remodeling could occur (in blue) directly on the phagophore membrane.

Lipids in the formation of autophagic membranes in plants | Page 9 of 13 Downloaded from https://academic.oup.com/jxb/advance-article-abstract/doi/10.1093/jxb/erx392/4626680 by Universite de Bordeaux user on 20 November 2017 2015). Additionally, the membrane composition of particular organelles can be profoundly altered upon stress. For instance, the plastid-resident lipid digalactosyldiacylglycerol (DGDG) relocates to the PM in response to phosphate starvation to preserve the integrity of the membrane while phospholipids are turned over to remobilize the phosphate pool [START_REF] Dubots | Role of phosphatidic acid in plant galactolipid synthesis[END_REF]. Therefore, membrane sources/platforms for the nucleation and elongation of the phagophore may depend on particular signals. (ii) Combined inputs from several organelles may be required to maintain the homeostasis of the overall endomembrane system. Upon continuous induction conditions, the massive accumulation of autophagic bodies re ects the drastic requirement of building blocks for the biogenesis of autophagosomes. The depletion of such an amount of lipid/membrane from a unique compartment could profoundly affect its structure and functions leading to dramatic physiological consequences. (iii) Differently composed phagophores and APs could co-exist in the cells depending on the type of selective autophagy, i.e. on the type of cargo they accommodate. The size but also the nature of the cargo could trigger the increased delivery and/or synthesis of speci c lipids. Additionally, lipids from the cargo itself could be utilized for the formation of the autophagic structures. A good example is the previously mentioned use of TAGs from LDs to promote the formation of autophagosomes speci c for the degradation of these very LDs during lipophagy in mammals [START_REF] Dupont | Neutral lipid stores and lipase PNPLA5 contribute to autophagosome biogenesis[END_REF]. (iv) All lipid species are not equally distributed among endomembranes. Therefore, it is plausible that inputs from multiple membrane sources are required to reach the speci c protein and lipid composition that promotes the particular structure and functions of the phagophore. For example, the maturation of autophagosomes by fusion with endosomes in mammals is necessary for the collection of components mediating their subsequent fusion with the lysosomes [START_REF] Pankiv | FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end-directed vesicle transport[END_REF][START_REF] Olsvik | FYCO1 contains a C-terminally extended, LC3A/B-preferring LC3-interacting region (LIR) motif required for efficient maturation of autophagosomes during basal autophagy[END_REF].

How are lipids mobilized towards the autophagy pathway?

Several ways of lipid mobilization to the PAS/phagophore/ autophagosome can be proposed and could co-exist (Fig. 3). First, the involvement of the ERES, ERGIC, COPII, COPI, and ATG9 reservoirs in autophagy in mammals and/or yeast suggests that vesicular traf cking is required for the nucleation of the phagophore (Carlsson and Simonsen, 2015;[START_REF] Davis | Sec24 phosphorylation regulates autophagosome abundance during nutrient deprivation[END_REF]. Further, ATG8-PE-containing membranes might also be produced at other locations than at the phagophore, including speci c COPII vesicles [START_REF] Ge | Phosphatidylinositol 3-kinase and COPII generate LC3 lipidation vesicles from the ER-Golgi intermediate compartment[END_REF] as well as endosomes marked with ATG16 (Knaevelsrud et al., 2013). These vesicles would then be transported to the PAS for expansion of the phagophore. However, the contribution of either the early secretory pathway or post-Golgi vesicles in autophagosome formation is not yet established in plants. Second, lipid synthesis and/or lipid remodeling could occur directly at the phagophore membrane. In fact, several lipidmodifying enzymes are present at the PAS in mammals and/ or in yeast: the PA-forming PLD1 (Dall [START_REF] Armi | The Phospholipase D1 Pathway Modulates Macroautophagy[END_REF], the PI3K complex (Reggiori and Klionsky, 2013) as well as the kinase catalysing the formation of PI(4,5)P 2 [START_REF] Tan | PtdIns(4,5)P 2 signaling regulates ATG14 and autophagy[END_REF]. Additionally, a recent report in mammals showed the co-localization of components of the ULK1/ATG1 complex with several lipid biosynthetic enzymes at speci c ER subdomains in cells treated with wortmannin (Nishimura et al., 2017). Third, membrane lipids could be directly transferred from a donor membrane to the phagophore at sites of contact between the two membranes. Membrane contact sites (MCSs) play critical roles in cell physiology by mediating the movement of small molecules from one compartment to the other [START_REF] Stefan | ER-PM connections: sites of information transfer and inter-organelle communication[END_REF]. The ER is the major site for lipid synthesis and thus MCSs engaging the ER may be particularly relevant in the transfer of lipids to other endomembranes [START_REF] Pérez-Sancho | Stitching organelles: organization and function of specialized membrane contact sites in plants[END_REF]. In fact, ERmitochondria and ER-PM contact sites are critical for the biogenesis of autophagosomes in mammals by providing platforms for the assembly of the phagophore (Hamasaki et al., 2013;Nascimbeni et al., 2017). ER-mitochondria junctions are proposed to channel phospholipids to the phagophore (Axe et al., 2008;Hamasaki et al., 2013) while ER-PM contacts are essential for autophagy-associated PI3P synthesis (Nascimbeni et al., 2017). In yeast, ER-mitochondria contacts (ER-mitochondria encounter structure, ERMESs) are also required for mitophagy but not for bulk autophagy suggesting that particular MCSs could play speci c roles depending on the type of selective autophagy [START_REF] Böckler | Mitochondrial ER contacts are crucial for mitophagy in yeast[END_REF]. As mentioned before, the phagophore forms in the close vicinity of speci c ER subdomains in Arabidopsis and contacts between the phagophore and the ER membrane were observed (Zhuang et al., 2013;Le Bars et al., 2014). Whether these contacts mediate direct shuttling of components between the two membranes remains to be addressed. Similarly, while ER-mitochondrion as well as ER-PM contacts also exist in plant cells [START_REF] Pérez-Sancho | Stitching organelles: organization and function of specialized membrane contact sites in plants[END_REF], their involvement in autophagy has not yet been documented. ER membranes also show tight connections with that of chloroplasts [START_REF] Pérez-Sancho | Stitching organelles: organization and function of specialized membrane contact sites in plants[END_REF]. Autophagy contributes to the degradation of chloroplastic proteins and even entire chloroplasts, which provides a tremendous source for nitrogen turnover upon stress [START_REF] Yang | New insight into the mechanism and function of autophagy in plant cells[END_REF]Izumi et al., 2017). Further, recent studies showed that autophagy can also participate in the Rubisco containing body (RCB) pathway that speci cally mediates the degradation of stromal proteins from the chloroplast [START_REF] Sakuraba | Delayed degradation of chlorophylls and photosynthetic proteins in Arabidopsis autophagy mutants during stress-induced leaf yellowing[END_REF]. ATG8 localizes on chloroplast protrusions and depletion of the ESCRT-associated protein CHMP1 results in the accumulation of arrested phagophores as well as RCBlike vesicles [START_REF] Spitzer | The endosomal protein CHARGED MULTIVESICULAR BODY PROTEIN1 regulates Page 12 of 13[END_REF]. Together this suggests that ER-chloroplast contact sites may participate in the selective degradation of chloroplasts by autophagy and/or in the RCB pathway.

Regulation of lipid supply to the autophagy pathway

Autophagosome formation occurs very rapidly upon autophagy induction [START_REF] Merkulova | Assessment and optimization of autophagy monitoring methods in Arabidopsis roots indicate direct fusion of autophagosomes with vacuoles[END_REF]. Further the use of concanamycin A, which stabilizes the autophagic bodies [START_REF] Bassham | Methods for analysis of autophagy in plants[END_REF], reveals the massive accumulation of APs in the vacuole when autophagy is induced by nutrient starvation (Le Bars et al., 2014). This re ects the requirement of a tremendous amount of membrane for the autophagy pathway. Therefore, proper autophagy relies on ef cient and continuous lipid delivery towards autophagosome formation that needs to be acutely regulated upon autophagy induction. This includes (i) sustained and/or increased synthesis of speci c proteins and lipids that participate in the formation of the APs, (ii) remodeling of membrane donor compartments while maintaining their homeostasis, (iii) ef cient and rapid organization of lipid transfer/vesicular traf cking to the phagophore, and (iv) one can propose that lipids of the outer membrane of the AP that fuse with the vacuolar membrane are recycled back and reused for a new round of AP formation. This recycling would also preserve the integrity of the vacuolar membrane upon massive input of AP lipids. At this point in time, nothing is known about the organization and the regulation of lipid supply to the autophagy pathway in plants mostly because of a lack of information about either the identity of the lipids involved in AP biogenesis or the machinery involved in their delivery at the site of phagophore initiation and elongation.

Concluding remarks and perspectives

While autophagy builds on the formation of a unique double membrane vesicle, very little is currently known on the lipids composing these structures. Future research should establish the lipid pro le of autophagic structures to address the functional relevance of these molecules in the shaping and the functions of the phagophore and autophagosomes. This would further provide grounds for in vitro reconstitution of the autophagy pathway; characterizing the interplay between autophagy-related lipids and ATG proteins is key to fully understanding the autophagy machinery. Delivery of lipids must be particularly regulated in both time and amplitude to provide building blocks for the formation of autophagosomes. Elucidating the source of lipids and the machinery put in place for their mobilization towards the autophagy pathway should identify targets for ne tuning of stress response programs in plants.

Introduction

Plants are sessile organisms subject to a plethora of stresses during their life cycle. These stresses include in particular abiotic stresses depending on climatic conditions, including drought and osmotic stresses, nutritional starvation in macro and micronutrients, temperature, intensity and quality of light, but also biotic stresses, including attacks by pathogenic and herbivory organisms as well as viruses. Furthermore, the phenology of plants plays a critical role in the plant life cycle. These periodic developmental events, including seed formation, anthers dehiscence, pollen grain development, tuberization, flowering, leafing and senescence, are dependent on environmental conditions and in fine on current global change.

To adapt to these multiple environmental and developmental constraints, plants have developed sophisticated defense and quality control mechanisms, including macroautophagy (« self-eating »). Macroautophagy (hereafter called autophagy) is a well-preserved mechanism mediating the degradation and recycling of intracellular compounds in eukaryotic organisms (mammals, fungi and plants) [START_REF] Gomez | Lipids in membrane dynamics during autophagy in plants[END_REF].

The autophagy pathway is subdivided into a six-act play requiring specific molecular actors including the core-component of the autophagy machinery, the AuTophaGy-related (ATG) proteins, as well as lipids and the product to be degraded, so-called cargo (Figure 1): i) recruitment of the ATG proteins for the establishement of the pre-autophagosomal structure (PAS) on specific subdomains of the endoplasmic reticulum (ER), ii) nucleation of an initial structure, termed the phagophore, an open cup-like double membrane, with highly curved edges termed phagophore, iii) expansion of the phagophore by addition of proteins and lipids, iv) closure of the phagophore, completing the formation of the autophagy-specialized double membrane vesicle, the so-called autophagosome (AP), v) fusion of the outer membrane of the AP with the vacuole and release of the inner membrane and their contents in the form of autophagic bodies (AB) in the vacuole and vi) degradation of the cargo by hydrolases (lipases, proteases); breakdown products are then recycled into the cytosol in building blocks (free fatty acids, amino acids, sugars) to maintain basal homeostasis (Figure 1) (Masclaux-Daubresse et al., 2017). Thus, the biogenesis of AP is a highly dynamic event of In nutrient rich conditions upstream regulators of autophagy (such as TOR/RAPTOR) inhibit the pathway. Upon autophagy induction, the inhibition is stopped, and the autophagosome biogenesis process initiates. The ATG1 initation complex, ATG9 vesicles and other early ATG components such as the PI3K complex relocalize to a site in close proximity to the endoplasmic reticulum (ER) to establish the pre autophagomal structure (PAS). The PAS is considered a lipo-proteic compartment from which an initial membrane emerges and constitutes the double membrane phagophore structure. Then, the ATG8 lipidation machinery (ATG5-ATG12 complex, ATG4, ATG3, ATG7) mediate the covalent conjugation of ATG8 protein to a phosphatidylethanolamine (PE) moiety. The incorporation of ATG8-PE along with other mechanisms lead to the expansion of the phagophore and an engulfment of autophagic cargo. Once the structure has reached its final size, the membranes at the rim of the phagophore eventually close to form a sealed autophagosome in a process implicating ESCRT complexes. Upon completion, ATG proteins dissociate from the autophagosome and an active removal of ATG8-PE moieties present in the outer membrane mediated by the cysteine protease ATG4 is required. Then, the outer membrane of the mature autophagosome fuses with the vacuolar membrane resulting in the delivery and subsequent degradation by vacuolar hydrolases of the autophagosme inner membrane (called « autophagic body» when present inside the vacuole) along with the autophagic cargo. Finally, the resulting macromolecules are recycled back into the cytosol.

membrane lipid remodeling which is finely regulated in time and space to support cell quality and adaptation to stresses (Masclaux-Daubresse et al., 2020). Among these molecular players, the ATG proteins are the most studied of the autophagic machinery. However, in plant organisms, little is still known compared to yeast and mammalian models. To date, more than fourty ATG genes have been identified in Arabidopsis thaliana and eighteen are notably involved in the formation of AP (Masclaux-Daubresse et al., 2020). Within the Swiss Army Knife of tools allowing the study of autophagy in plant cells, the study of atg mutants is an essential prerequisite for understanding the morphology of the autophagy pathway. The majority of atg mutants exhibit a blockage of the autophagic machinery associated with high sensitivity or lower resistance to environmental constraints (See Section 2).

When the living conditions for the plant are adequate, ATG proteins are located in the cytosol. However, when the environmental parameters become difficult, ATG proteins are recruited to specific sites within the cell where AP formation occurs (Figure 1). The induction of autophagy in Arabidopsis is initiated by the dephosphorylation of the ATG1 kinase complex including the proteins ATG1a-c, ATG13a-b, ATG11 and ATG101 [START_REF] Gomez | Lipids in membrane dynamics during autophagy in plants[END_REF]Reggiori and Klionsky 2013;[START_REF] Masclaux-Daubresse | Regulation of nutrient recycling via autophagy[END_REF][START_REF] Kamada | Tor-mediated induction of autophagy via an Apg1 protein kinase complex[END_REF]. Then, the phagophore, is initiated de novo by nucleation on an initial membrane compartment (Soto-Burgos et al., 2018;Carlsson and Simonsen, 2015) (Figure 1). The nucleation and elongation of the PAS by addition of lipids involves different actors of the autophagic machinery: the ATG9 complex containing ATG2, ATG9, ATG18a-i, as well as the VACUOLAR PROTEIN SORTING 34/PHOSPHATIDYLINOSITOL-3 KINASE (VPS34/PI3K) complex comprising ATG14, ATG6, VPS15 and VPS34. The VPS34 complex is notably involved in the synthesis of phosphatidylinositol-3-phosphate (PI3P), an inverted conical lipid crucial in AP formation (see section 1).

Subsequently, two conjugation systems are also involved in the expansion and closure of the phagophore: the ATG8-phosphatidylethanolamine (PE) conjugation system comprising ATG8a-i, ATG3, ATG4ab and ATG7 as well as the ATG5-ATG12 conjugation system including ATG5, ATG12ab, ATG7, ATG16 and ATG10 [START_REF] Masclaux-Daubresse | Regulation of nutrient recycling via autophagy[END_REF][START_REF] Gomez | Lipids in membrane dynamics during autophagy in plants[END_REF] (Figure 1). Upon completion of the AP, ATG4 cleaves the bond between ATG8 and PE releasing ATG8 from the outer membrane of the AP (Woo et al., 2014;Yoshimoto et al., 2004).

Autophagy can degrade a wide range of cargo, ranging from proteins to entire organelles, either randomly engulfed by the AP or specifically targeted by the autophagy machinery during so-called selective autophagy. We then speak of chlorophagy for the selective degradation of chloroplast [START_REF] Wada | Autophagy plays a role in chloroplast degradation during senescence in individually darkened leaves[END_REF], reticulophagy for the reticulum [START_REF] Bernales | Autophagy Counterbalances Endoplasmic Reticulum Expansion during the Unfolded Protein Response[END_REF][START_REF] Yorimitsu | Endoplasmic Reticulum Stress Triggers Autophagy*[END_REF], pexophagy for the peroxisome [START_REF] Shibata | Highly oxidized peroxisomes are selectively degraded via autophagy in Arabidopsis[END_REF], mitophagy for the mitochondria [START_REF] Ashrafi | The pathways of mitophagy for quality control and clearance of mitochondria[END_REF]Li et al., 2014), ribophagy for the ribosomes [START_REF] Kraft | Mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease[END_REF] or proteaphagy for the 26S proteasome [START_REF] Marshall | Autophagic Degradation of the 26S Proteasome Is Mediated by the Dual ATG8/Ubiquitin Receptor RPN10 in Arabidopsis[END_REF]. Adapter or receptor proteins that bind to the cargo achieve selective autophagy. Among them, the NEIGHBOR OF BRCA1 GENE 1 (NBR1) protein is notably one of the most studied [START_REF] Svenning | Plant NBR1 is a selective autophagy substrate and a functional hybrid of the mammalian autophagic adapters NBR1 and p62/SQSTM1[END_REF] and the JOKA2 homologue [START_REF] Zientara-Rytter | Identification and functional analysis of Joka2, a tobacco member of the family of selective autophagy cargo receptors[END_REF] of NBR1 in tobacco is also degraded in the vacuole when autophagy increases, thus allowing autophagic flux to be followed.

In recent years, autophagy has emerged has a central adaptation program to various stresses.

Autophagy integrates multiple environmental and developmental cues, mediating the degradation of random portion of the cytosol as well as selectively target specific cargo to (1) maintain quality in the cell by removing damaged or unwanted intracellular material and organelles; (2) degrade proteins/machinery to reshape metabolism, recycle nitrogen sources and promote survival. Autophagy relies on the formation of the AP which engulfs and traffics cargo to the vacuole for degradation. The molecular composition of the AP (lipids, proteins) dictates rates and types of autophagy -with adapters interacting with specific cargo for selection-as well as the size of the AP and thus its capacity for the volume of cargo to be trafficked. Size and numbers of APs are therefore a rate limiting step for autophagy activity, and both are regulated by the availability and the nature of lipids composing AP membranes acting as both building blocks as well as key components of the structure and function of AP membranes. In turn autophagy mediates the regulation of lipid metabolism upon various stresses. In this review we will see how lipids are fundamental actors of the autophagy machinery, how autophagy controls plant adaptation to environmental stresses and explore the crosstalk between lipids and autophagy in these processes.

II-1. Lipids are fundamental actors of the autophagy machinery

Morphology and membrane dynamics in AP formation

Autophagosomes, the autophagy specialized vesicles are unique in the endomembrane system; first because they are made of a double lipid bilayer, second because they do not bud from a pre-existing membrane. Instead, they form de novo in a multistep process that requires numerous membrane remodeling events. Upon induction, autophagy morphologically starts with the nucleation of an initial membrane structure called phagophore (or isolation membrane). The phagophore then expands by addition of lipids and finally seals to form a double membrane vesicle, the AP. Upon completion, the AP traffics to the vacuole and its outer membrane fuse with the tonoplast releasing the inner membrane and cargo inside the vacuole for degradation.

Autophagy is initiated by the recruitment of the core autophagy machinery composed of ATG proteins (Feng et al., 2014). At steady state, ATG proteins reside in the cytoplasm and upon autophagy induction, they are recruited to the site of autophagosome formation. These ATG proteins work together to assemble an initial lipo-proteic condensate called preautophagosomal structure (PAS) [START_REF] Fujioka | Phase separation organizes the site of autophagosome formation[END_REF] that defines the site of autophagosome formation. Yeast displays a single PAS structure formed in close proximity to the vacuole [START_REF] Suzuki | The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation[END_REF]. Mammals display multiple PAS structures that seem to emerge from phosphatydilinositol-3-phosphate (PI3P) enriched subdomains of the endoplasmic reticulum (ER) (Axe et al., 2008). Likewise, plants exhibit multiple PAS structures that are also formed in close apposition to specific subdomains of the ER (Le Bars et al., 2014;[START_REF] Zhuang | Autophagosome Biogenesis and the Endoplasmic Reticulum: A Plant Perspective[END_REF].

In both organisms, membrane contact sites between the ER and the plasma membrane (PM) have also been implicated in the AP biogenesis indicating a functional contribution of the plasma membrane in autophagy [START_REF] Ravikumar | Plasma membrane contributes to the formation of pre-autophagosomal structures[END_REF]Wang et al., 2019). Following the formation of the early PAS structure, ATG proteins are sequentially recruited to nucleate the phagophore.

The exact mechanism of phagophore nucleation is not well understood, although multiple hypotheses exist concerning the source of this initial membrane. The ATG1 kinase complex (ULK1 in mammals) is part of the autophagy initiation complex and regulates the activity as well as the recruitment of downstream ATG proteins required for autophagosome biogenesis. ATG9, the only transmembrane protein among ATGs, resides in vesicles called "ATG9 vesicles" that, upon autophagy induction are recruited by the ATG1 complex to the PAS [START_REF] Papinski | Early steps in autophagy depend on direct phosphorylation of Atg9 by the Atg1 kinase[END_REF][START_REF] Young | Starvation and ULK1dependent cycling of mammalian Atg9 between the TGN and endosomes[END_REF] in yeast and mammals. ATG9 vesicles as well as other vesicles such as endosomes or the ER exit sites (ERES) -derived COPII vesicles coalesce at the PAS and are thought to serve as membrane seeds for the nucleation of the initial phagophore membrane [START_REF] Shima | COPII vesicles contribute to autophagosomal membranes[END_REF]Tan et al., 2013;Graef et al., 2013;Ge et al., 2017;[START_REF] Gómez-Sánchez | Atg9 establishes Atg2-dependent contact sites between the endoplasmic reticulum and phagophores[END_REF]. In plants however, ATG9 seems to have a distinct function than that of its yeast and mammalian counterparts. In Arabidopsis, ATG9 is not strictly required for the formation of autophagic structures, yet in the atg9 knock-out mutant they display an abnormal size and shape; further, autophagy flux is significantly reduced in atg9, suggesting that ATG9 may have alternative or additional functions in plants compared to other organisms (Zhuang et al., 2017).

The ATG1 complex is also implicated in the activation of the autophagy specific PI3 kinase complex (PI3K) [START_REF] Russell | ULK1 induces autophagy by phosphorylating Beclin-1 and activating Vps34 lipid kinase[END_REF]. The PI3K complex mediates the production of PI3P at the PAS and further recruit PI3P-effectors, such as ATG18 (Proikas-Cezanne et al., 2015), as well as the ATG12-ATG5-ATG16 complex (Romanov et al., 2012). This local enrichment of PI3P at the PAS is a hallmark of autophagy as it occurs in mammals, yeast and plants. In plants, both the PI3K complex (Liu et al., 2020) as well as the ATG1 complex (Suttangkakul et al., 2011) have been identified and implicated in the autophagy pathway.

Moreover, components of both complexes were also found to be degraded via autophagy indicating that a tight regulation loop could limit the extent of autophagy activity in plant cells (Liu et al., 2020;Suttangkakul et al., 2011). Interestingly, mutants of the initiation complex were still capable of producing the ATG-PE conjugate, although they did not form mature autophagosomes (Suttangkakul et al., 2011). This could mean that in plants, the initiation complex acts in stages succeeding ATG8 lipidation, i.e., in latter stages of autophagosome formation. Morphologically, the phagophore is observed as a cup with highly curved edges.

This particular shape has been described in all organisms (Axe et al., 2008;[START_REF] Suzuki | Fine mapping of autophagy-related proteins during autophagosome formation in <em>Saccharomyces cerevisiae</em&gt[END_REF]Zhuang et al., 2013) suggesting that it plays a key role in the process, most likely by supporting effective engulfment of the cargo. The maintenance of this particular shape seems to implicate the actin cytoskeleton as mutants defective in this process show a reduced number of autophagosomes [START_REF] Aguilera | The actin cytoskeleton participates in the early events of autophagosome formation upon starvation induced autophagy[END_REF]Wang et al., 2016).

Surrounding the cargo to be degraded, the phagophore expands through the delivery of lipids and membranes while keeping its unique structure and ensuring the engulfment of cargo. The highly curved edges of the phagophore are considered of outermost importance for the growth of the structure. Phagophore growth depends on the association of peripheral proteins (including ATG proteins), and many of these proteins sense membrane curvature as cues for their recruitment to the autophagy structure (Nguyen et al., 2017). In fact, some of these proteins were shown to localize precisely to the rim of the phagophore to support the lipidation of ATG8 [START_REF] Dancourt | Lipidation of the autophagy proteins LC3 and GABARAP is a membrane-curvature dependent process[END_REF]. ATG8 is a central protein in autophagy which conjugation to phosphatidylethanolamine (PE) is a prerequisite for its incorporation into autophagic structures and for the progression of autophagosome biogenesis. The incorporation of ATG8-PE into the phagophore is thought to contribute to the growth of the membrane as downregulation in ATG8 gene expression lead to autophagosomes of smaller size (Xie et al., 2008). Mutants affected in the lipidation of ATG8 show a complete absence of autophagosome or autophagic bodies in plants cells (Yoshimoto et al., 2004). In addition, ATG8 is critical for selective autophagy by mediating the targeting and recruitment of specific cargo through direct protein/protein interaction (Kellner et al., 2017).

Once the phagophore has reached its final size, the membranes at the rim of the phagophore fuse together, sealing the structure that becomes a double membrane vesicle, the autophagosome (AP). The sealing of the phagophore structure is directed by the endosomal system complex required for transport (ESCRT) in mammals (Takahashi et al., 2018;[START_REF] Yu | The coordination of membrane fission and fusion at the end of autophagosome maturation[END_REF]) and also in plants [START_REF] Katsiarimpa | The deubiquitinating enzyme AMSH1 and the ESCRT-III subunit VPS2.1 are required for autophagic degradation in Arabidopsis[END_REF]. Upon completion, many ATG proteins dissociate from the autophagosome and are recycled back to the cytosol (Koyama-Honda et al., 2013). From then, mature autophagosomes dissociate from the ER and traffic towards the vacuole for fusion. The outer membrane of autophagosomes fuses with that of the vacuole which results in the delivery of the internal membrane along with the cargo inside of the vacuole for degradation. To allow proper fusion, a tether between autophagosomes and the vacuole/lysosome membranes is required. Multiple SNARE (soluble NSF attachment protein receptors) complexes have been implicated in this process in yeast and mammals (reviewed in [START_REF] Nakamura | New insights into autophagosome-lysosome fusion[END_REF][START_REF] Zhao | Autophagosome maturation: An epic journey from the ER to lysosomes[END_REF]). In plants, the molecular machinery implicated in this process is not well characterized, but some actors have been identified. The FYVE domain protein required for endosomal sorting (FREE1), which is a member of the endosomal sorting complex required for transport (ESCRT), also plays a role in the fusion of autophagosomes to vacuoles (Gao et al., 2015). Additionally, the VTI (11,13) proteins, homologs of proteins of the mammalian SNARE complex, have been identified in Arabidopsis, and their mutants displayed phenotypes reminiscent of autophagy mutants, such as accelerated senescence suggesting a possible role of this complex in plant autophagy (Surpin et al., 2003).

In conclusion, the autophagosome biogenesis process requires intense membrane remodeling events to (1) nucleate a membrane de novo, (2) shape the membrane to a specific architecture,

(3) expand this membrane by articulating multiple sources of lipid delivery while keeping structure and function, (4) regulate the formation and dissociation of membrane contact sites, (5) control membrane fusion with the rim of the phagophore and then with the tonoplast in a timely manner to ensure appropriate size of the AP and avoid premature fusion with the vacuole thus ensuring delivery of cargo for degradation. Further, by promoting the expansion/shaping of the phagophore, these events also control the size and the number of autophagosomes thus modulating the rate and amplitude of autophagic activity.

Lipids control the architecture and functions of biological membranes

As mentioned above, autophagosome formation is carried out by the core ATG machinery, but in the recent years the fundamental role of lipids in this process has started to emerge, particularly in yeast and mammals. Lipids are not only passive constituents of biological membranes; instead, their specific biochemical and biophysical properties determine critical features of membranes (Harayama and Riezman 2018). The chemical structure of lipids, such as the size of their polar head as well as the unsaturation degree of their acyl chains have a great impact on their shape. For example, lipids with a small polar head but highly unsaturated acyl chains will display a conical shape; and on the opposite side, lipids with big polar heads and saturated acyl chains exhibit an inversed cone shape (Bigay & Antonny, 2012). Hence, when these lipids are incorporated and accumulate into membranes, they are able to create curvatures within these membranes. Furthermore, lipids with saturated acyl chains pack tightly together within membranes, while lipids with highly unsaturated acyl chains do the opposite. Thus, lipids with highly unsaturated acyl chains can create packing defects in membranes that can in turn be utilized by membrane anchored proteins for their incorporation into these membranes (Harayama and Riezman 2018). In this manner, lipid geometry (i.e., shape) determines critical features such as membrane curvature as well as the lipid density within each membrane. In addition to their structural roles, lipids are fundamental contributor of membrane activities by mediating the recruitment of specific proteins with lipidbinding domains or through electrostatic interactions. The nature, quantity and association of lipids within membranes thus define a unique identity for each and every biological membrane dictating membrane properties such as curvature and electrostatics and attracting selected set of proteins that support membrane functions. In that context, the following part of the review will focus on the role of lipids in the formation of autophagy membranes. Although most of the research in this subject has been performed in yeast and mammals, they point out to the importance of lipids for autophagy in general. The lipid composition of autophagic structures and their function may differ from one organism to another, thus we must be careful as all of these discoveries cannot be directly extrapolated to plants. However, these findings suggest that lipids are surely critical players for plant autophagy as well.

Lipids are key contributors of AP membrane formation

Autophagosome formation is a membrane-based process and as such, lipids are critical, at the very least by providing the building blocks required for the formation of the autophagic membranes. In fact, a considerable amount of lipids is required to support phagophore expansion and autophagosome formation, whether these lipids come from pre-existing organelles or are produced at the phagophore membrane. In mammals, upon nutrient starvation-induced autophagy, the number of autophagosomes present in one cell at a given time can go up to about 100 structures per cell [START_REF] Guo | AP1 is essential for generation of autophagosomes from the trans-Golgi network[END_REF][START_REF] Hailey | Mitochondria supply membranes for autophagosome biogenesis during starvation[END_REF]. In plants Multiple membrane sources contribute first for the establishment of the initial autophagic membrane as well as for its expansion. Different types of vesicles (notably ATG9 vesicles but also endosomes or COPII vesicles) have been proposed as membrane seeds for the nucleation of the initial structure. Lipid transfer proteins implicated in autophagy (such as ATG2) mediate the direct transfer of lipids from other organelles in contact sites such as ERphagophore contact sites. Finally, on-site lipid synthesis is also an important mechanism for phagophore expansion, for example localized PI3P synthesis allows for the recruitment of downstream effectors of the pathway leading to growth of the structure.

root cells, upon nutrient starvation during a few hours, the number of autophagic structures observable inside of the vacuole (in plants concomitantly treated with concanamycin A to accumulate autophagic bodies) can go up to more than 100 APs per cell as well (Figure 2).

Knowing the size and number of autophagosomes present per cell in mammals, Melia et al., made a clever estimation of the number of lipids that would be required to mobilize in order to create all the membranes required for autophagosome formation in starvation conditions (Melia et al., 2020). Their estimation was that in these conditions, cells would need the mobilization of 1000 000 000-plus lipids to support AP growth. This means that a considerable amount of lipids needs to be remobilized (or synthesized) to ensure the autophagic process.

The fact that APs are devoid of transmembrane proteins has made it difficult to trace the origin of the phagophore lipid/membrane. Recent advances have allowed the proposal of the main mechanisms by which lipids are remobilized to the formation of these autophagic structures: vesicle mediated delivery, protein mediated transport of lipids and on-site lipid synthesis (Figure 3). Such mechanisms and how they affect the autophagic pathway are described below.

1.3.1 Vesicle mediated delivery ATG9, the sole transmembrane protein among ATGs, cycles between different vesicular pools and is required at the PAS for the formation of autophagic structures. In yeast, Atg9 exists predominantly in 30-60nm diameter cytoplasmic vesicles (called Atg9 vesicles) derived from the Golgi apparatus (Mari et al., 2010). Upon autophagy activation, Atg9 vesicles are directed towards the site of autophagosome formation and their membranes are thought to serve as seeds for the nucleation of the initial autophagic membrane together with additional membrane sources (Yamamoto et al., 2012). In addition to provide an initial platform for the formation of autophagic structures, ATG9 vesicles have important roles in other aspects of the process. A recent outstanding study preformed in yeast showed that ATG9 vesicles can be used as substrates for the PI3K complex for in situ PI3P production (Sawa-Makarska et al., 2020). The same study showed that this in situ PI3P production mediated the recruitment of downstream PI3P-effectors as well as the conjugation machineries which allowed for the ATG8 lipidation to occur in ATG9 vesicles (Sawa-Makarska et al., 2020). In this manner, this study showed that ATG9 vesicles constitute a platform for the recruitment of autophagic machinery and thus are critical for the nucleation and expansion of an initial autophagic membrane in yeast. A difference between yeast and mammals is that in the latter, Atg9 vesicles do not fuse and coalesce with autophagic structures, they only transiently interact with them (Orsi et al., 2012). However, in both organisms Atg9 vesicles mediate the transport of essential elements required for autophagosome formation. In fact, it was shown that Atg9 vesicles serve as a vehicle for the delivery of a class III phosphatidylinositol-4-kinase PI4KIIIβ to the autophagy initiation membrane in mammals (Judith et al., 2019). The trafficking of yeast Atg9 vesicles and the subsequent autophagosome formation depends on the activity of this PI4K, indicating a tight connection between the Atg9 vesicles and the phosphoinositide PI4P.

Plant ATG9 also reside in vesicles that relocalize to autophagosome formation site upon autophagy induction (Zhuang et al., 2017). However, plant ATG9 seems to have distinct functions that its counterparts in mammals or yeast. In yeast and mammals, mutation in ATG9 results in an inability to form autophagosomes [START_REF] Lamb | The autophagosome: origins unknown, biogenesis complex[END_REF]Yamamoto et al., 2012). In contrast, ATG9 plant homolog is actively implicated in autophagy but not mandatory, as it was shown that the atg9 mutant shows a decrease, but not a total block in the autophagic flux (Shin et al., 2014). ATG9 was further described as required for the formation of autophagosomes from ER membranes (Zhuang et al., 2017). In this same study, authors found that atg9 mutants are still able to form GFP-ATG8a labelled structures with direct connection with the ER; however, these structures are abnormally large and misshaped (Zhuang et al., 2017). Taken together, this information strongly suggests that ATG9 is not the main source of lipids for autophagosome formation, and that, in plants, ATG9 vesicles may have a role in later stages of the process. Notably a recent study determined the structure of Arabidopsis ATG9 (ATG9) by cryo-EM, and showed a membrane-bound core region consisting of six transmembrane domains flanked by disordered N-and C-terminal cytoplasmic regions [START_REF] Lai | Subnanometer resolution cryo-EM structure of Arabidopsis thaliana ATG9[END_REF]. It was also found that, like its yeast counterpart, plant ATG9 self-interacts, and upon trimer formation, the C-terminal folds into a more stable conformation, which could in turn provide a scaffold for subsequent protein interactions [START_REF] Lai | Subnanometer resolution cryo-EM structure of Arabidopsis thaliana ATG9[END_REF]. Finally, both yeast and mammalian ATG9 were recently found to be capable of translocating phospholipids between membrane leaflets in vitro [START_REF] Matoba | Atg9 is a lipid scramblase that mediates autophagosomal membrane expansion[END_REF]. This means that ATG9 is locally capable of modifying the leaflet lipid composition of autophagic membranes, indicating a possible role of this protein in the remodeling/shaping of autophagy membranes.

Other types of vesicles have also been implicated in the formation of autophagic structures in yeast and mammals. The Endoplasmic Reticulum -Golgi intermediate compartment (ERGIC)-derived COPII vesicles have been proposed as other membrane seeds implicated in the nucleation of the phagophore as they have been shown to relocalize to the site of autophagosome formation [START_REF] Ge | Remodeling of ER-exit sites initiates a membrane supply pathway for autophagosome biogenesis[END_REF]Graef et al., 2013;Tan et al., 2013). COPII vesicles play an important role in the transport of proteins and membranes from the ER to the Golgi apparatus. Mutants deficient in COPII vesicle formation cannot form autophagosomes [START_REF] Ishihara | Autophagosome requires specific early Sec proteins for its formation and NSF/SNARE for vacuolar fusion[END_REF]. Furthermore, COPII vesicles have been shown to mediate the transport of an ER-localized protein implicated in autophagosome formation (mutants displayed less autophagosomes) to the site of autophagosome biogenesis [START_REF] Lemus | An ER-Localized SNARE Protein Is Exported in Specific COPII Vesicles for Autophagosome Biogenesis[END_REF]. Moreover, it has been proposed that COPII vesicles become part of autophagosome membranes upon the transport of proteins to autophagosomes [START_REF] Shima | COPII vesicles contribute to autophagosomal membranes[END_REF]Tan et al., 2013). Another type of vesicles implicated in autophagy are endosomes. ATG9 has been shown to traffic between the PM and the trans golgi network (TGN) via endosomes that eventually coalesce to provide an initial membrane for autophagy (Puri et al., 2013;Shirahama-Noda et al., 2013;[START_REF] Young | Starvation and ULK1dependent cycling of mammalian Atg9 between the TGN and endosomes[END_REF]. This highlights the potential importance of multiple organelles in the formation of autophagic structures. In plants, very little is known at this time about the contribution of intracellular vesicles to the delivery of lipids for autophagosome formation although endosomal compartments have been proposed to participate in autophagy suggesting a potential crosstalk between endocytosis and AP formation (Zhuang et al., 2015).

Action of lipid transfer proteins

As mentioned previously, multiple organelles, especially the ER, have been shown to contribute to the formation of autophagic membranes. The ER is the central organelle where most of the lipid synthesis occurs (Van Meer & Sprong 2004), and as such, represents a potential critical source of lipids for the formation of autophagosomes. In fact, besides vesiclemediated delivery, lipids required for autophagosome formation can also be transferred to the autophagic structures directly by lipid transfer proteins (LPTs) [START_REF] Wong | Lipid transfer proteins: the lipid commute via shuttles, bridges and tubes[END_REF]. These proteins benefit from the numerous membrane tethers that we have described above to transfer lipids from one organelle to phagophores. The ATG18-ATG2-ATG9 complex has been shown to allow the establishment of contact sites between the ER and the edges of the growing phagophore [START_REF] Gómez-Sánchez | Atg9 establishes Atg2-dependent contact sites between the endoplasmic reticulum and phagophores[END_REF]. Interestingly, ATG2 has also been shown to localize to mitochondria-associated ER membranes to allow phagophore progression [START_REF] Tang | Atg2A/B deficiency switches cytoprotective autophagy to non-canonical caspase-8 activation and apoptosis[END_REF]; in mammals ATG2 also displays a dual localization on both autophagic membranes and lipid droplets (Nishimura et al., 2017). These results indicate that the ATG18-ATG2-ATG9 complex is able to tether different membranes to that of the growing phagophore, presumably to use them as lipid sources for phagophore expansion [START_REF] Chowdhury | Insights into autophagosome biogenesis from structural and biochemical analyses of the ATG2A-WIPI4 complex[END_REF]Kotani et al., 2018). Besides its role in creating membrane tethers between different organelles, a series of recent studies determined another role for ATG2 in AP biogenesis. ATG2 was recently characterized as having lipid transfer capabilities in mammals [START_REF] Maeda | The autophagic membrane tether ATG2A transfers lipids between membranes[END_REF] as well as in S. pombe [START_REF] Osawa | Atg2 mediates direct lipid transfer between membranes for autophagosome formation[END_REF]. In S. pombe, Atg2 displays a hydrophobic cavity capable of accommodating phospholipid acyl chains that was found to act as a bridge allowing the transfer of lipids [START_REF] Osawa | Atg2 mediates direct lipid transfer between membranes for autophagosome formation[END_REF]. In this manner, mammalian ATG2 was found to bind up to 20 different lipid molecules at a given time, and capable of lipid transfer into liposomes in vitro [START_REF] Valverde | ATG2 transports lipids to promote autophagosome biogenesis[END_REF]. This places ATG2 as a major actor in the expansion of phagophores through its lipid transfer capacities, and highlights the importance of such mechanisms in the autophagic process.

Similarly, GRAMD1A (GRAM domain-containing) is another LPT characterized as implicated in autophagosome formation. GRAMD1A is a cholesterol transfer protein that was seen to relocalize to the site of autophagosome formation in a PI3P-dependent manner. Similar to ATG2, GRAMD1A inhibition lead to an absence of autophagosome formation, indicating its crucial role in the process [START_REF] Laraia | The cholesterol transfer protein GRAMD1A regulates autophagosome biogenesis[END_REF]. Finally, VPS13A and VPS13C were also described as LPTs responsible of lipid transfer between the ER and other organelles [START_REF] Kumar | VPS13A and VPS13C are lipid transport proteins differentially localized at ER contact sites[END_REF]. Interestingly, VPS13A downregulation leads to an impaired autophagic flux [START_REF] Muñoz-Braceras | TipC and the chorea-acanthocytosis protein VPS13A regulate autophagy in Dictyostelium and human HeLa cells[END_REF]. This could be explained by an inability in forming correct autophagic structures due to the absence of the lipid transfer activities of VPS13A as authors observed a reduced rate of ATG8 lipidation in VPS13A knockdown cells [START_REF] Muñoz-Braceras | TipC and the chorea-acanthocytosis protein VPS13A regulate autophagy in Dictyostelium and human HeLa cells[END_REF].

The identification of these proteins greatly improved our understanding of the mechanisms leading to phagophores growth. More studies are nevertheless needed to fully understand these lipid-transfer models, such as determining the lipid preferences of these proteins as to having a better image of their contribution to the lipid composition of phagophores. Moreover, in plants direct implication of LPTs in autophagy has so far not been described. Plant ATG2 has been identified and is strictly required for autophagy; furthermore, ATG2 was implicated in maintaining ROS homeostasis [START_REF] Yamauchi | Autophagy controls reactive oxygen species homeostasis in guard cells that is essential for stomatal opening[END_REF], as well as chlorophyll degradation upon abiotic stress [START_REF] Jiang | Autophagy-Related 2 Regulates Chlorophyll Degradation under Abiotic Stress Conditions in Arabidopsis[END_REF]. The molecular function of ATG2 in plant autophagy has not yet been documented, but interestingly, plant atg2 mutants are capable of forming small-size ATG8-positive structures which presumably correspond to phagophores or immature autophagic structures unable to fuse with the vacuole and thus accumulating in the cytosol (Kang et al., 2018). This, as well as the low level of similarities between plant ATG2 and its mammalian (ATG2A) or yeast counterparts (27% in both cases) suggest that in plants, ATG2 may have different or additional functions and would seem to be required for different step(s) in the process.

On site lipid synthesis

In addition to vesicular trafficking and lipid transfer, lipids required for autophagosome formation can also be synthesized directly on autophagic membranes. For example, and as mentioned above, the localized production of PI3P by the PI3K complex at the phagophore structure is a prerequisite for autophagosome formation by mediating the recruitment of many ATG proteins that bind to this lipid (Nascimbeni et al., 2017). This PI3P localized production is a conserved hallmark of autophagy, however, how PI3P is distributed within the autophagic membranes differs between organisms. In yeast PI3P is present in both leaflets of the autophagosome membrane, with a slight accumulation in the luminal leaflet whereas in mammals, PI3P was found exclusively in the cytosolic leaflet of autophagosomes (Cheng et al., 2014). The fact that PI3P is present in one leaflet more than another might play a role in the recruitment of downstream effectors in specific parts of the autophagic structures. Besides the localized PI3P production, other lipid synthesizing enzymes are also recruited to the autophagic structures, supposedly for lipid production to promote AP formation. Both the phospholipase D (PLD) as well as the PI4 Kinase enzymes colocalize at autophagic structures and although their exact functions in autophagy are still to be determined (Holland et al., 2016;Judith et al., 2019). Furthermore, the lipid PI4P is unevenly distributed in both leaflets of phagophore membranes from yeast and mammals, suggesting that the action of PI4K is tightly regulated in space (Kurokawa et al., 2019). Another example is the phosphatidylinositol synthase (PIS1) enzyme, which was shown to accumulate at the site of phagophore nucleation on ER subdomains of mammalian cells, and to colocalize with components of the initiation machinery (Nishimura et al., 2017). Thus, PI seems to function in the recruitment of the initiation complex in a step preceding the localized synthesis of PI3P (Nishimura et al., 2017).

PIS1 was later found to colocalize with the ER-phagophore contact protein VMP1, as well as with the choline/ethanolamine phosphotransferase enzyme [START_REF] Tábara | VMP1 Establishes ER-Microdomains that Regulate Membrane Contact Sites and Autophagy[END_REF][START_REF] Tábara | Vacuole membrane protein 1 marks endoplasmic reticulum subdomains enriched in phospholipid synthesizing enzymes and is required for phosphoinositide distribution[END_REF]. Thus, different phospholipid synthesizing enzymes have been shown to localize to sites where the autophagosome and the ER make contact, probably to support the growth of the structure. Other lipid synthesizing enzymes have been shown to act directly at autophagic structures. For instance, the acyl-CoA synthetase Faa1 relocalizes to autophagic structures and was shown to activate fatty acids that will later be channeled and contribute to the growth of the phagophore ( (Schütter et al., 2020) ; Figure 3). Moreover, PCYT1A a phosphate citydyltransferase which synthesizes phosphatidylcholine (PC) was found to be required for autophagosome formation, suggesting that PC is required for autophagy; and furthermore, authors showed that newly synthesized PC was incorporated into autophagosomes (Andrejeva et al., 2020) and thus contributes to the bulk of lipids composing autophagic membranes.

In plants, much less is known concerning the lipid synthesis required for the formation of autophagosomes. However, the PI3K complex was shown to co-localize with autophagosomes as well as to autophagic bodies within the vacuole. This suggests that the complex is indeed recruited to autophagic structures to produce PI3P and allow autophagy progression (Liu et al., 2020). Furthermore, the fact that the PI3K complex stays to autophagic structures up until their vacuolar degradation could mean that PI3P is required for multiple steps in the process (Liu et al., 2020). Another possibility could be that the complex is degraded through the autophagic pathway as a way to finely regulate autophagic activity, as it has been proposed for the initiation complex (Suttangkakul et al., 2011).

Taken together, multiple lipid-synthesizing enzymes have been implicated in the formation of autophagic structures, whether it is to support the flux of lipids required for the growth of the structure, but also to modulate the specific lipid composition required for the progression of these structures. These three mechanisms (i.e., vesicle mediated delivery, direct lipid transfer and on-site synthesis, see Figure 3) highlight the fact that a plethora of lipids need to be remobilized for the autophagosome formation, and any de-regulation in these mechanisms hampers the overall autophagic process. However, not all lipids contribute in the same manner to this process. As mentioned above, some lipids are utilized as building blocks for the structure, while others allow for the recruitment of downstream effectors. This emphasizes the high level of regulation that governs these mechanisms. In plants, although these mechanisms have not yet been documented, they are surely required for the process.

Lipids as critical components of the structure and identity of the autophagy membranes

Lipids are not only building blocks for the formation of membranes, their distribution within each membrane as well as their different ratios contribute to establish the particular membrane identity of each organelle (Bigay & Antonny 2012). The composition of biological membranes dictates many of its properties such as its curvature, fluidity and electrostatic field.

In that context, lipids composing the phagophore likely play key roles in the activity of this organelle. The exact membrane composition of autophagic structures has remained elusive in all organisms. Nevertheless, it was recently reported that in yeast, Atg8-containing membranes (that could represent phagophores, autophagosomes, and/or autophagic bodies within the vacuole) have a phospholipid composition of 38% PC, 37% PI, 19% PE, 3% PS and 3% PA (Schütter et al., 2020). Compared to other endomembranes, ATG8-labeled membranes thus display an enrichment in phosphatidylinositol (PI), the precursor of PI3P, which we know regulates different steps of the autophagic process. Recent reports have shown that PC is synthesized de novo and incorporated into autophagic structures (Andrejeva et al., 2020) which is in coherence with PC being the most abundant lipid in these structures (38%). We can also see that these membranes contain a considerable amount of PE, a lipid known to be required for autophagy and which role will be described below. The specific lipid composition of autophagosome membranes is thought to provide critical input for their formation and activity as well as their global architecture. The molecular functions of lipids in autophagy are still poorly described in plants where the nature, dynamics and proportion of these molecules are still largely unknown. In the following section, we will thus detail the recent advances in understanding the functions of different lipids on autophagic membranes mostly based on studies in yeast and mammals. These studies brought critical insights into the machinery of autophagy, and as such can shed light into how this process could occur in plants, even though, as mentioned previously, there might be fundamental differences in the process between plants and other organisms.

1.4.1 Lipid/protein binding: lipids mediate the transient association of ATG proteins with the phagophore Upon autophagy induction ATG proteins are very rapidly recruited from the cytosol, where they normally reside, to the lipo-proteic PAS structure to nucleate the initial autophagic membrane. The recruitment of ATG proteins relies on protein-protein interactions but also on the binding of ATG proteins to lipids or to specific lipid-mediated membrane properties. In fact, several ATG proteins -from different ATG complexes-contain either lipid-binding motifs or positively charged regions that allow them to bind lipids through electrostatic interactions. In addition, other ATG proteins are able to sense membrane features such as membrane curvature or lipid packing defects (Table 1; note that this list is not exhaustive, examples have been chosen to illustrate the different steps of autophagosome formation in which interactions with lipids are important).

For example, yeast Atg1 displays an early autophagy targeting (EAT) domain that is able to sense membrane curvature with a preference for highly curved membranes [START_REF] Chan | Kinase-inactivated ULK proteins inhibit autophagy via their conserved C-terminal domains using an Atg13-independent mechanism[END_REF]Ragusa et al., 2012). In the same manner, ATG14, a member of the PI3KC complex in mammals is able to recognize membrane curvature through its Barkor/Atg14(L) Autophagosome Targeting Sequence (BATS) domain [START_REF] Fan | Autophagosome targeting and membrane curvature sensing by Barkor/Atg14(L)[END_REF]. These membranesensing capacities of ATG proteins allow them to relocalize specifically at the highly curved edges of the phagophore and thus contribute to the high spatiotemporal regulation that governs the autophagosome biogenesis process. The recruitment of the PI3KC complex to membranes with high curvature allows for the localized production of PI3P required for the progression of AP formation. In turn, PI3P allows for the recruitment of what are called "PI3Peffectors" such as the ATG18 proteins. Yeast Atg18 is a seven bladed β-propeller protein and binds PI3P through an FRRG motif [START_REF] Rieter | Atg18 function in autophagy is regulated by specific sites within its β-propeller[END_REF]. The Atg18 protein partner Atg2, was found to bind to PI3P containing liposomes and this interaction was increased in ergosterolcontaining liposomes (ergosterol creates lipid packing defects in membranes) suggesting that Atg2 is also capable of sensing packing defects [START_REF] Gómez-Sánchez | Atg9 establishes Atg2-dependent contact sites between the endoplasmic reticulum and phagophores[END_REF]. In mammals, Table 1. Most important regulators in autophagosome biogenesis in: Saccaromyces cerevisiae (Sc), Homo sapiens (Hs), and Arabidopsis thaliana (At) ATG18 proteins bind PI3P trough two distinct recognition sites: the FRRG motif as it occurs in yeast, and through the WD-repeat domain Interacting with phosphoinositides [START_REF] Baskaran | Two-site recognition of phosphatidylinositol 3-phosphate by PROPPINs in autophagy[END_REF]. Further, it has been reported that mammalian ATG16L was able to bind phosphoinisitides, notably PI3P, through its coiled-coil domain (CCD) and the deletion of this domain resulted in an incapability to lipidate ATG8 [START_REF] Dudley | Intrinsic lipid binding activity of ATG16L1 supports efficient membrane anchoring and autophagy[END_REF]. Another protein implicated in the conjugation of ATG8 to PE, Atg3, contains an amino-terminal amphiphatic helix and was found to promote ATG8 lipidation in vitro in liposomes ATG3 functions at membranes with a high curvature (Nath et al., 2014).

Thus, it would seem that ATG8 lipidation, as well as other hallmarks of autophagy, is spatially regulated by a wide range of lipid/protein interactions, many of them involving PI3P but also mediated by membrane features such as membrane curvature of packing defects. In plants, PI3P is also critical for autophagy, as mutants for genes part of this complex display a hampered autophagosome formation (Liu et al., 2009). The autophagy specific PI3K complex as well as plant PI3P-effectors such as SH3P2 or ATG18 have also been characterized and have provided evidence of the importance of PI3P for plant autophagy (Liu et al., 2020;Zhuang et al., 2017;Zhuang et al., 2013). For example, the PI3P-binding SH3P2 protein was found to promote PI3K foci formation and to interact with ATG8 and was thus proposed as playing important roles in autophagosome progression (Zhuang et al., 2013). Moreover, the FYVE domain required for endosomal sorting 1 (FREE1) protein that we mentioned above in this review, was found to interact with SH3P2 and ATG6, a member of the PI3K complex (Gao et al., 2015). Finally, ATG5 was found to localize specifically to the rim of phagophore structures, suggesting that some specific membranes features such as curvature dictate the localization of the ATG5-ATG12 complex as well (Le Bars et al., 2014).

Another phosphoinositide important for the formation of autophagosomes is PI4P. In both mammals and yeast, PI4P plays important roles in the trafficking of ATG9 vesicles (Judith et al., 2019;Wang et al., 2012). Both PI3P and PI4P have been shown to mediate multiple steps in the autophagic pathway. For example, a clearance of PI3P from autophagosomes is required for autophagy, as the loss of function of the myotybularin PI3P phosphatase Ymr1 resulted in a continuous association of ATG proteins with completed APs which failed to fuse with vacuole in yeast (Cebollero et al., 2012). This phenomenon has also been observed in C. elegans [START_REF] Wu | PI3P phosphatase activity is required for autophagosome maturation and autolysosome formation[END_REF]. However, PI3P has also a role in AP maturation as it facilitates the recruitment and stabilization of specific proteins required for its fusion [START_REF] Bas | Reconstitution reveals Ykt6 as the autophagosomal SNARE in autophagosomevacuole fusion[END_REF]Gao et al., 2018) highlighting that these events are highly regulated in a spatiotemporal manner.

Moreover, a deficiency of the yeast SAC1 phosphatase, responsible for cleaving the phosphate moiety of PI4P, lead to an abnormal accumulation of PI4P in autophagosomes and resulted in a failure to recruit the SNARE complexes required for autophagosome fusion with the vacuole (Zhang et al., 2020). Another recent publication also identified the SAC1 interactor TMEM39A which promotes the intracellular trafficking of SAC1 to regulate the spatial distribution and levels of PI4P [START_REF] Miao | The ER-Localized Transmembrane Protein TMEM39A/SUSR2 Regulates Autophagy by Controlling the Trafficking of the PtdIns(4)P Phosphatase SAC1[END_REF]. These results suggest that similarly to PI3P, the PI4P pool at autophagic structures is under highly regulated turnover to support the proper functioning of the autophagic process.

Interestingly, both of PI3P and PI4P display asymmetrical distributions on autophagic membranes from yeast and mammals [START_REF] Cheng | Yeast and mammalian autophagosomes exhibit distinct phosphatidylinositol 3-phosphate asymmetries[END_REF]Kurokawa et al., 2019). The geometry of both these lipids (with a polar head larger than its acyl chains) confers them an inverted cone shape. When lipids with an inverted cone shape cluster within membranes, they can deform membranes and create curvature (Bigay & Antonny 2012). Thus, the asymmetrical distribution of PI3P or PI4P within phagophores membranes could contribute to its highly curved architecture. Another critical lipid for autophagy is phosphatidylethanolamine (PE).

Conjugation of the ATG8 protein to PE allows for its incorporation into autophagic membranes.

The incorporation of ATG-PE to the phagophore is thought to support the expansion of the growing structure (Xie et al., 2008). Furthermore, the incorporation of ATG8-PE contributes to the membrane curvature of the phagophore (Knorr et al., 2014). Finally, other phosphoinositides such as PI(4,5)P2 and PI(3,5)P2 have been shown to recruit proteins implicated in autophagy. PI(4,5)P2 was found to mediate the recruitment of the endomembrane trafficking regulator SNX18, which further recruited ATG16L to perform ATG8 lipidation but also contributes to the trafficking of ATG9 vesicles (Knaevelsrud et al., 2013;[START_REF] Søreng | SNX18 regulates ATG9A trafficking from recycling endosomes by recruiting Dynamin-2[END_REF]. The implication of PI(3,5)P2 in autophagy has also been reported although the exact molecular mechanism remains elusive. A reduced phosphoinositol-3,5-kinase Fab1 activity lead to an impairment in autophagosome/lysosme fusion [START_REF] Rusten | ESCRTs and Fab1 Regulate Distinct Steps of Autophagy[END_REF]. Nevertheless, an impairment of a phosphoinositol-3,5-phosphatase INPP5E also lead to this same phenotype (Hasegawa et al., 2016). This indicates that the modulation of PI3,5P2 levels on autophagic structures is tightly regulated to allow proper fusion with lysosomes.

At present, several lipids have been reported to have a critical role in the progression of autophagy and their function as binding modules for the recruitment of ATG proteins has been proposed. However, information about their quantity, dynamics and repartition within the membranes of autophagosomes remain scarce. How these impact on the shaping and ultrastructure of the phagophore thus remain a fundamental open question that future research will need to address to resolve the puzzled mechanisms of autophagosome formation.

Lipids are implicated in the regulation of autophagy

In addition to their function as docking sites for the recruitment of ATG proteins, some lipids play key roles in the regulation of autophagy. Phosphatidic acid (PA) was shown to modulate autophagy in mammalian cells. The PA-synthesizing enzyme PLD1 relocalizes to autophagic structures and its absence greatly impairs autophagic activity (Dall [START_REF] Armi | The Phospholipase D1 Pathway Modulates Macroautophagy[END_REF].

Moreover, the PA-binding protein HSBP13a has been shown to negatively affect autophagy by modulating the activity of PLD1 and PA levels (Holland et al., 2016). These results suggest that the synthesis of PA is required at the site of autophagosome formation. Although recent evidence suggest that PA is present at low concentration on the membranes of autophagy structures in yeast (Schütter et al., 2020), its function in the structure or activities of autophagy membrane remains largely unknown. Besides its role as a structural component, PA is also known to regulate autophagy by stimulating the mTOR pathway which suppresses autophagy [START_REF] Sun | mTOR signaling: PLD takes center stage[END_REF]. mTOR is a master regulator capable of transducing nutrient signals and to regulate several cellular functions, and one of mTORs function is to inhibit the autophagy pathway [START_REF] Sun | mTOR signaling: PLD takes center stage[END_REF]. This implicates two distinct functions of PA concerning autophagy. One possible explanation is that different pools of PA are responsible for these distinct functions, one pool of PA might be incorporated into autophagosomes, while the other pool could be responsible for interacting with mTOR to suppress autophagic activity. Further studies await to reveal the molecular role of PA in the fine-tuning of autophagy activity.

Another class of lipids implicated in the regulation of autophagy are sphingolipids. Two sphingolipids have been shown to play important roles in autophagy regulation: ceramide and sphigosine-1-phosphate (S1P). Conversely to PA, ceramides inhibition of Akt, a mTOR regulator, results in mTOR inactivation and thus in autophagy activation [START_REF] Pattingre | Role of JNK1-dependent Bcl-2 Phosphorylation in Ceramide-induced Macroautophagy[END_REF].

The activity of the S1P synthesizing enzyme, the Sphingosine Kinase, was shown to also induce autophagy, through an Akt-independent deregulation of the mTOR pathway [START_REF] Lavieu | Regulation of Autophagy by Sphingosine Kinase 1 and Its Role in Cell Survival during Nutrient Starvation *[END_REF]. Multiple fatty acids have also been shown to regulate autophagy. The highly unsaturated docosahexaenoic acid (DHA) (22 carbons and 6 unsaturations, 22:6) was found to induce autophagy in cancer cell lines by AMP-activated protein kinase (AMPK)-mediated inhibition of mTOR [START_REF] Jing | Docosahexaenoic acid induces autophagy through p53/AMPK/mTOR signaling and promotes apoptosis in human cancer cells harboring wild-type p53[END_REF]. Interestingly, it was also found that palmitate (16:0) and oleate (18:1) induced autophagy in mammalian cells, although through distinct mechanisms.

Palmitate supplementation activated autophagy via the canonical PI3K-induced autophagy pathway [START_REF] Niso-Santano | Unsaturated fatty acids induce non-canonical autophagy[END_REF]. Oleate on the other hand, was able to induce autophagy even in cells defective for the PI3K complex, indicating that oleate activates a non-canonical autophagic pathway [START_REF] Niso-Santano | Unsaturated fatty acids induce non-canonical autophagy[END_REF]. Finally, a pharmacological inhibition of cholesterol in mammalian cells lead to an activation of autophagy due to the inactivation of the mTOR pathway [START_REF] Vilimanovich | Statin-mediated inhibition of cholesterol synthesis induces cytoprotective autophagy in human leukemic cells[END_REF].

Throughout this section, we have seen that lipids are key actors in the autophagic process.

They not only contribute to defining the growth and particular shape of autophagic structures, but also serve as anchors for the recruitment of many membrane-associated proteins that drive this process. In plants, little is known about the lipids that are present on autophagic structures as well as their functions in the process. Different types of stress can induce autophagy, with a specific autophagic response for each of them (Stephani & Dagdas, 2020). It is likely that different types of stress induce the formation of distinct populations of autophagosomes, whose lipid composition might as well vary depending on the stress. We know that autophagy is critical for plants response and adaptation to stressful environments, yet little is known about the lipid composition and functions in the formation of autophagic structures upon each stress and/or on the type of cargo to be engulfed. Future studies are required to unravel the nature, dynamics and functions of lipids in plant autophagosomes. These should provide valuable insights into the understanding of how lipids functional contribute to the autophagic-mediated addptation of plant to environmental stresses.

II-2. Autophagy controls plant responses to environmental stresses

Autophagy is induced by multiple stresses and post-translational regulated

Maintaining the constants of the internal environment, also called homeostasis, is a complex process in eukaryotic organisms that requires multiple controls over time and space. Plants are subjected throughout their life cycle to multiple environmental and developmental disturbances that affect homeostasis. In addition, the acceleration of global warming since the beginning of the first industrial revolution is also an important process that affects the homeostasis of the plant. Thus, faced with these multiple stresses, plants have developed stringent and specific quality control mechanisms such as autophagy. The simplest and most classic way to follow the effect of environmental parameters on the mechanism of autophagy is to observe the formation of AP in the form of punctas by confocal microscopy using preferentially fluorescent-tagged proteins (GFP) coupled to a autophagy marker proteins (ATG8 for example) or by directly labeling AP with acidotropic dyes (for example, monodansylcadaverine (MDC), monodansylpentane (MDH), acridine orange, etc.) but with a lower sensitivity [START_REF] Klionsky | Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)[END_REF]. The use of concanamycin A, an inhibitor of the degradation of AB in the vacuole, can also be used to visualize the accumulation of autophagy structures. Using these techniques, the rate of AP formation and their number has been shown to be significantly induced by a plethora of environmental stresses [START_REF] Chen | Autophagy contributes to regulation of the hypoxia response during submergence in Arabidopsis thaliana[END_REF]Li et al., 2014;[START_REF] Marshall | Autophagic Degradation of the 26S Proteasome Is Mediated by the Dual ATG8/Ubiquitin Receptor RPN10 in Arabidopsis[END_REF] (Figure 4).

Induction of autophagy in response to environmental or developmental cues is finely

regulated by multiple mechanisms. Indeed, several studies have shown that the relative levels of expression of ATG genes were modified during nitrogen and carbon deficiency [START_REF] Pei | Identification of autophagy-related genes ATG4 and ATG8 from wheat (Triticum aestivum L.) and profiling of their expression patterns responding to biotic and abiotic stresses[END_REF][START_REF] Zhai | Autophagy, a Conserved Mechanism for Protein Degradation, Responds to Heat, and Other Abiotic Stresses in Capsicum annuum L[END_REF][START_REF] Rose | Starvation-induced expression of autophagy-related genes in Arabidopsis[END_REF][START_REF] Garapati | Transcription Factor Arabidopsis Activating Factor1 Integrates Carbon Starvation Responses with Trehalose Metabolism[END_REF], osmotic stresses (Liu et al., 2009;[START_REF] Pei | Identification of autophagy-related genes ATG4 and ATG8 from wheat (Triticum aestivum L.) and profiling of their expression patterns responding to biotic and abiotic stresses[END_REF]Wang et al., 2015;[START_REF] Xia | Genome-wide identification, classification, and expression analysis of autophagy-associated gene homologues in rice (Oryza sativa L.)', DNA research : an international journal for rapid publication of reports on genes and genomes[END_REF][START_REF] Zhai | Autophagy, a Conserved Mechanism for Protein Degradation, Responds to Heat, and Other Abiotic Stresses in Capsicum annuum L[END_REF], heat and cold stresses [START_REF] Zhou | NBR1-Mediated Selective Autophagy Targets Insoluble Ubiquitinated Protein Aggregates in Plant Stress Responses[END_REF][START_REF] Zhou | Role and regulation of autophagy in heat stress responses of tomato plants[END_REF][START_REF] Pei | Identification of autophagy-related genes ATG4 and ATG8 from wheat (Triticum aestivum L.) and profiling of their expression patterns responding to biotic and abiotic stresses[END_REF][START_REF] Xia | Genome-wide identification, classification, and expression analysis of autophagy-associated gene homologues in rice (Oryza sativa L.)', DNA research : an international journal for rapid publication of reports on genes and genomes[END_REF][START_REF] Zhai | Autophagy, a Conserved Mechanism for Protein Degradation, Responds to Heat, and Other Abiotic Stresses in Capsicum annuum L[END_REF], light quality and intensity [START_REF] Dündar | Autophagy-deficient Arabidopsis mutant atg5, which shows ultraviolet-B sensitivity, cannot remove ultraviolet-B-induced fragmented mitochondria[END_REF][START_REF] Xia | Genome-wide identification, classification, and expression analysis of autophagy-associated gene homologues in rice (Oryza sativa L.)', DNA research : an international journal for rapid publication of reports on genes and genomes[END_REF], hypoxia [START_REF] Chen | Autophagy contributes to regulation of the hypoxia response during submergence in Arabidopsis thaliana[END_REF], biotic stress [START_REF] Pei | Identification of autophagy-related genes ATG4 and ATG8 from wheat (Triticum aestivum L.) and profiling of their expression patterns responding to biotic and abiotic stresses[END_REF], viruses infection [START_REF] Hafrén | Selective autophagy limits cauliflower mosaic virus infection by NBR1-mediated targeting of viral capsid protein and particles[END_REF][START_REF] Zhou | Autophagy contributes to regulate the ROS levels and PCD progress in TMV-infected tomatoes[END_REF][START_REF] Li | Beclin1 restricts RNA virus infection in plants through suppression and degradation of the viral polymerase[END_REF][START_REF] Haxim | Autophagy functions as an antiviral mechanism against geminiviruses in plants[END_REF][START_REF] Miozzi | Transcriptomics of the Interaction between the Monopartite Phloem-Limited Geminivirus Tomato Yellow Leaf Curl Sardinia Virus and Solanum lycopersicum Highlights a Role for Plant Hormones, Autophagy and Plant Immune System Fine Tuning during Infection[END_REF][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], plant reproductive development [START_REF] Berardino | Autophagy controls resource allocation and protein storage accumulation in Arabidopsis seeds[END_REF][START_REF] Chung | The ATG Autophagic Conjugation System in Maize: ATG Transcripts and Abundance of the ATG8-Lipid Adduct Are Regulated by Development and Nutrient Availability[END_REF], senescence [START_REF] Breeze | High-Resolution Temporal Profiling of Transcripts during Arabidopsis Leaf Senescence Reveals a Distinct Chronology of Processes and Regulation[END_REF] and plant root development [START_REF] Wojciechowska | Occurrence of autophagy during pioneer root and stem development in Populus trichocarpa[END_REF]. In addition to this transcriptional regulation, post-translation modifications are also critical for the control of autophagy activity. When growing conditions are optimal, the ATG1 kinase complex is phosphorylated, in particular by the evolutionary conserved complex TARGET OF RAPAMYCIN (TOR), which inhibits the initiation of autophagy (Liu and Bassham 2010) (Figure 1) The TOR complex is made up of TOR itself, a serine/threonine kinase, as well as two partners: the REGULATORY ASSOCIATED PROTEIN OF TOR (RAPTOR) which transfers substrates to TOR and LETHAL WITH SEC THIRTEEN 8 (LST8) which stabilizes the complex [START_REF] Pu | TOR-Dependent and -Independent Pathways Regulate Autophagy in Arabidopsis thaliana[END_REF]. The Arabidopsis tor knockout mutant is embryo-lethal and the RNAi lines of TOR present a constitutive autophagy phenotype (Liu and Bassham 2010;[START_REF] Menand | Expression and disruption of the Arabidopsis TOR (target of rapamycin) gene[END_REF]. Recently, Van Leene et al., also showed that TOR could control the induction of autophagy in plants via the hyperphosphorylation of ATG13 [START_REF] Van Leene | Capturing the phosphorylation and protein interaction landscape of the plant TOR kinase[END_REF]. Following environmental stress, autophagy is activated by inactivation of the TOR complex by the SNF1-RELATED PROTEIN KINASE 1 (SnRK1) complex, or by direct phosphorylation of ATG1 during nutrient deprivation (Soto-Burgos et al., 2018). The heterotrimeric SnRK1 complex is composed of an α catalylic subunit (KIN10 and KIN11) and two groups of regulatory subunits: the classical subunits (β1, β2 and γ) and the plant-specific subunits (β3 and βγ) [START_REF] Chen | The AMP-Activated Protein Kinase KIN10 Is Involved in the Regulation of Autophagy in Arabidopsis[END_REF]. During abiotic stress, overexpression of KIN10 leads to the induction of autophagy while autophagy is blocked in the kin10 mutant meaning that SnRK1 is required for the induction of autophagy [START_REF] Chen | The AMP-Activated Protein Kinase KIN10 Is Involved in the Regulation of Autophagy in Arabidopsis[END_REF]Soto-Burgos and Bassham 2017).

Transcriptional regulation of autophagy also involves WRKY, one of the largest family of transcription factors in plants. This family is primarily involved in resistance to pathogens [START_REF] Rushton | WRKY transcription factors[END_REF]. Zheng and colleagues have shown that the WRKY33 transcription factor plays an important role in resistance to necrotrophic pathogens [START_REF] Zheng | Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens[END_REF]. The wkry33 mutant is notably hypersensitive to the necrotrophic fungal agents Botrytis cinerea and Alternaria brassicicola but not to the hemi-biotrophic bacteria Pseudomonas syringae DC3000 [START_REF] Zheng | Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens[END_REF]. In addition, overexpression of WRKY33 promotes resistance to B. cinerea and A. brassicicola [START_REF] Zheng | Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens[END_REF]. By a yeast double hybrid study, Lai et al.,also showed that the transcription factor WRKY33 interacted with ATG18a [START_REF] Lai | A critical role of autophagy in plant resistance to necrotrophic fungal pathogens[END_REF]. Finally, Zhou et al., also demonstrated that silencing of WRKY33a and WRKY33b in tomato during heat stress causes a concomitant decrease in ATG genes expression and AP formation [START_REF] Zhou | Role and regulation of autophagy in heat stress responses of tomato plants[END_REF].

A recent study focused on the plant BAX INHIBITOR-1 (BI-1) protein, a highly conserved protein which suppresses cell death by modifying lipid dynamics, ROS level and the control of calcium flux [START_REF] Ishikawa | Bax inhibitor-1: a highly conserved endoplasmic reticulum-resident cell death suppressor[END_REF][START_REF] Ihara-Ohori | Cell Death Suppressor Arabidopsis Bax Inhibitor-1 Is Associated with Calmodulin Binding and Ion Homeostasis[END_REF]. Xu and colleagues demonstrated that the BI-1 protein interacts with ATG6 in vivo and in vitro [START_REF] Xu | Plant Bax Inhibitor-1 interacts with ATG6 to regulate autophagy and programmed cell death[END_REF].

Using a Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system, the authors also showed that the silencing of BI-1 in Nicotiana benthamiana reduced the autophagic activity induced by the Tobacco mosaic virus (TMV) and methyl violagen (MV) while the overexpression of BI-1 increases autophagy activity [START_REF] Xu | Plant Bax Inhibitor-1 interacts with ATG6 to regulate autophagy and programmed cell death[END_REF]. The authors conclude that BI-1 positively regulates autophagy and exhibits a dual autophagy-dependent manner prosurvival and prodeath roles [START_REF] Xu | Plant Bax Inhibitor-1 interacts with ATG6 to regulate autophagy and programmed cell death[END_REF]. photosynthetic GAPDHs (GAPA1, GAPA2, GAPB), ii) cytosolic glycolytic GAPDHs (GAPC1 and GAPC2), iii) plastidic glycolytic GAPDHs (GAPCp1 and GAPCp2) and iv) NADPdependent non-phosphorylating cytosolic GAPDH (NP-GAPDH) [START_REF] Zaffagnini | Plant cytoplasmic GAPDH: redox post-translational modifications and moonlighting properties[END_REF][START_REF] Henry | Beyond Glycolysis: GAPDHs Are Multi-functional Enzymes Involved in Regulation of ROS, Autophagy, and Plant Immune Responses[END_REF]. This enzyme, who also catalyzed the conversion of glyceraldehyde 3-phosphate to 1,3 biphosphoglycerate during the glycolysis, interacts with ATG3 in Nicotiana benthamiana [START_REF] Han | Cytoplastic Glyceraldehyde-3-Phosphate Dehydrogenases Interact with ATG3 to Negatively Regulate Autophagy and Immunity in <em>Nicotiana benthamiana</em&gt[END_REF]. Using a TRV-based VIGS system, the authors notably showed that the silencing of GAPC1, GAPC2 and GAPC3 activated an ATG3-dependent autophagy mechanism and enhanced N gene-mediated cell death and plant resistance against TMV, Pseudomonas syringae pv tomato DC3000 and P. syringae pv tabaci in N. benthamiana [START_REF] Han | Cytoplastic Glyceraldehyde-3-Phosphate Dehydrogenases Interact with ATG3 to Negatively Regulate Autophagy and Immunity in <em>Nicotiana benthamiana</em&gt[END_REF]. Henry and colleagues have also shown the same kind of responses in A. thaliana

for GAPDH knockout lines subject to biotic stress with P. syringae DC3000 [START_REF] Henry | Beyond Glycolysis: GAPDHs Are Multi-functional Enzymes Involved in Regulation of ROS, Autophagy, and Plant Immune Responses[END_REF].

Conversely, overexpression of GAPCs in N. benthamiana suppresses autophagy suggesting that GAPCs is a negative regulator of autophagy [START_REF] Han | Cytoplastic Glyceraldehyde-3-Phosphate Dehydrogenases Interact with ATG3 to Negatively Regulate Autophagy and Immunity in <em>Nicotiana benthamiana</em&gt[END_REF]. Furthermore, Haxim and colleagues have identified by yeast-two hybrid but also by bimolecular fluorescence complementation (BiFC) in N. benthamiana that the virulence factor βC1 produce by the Cotton leaf curl Multan betasatellite (CLCuMuB) interacts with ATG8f [START_REF] Haxim | Autophagy functions as an antiviral mechanism against geminiviruses in plants[END_REF]. The βC1 factor also interacts also with GAPC and disrupt GAPCs-ATG3 interactions that consequently lead to an activation of the autophagy pathway [START_REF] Ismayil | Cotton leaf curl Multan virus βC1 Protein Induces Autophagy by Disrupting the Interaction of Autophagy-Related Protein 3 with Glyceraldehyde-3-Phosphate Dehydrogenases[END_REF]. A βC1 variant (βC1 3A ) that has a reduced affinity for GAPC failed to induce autophagy and consequently, plants infected by this variant shows stronger disease symptoms [START_REF] Ismayil | Cotton leaf curl Multan virus βC1 Protein Induces Autophagy by Disrupting the Interaction of Autophagy-Related Protein 3 with Glyceraldehyde-3-Phosphate Dehydrogenases[END_REF].

Autophagy is critical for plant response to abiotic, biotic and virus stresses

To support their optimal development, plants need to draw essential nutriments from the soil, including macronutrients (C, H, O, N, P, S, Mg, Ca) as well as micronutrients (Fe, Zn, Cu, Ni, Mo, Mn, Cl, B). However, due to their inability to move, plants can have nutrient-deficient phases of their life cycle when the soil is poor (Masclaux-Daubresse et al., 2017). Thus, the first studies focusing on abiotic stress and autophagy relationship were carried out via nutrient deficiencies by analyzing knock-out mutants of the autophagy machinery (atg mutants), which, for the most part, show a complete block in autophagy activity. Doelling and colleagues showed that the atg7-1 mutant is hypersensitive to nitrogen and carbon deficiency (Doelling et al., 2002). Likewise, the atg9-1 mutant exhibits a chlorosis phenotype in the cotyledons as well as in the rosette leaves in comparison with the WT in nitrogen and carbon starvation [START_REF] Hanaoka | Leaf Senescence and Starvation-Induced Chlorosis Are Accelerated by the Disruption of an Arabidopsis Autophagy Gene[END_REF]. Similar results were also obtained with the atg4a4b-1 (Yoshimoto et al., 2004), atg5-1 (Thompson et al., 2005), atg7-2 (Li et al., 2014), atg9-4 [START_REF] Floyd | Evidence for autophagy-dependent pathways of rRNA turnover in Arabidopsis[END_REF], atg10-1 [START_REF] Phillips | The ATG12-conjugating enzyme ATG10 Is essential for autophagic vesicle formation in Arabidopsis thaliana[END_REF], atg11-1 and atg11-2 (Li et al., 2014), atg12a-1/atg12b-1 (Chung et al., 2010), atg13a-1/atg13b-2 and atg13a-2/atg13b-2 (Suttangkakul et al., 2011) and atg18a (Xiong et al., 2005) mutants in nitrogen and carbon depletion. In addition, overexpression of the SnRK1 α catylic subunit KIN10 enhanced the phosphorylation of ATG1a under nutrient starvation suggesting a positive regulation role of autophagy by KIN10 [START_REF] Chen | The AMP-Activated Protein Kinase KIN10 Is Involved in the Regulation of Autophagy in Arabidopsis[END_REF]. Recently, Shinozaki et al., showed in particular that the atg5 and atg7 mutants presented difficulties to grow as well as an advanced chlorosis phenotype in zinc deficient condition [START_REF] Shinozaki | Autophagy Increases Zinc Bioavailability to Avoid Light-Mediated Reactive Oxygen Species Production under Zinc Deficiency[END_REF].

The durations of drought as well as their severity are gradually increasing with global warming. Thus, understanding the responses of photosynthetic organisms to osmotic and drought stress becomes a major issue for the scientific community as well as for the agriculture world. At the end of the 2000s, initial results highlighted the importance of autophagy in plants following osmotic stress. Simply put, plants with a reduced or absent autophagy activity mostly show an increased sensitivity to drought. Liu et al., showed that the RNAi ATG18a mutant exhibits hypersensitivity to salt, mannitol and drought treatments (Liu et al., 2009). Zhou and colleagues presented similar results with the atg5, atg7 and nbr1 mutants [START_REF] Zhou | NBR1-Mediated Selective Autophagy Targets Insoluble Ubiquitinated Protein Aggregates in Plant Stress Responses[END_REF].

These mutants exhibit hypersensitivity to drought compared to WT as well as hypersensitivity to salt [START_REF] Zhou | NBR1-Mediated Selective Autophagy Targets Insoluble Ubiquitinated Protein Aggregates in Plant Stress Responses[END_REF]. The atg2, atg5, atg7 mutants present more difficulty to germinate in the presence of mannitol in comparison with WT whereas the atg9 mutant and the ATG8 overexpressor line germinate more quickly than WT [START_REF] Luo | Autophagy Is Rapidly Induced by Salt Stress and Is Required for Salt Tolerance in Arabidopsis[END_REF]. In addition, the percentage of fresh weight of reduced sugars and total soluble sugars decreased in the atg mutants after treatment with salt while it increased in the ATG8 overexpressor line [START_REF] Luo | Autophagy Is Rapidly Induced by Salt Stress and Is Required for Salt Tolerance in Arabidopsis[END_REF]. Moreover, Michaeli et al., also showed that the knock-down of ATG8-INTERACTING PROTEIN (ATI1) exhibited an increased sensitivity to salt suggesting a role in the elimination of damaged proteins during salt stress (Michaeli et al., 2014). In contrast to autophagy mutants, plants with higher autophagy flux mostly show an increased resistance to water scarcity.

Previously, a physiological study of GFP-AtATG8f-HA showed that the overexpressor was sensitive of mild salt with fainter green leaves, an increase in electrolyte leakage but with longer primary roots than WT [START_REF] Slavikova | An autophagyassociated Atg8 protein is involved in the responses of Arabidopsis seedlings to hormonal controls and abiotic stresses[END_REF]. Slavikova et al., propose that the arrest of wildtype primary root growth under salt stress is associated with programmed cell death (PCD) of the primary root meristem. Thus, the expression of this ATG8 construct could negatively affects PCD by stimulating autophagy [START_REF] Slavikova | An autophagyassociated Atg8 protein is involved in the responses of Arabidopsis seedlings to hormonal controls and abiotic stresses[END_REF]. The authors conclude that inactivation of basal autophagy is necessary for the inhibition of primary root growth under salt stress [START_REF] Slavikova | An autophagyassociated Atg8 protein is involved in the responses of Arabidopsis seedlings to hormonal controls and abiotic stresses[END_REF]. Similarly, the overexpression of KIN10, which induces autophagy, promotes drought tolerance compared to WT and KIN10-RNAi lines [START_REF] Chen | The AMP-Activated Protein Kinase KIN10 Is Involved in the Regulation of Autophagy in Arabidopsis[END_REF].

Heterologous expression of ATG3a and ATG3b from Malus domestica in Arabidopsis thaliana enhances tolerance to salt and mannitol with an higher fresh weights and longer roots compared to WT [START_REF] Wang | Apple autophagy-related protein MdATG3s afford tolerance to multiple abiotic stresses[END_REF]. Likewise, heterologous expression of apple ATG18a (MdATG18a) in tomato plants (Solanum lycopersicum) promotes drought tolerance characterized by a better survival rate and a higher water content (RWC) as well as a lower malondialdehyde (MDA) content and electrolyte leakage compare to WT [START_REF] Sun | Improvement of drought tolerance by overexpressing MdATG18a is mediated by modified antioxidant system and activated autophagy in transgenic apple[END_REF].

Furthermore, heterologous expression of ATG8f from Musa acuminata (banana) in Arabidopsis improved also drought stress resistance (Li et al., 2019). Finally, Bao and colleagues recently identified a DUF641 family of plant-specific proteins called COST1

(constitutively stressed 1) that inhibit autophagy under normal growth conditions by directly interacting with ATG8. In particular, the authors show that the cost1 mutant, which showed less autophagy, exhibits a better drought resistance phenotype while the COST1 overexpressor, in which autophagy is reduced, presents hypersensitivity to drought [START_REF] Bao | COST1 regulates autophagy to control plant drought tolerance[END_REF].

In addition to nutritional deficiencies and osmotic/water stresses, plants are also subject to rather significant temperature variations depending on the season. Furthermore, with the accentuation of the effect of the global warming, the periods of exposure to low or high temperature are likely to increase during the 21 st century with potential negative effects on crop productivity. For example, high temperatures promotes the denaturation and misfolding of proteins inducing their degradation via quality control pathways such as autophagy. First of all, the atg5-1, atg7-2 and nbr1-1 mutants are hypersensitive to heat stress in comparison with

WT after incubation at 37 °C or 45 °C [START_REF] Zhou | NBR1-Mediated Selective Autophagy Targets Insoluble Ubiquitinated Protein Aggregates in Plant Stress Responses[END_REF][START_REF] Yang | Activation of autophagy by unfolded proteins during endoplasmic reticulum stress[END_REF]. Furthermore, Jiang et al.,also showed that the atg2-1 mutant had a slight phenotype at 30 °C with a lower fresh weight and chlorophyll amount compared to WT [START_REF] Jiang | Autophagy-Related 2 Regulates Chlorophyll Degradation under Abiotic Stress Conditions in Arabidopsis[END_REF]. However, the atg2-1 mutant as well as the atg5-1, atg7-2 and atg10-1 mutants show an alteration in pollen development as well for the anthers dehiscence after treatment at 30 °C [START_REF] Dündar | Autophagy mitigates high-temperature injury in pollen development of Arabidopsis thaliana[END_REF].

Recently, Thirumalaikumar et al., demonstrated that NBR1 played an important role in the defense of plants against repeated heat stress, in particular by controlling the levels of HSP90.1 (heat shock protein 90.1) and ROF1 (rotamase FKBP 1) [START_REF] Thirumalaikumar | Selective autophagy regulates heat stress memory in Arabidopsis by NBR1-mediated targeting of HSP90 and ROF1[END_REF]. NBR1 mediates the degradation of HSP90.1 and ROF1 via autophagy repressing the heat stress response by lowering the expression of HSP genes regulated by the transcription factor HSFA2 (heat shock factor A2) [START_REF] Nishizawa | Arabidopsis heat shock transcription factor A2 as a key regulator in response to several types of environmental stress[END_REF][START_REF] Charng | A heat-inducible transcription factor, HsfA2, is required for extension of acquired thermotolerance in Arabidopsis[END_REF][START_REF] Thirumalaikumar | Selective autophagy regulates heat stress memory in Arabidopsis by NBR1-mediated targeting of HSP90 and ROF1[END_REF]. The same team also showed that autophagy was responsible for the accumulation of proteogenic dipeptides in response to heat stress [START_REF] Thirumalaikumar | Autophagy is responsible for the accumulation of proteogenic dipeptides in response to heat stress in Arabidopsis thaliana[END_REF]. Finally, to our knowledge, the study of autophagy in cold stress conditions has been little (Izumi et al., 2017) or even completely unexplored and therefore constitutes an interesting line of research for a better understanding of the mechanisms binding abiotic stresses and autophagy.

During their life cycle, plants are also subjected to more or less long photoperiods depending on the seasons but also to variable light spectral qualities including visible light (400-700 nm)

and ultraviolet-B (UV-B; 280-315 nm) which is the most energetic wavelenght light reaching the surface of the Earth (Izumi et al., 2017;[START_REF] Nakamura | Autophagy contributes to quality control of leaf mitochondria[END_REF]. A strong and prolonged exposure to visible light or UV-B can be responsible for damage in plants by degrading macromolecules (DNA, proteins and lipids). Recently it was shown that the atg2, atg5, atg7, atg10, atg12a and atg18a mutants as well as the phr1 (cyclobutane pyrimidine dimer-specific photolyase) mutant were hypersensitive to a treatment with UV-B leading to a half reduction in the activity of PSII in comparison with the WT (Izumi et al., 2017;[START_REF] Nakamura | Autophagy contributes to quality control of leaf mitochondria[END_REF]. Jiang et al. ,also showed that a high visible light treatment induced a necrosis phenotype in the atg2-1 mutant [START_REF] Jiang | Autophagy-Related 2 Regulates Chlorophyll Degradation under Abiotic Stress Conditions in Arabidopsis[END_REF]. The photoperiod is also a vital parameter for the development of the plant. Indeed, at the winter solstice, the night periods are much longer (short day) while at the summer solstice the periods of sunshine will be longer (long day). Thus, the photoperiod can therefore have an influence on the homeostasis of the plant and in fine on autophagy machinery. For example, the atg7-1 mutant cultivated in short days exhibits accelerated 2002). Similar results were also obtained with the atg5-1 mutant (Thompson et al., 2005) and the double mutants atg13a/atg13b (Suttangkakul et al., 2011).

Throughout their development, plants have to face diverse type of harmful invaders, including bacteria, fungi and viruses. They have developed a number of strategies to be able to survive to these environmental stresses. The implication of autophagy in plant immunity cannot be generalized because it can act either as a pro-and or anti-pathogens process depending on the kind of pathogen, its lifestyle and its infection strategy [START_REF] Lenz | Autophagy controls plant basal immunity in a pathogenic lifestyle-dependent manner[END_REF]. In order to block attacks by pathogens, plants use two types of immune system defense: a basal response and a specific response [START_REF] Wang | ATG2, an autophagy-related protein, negatively affects powdery mildew resistance and mildew-induced cell death in Arabidopsis[END_REF]Zhang et al., 2020;[START_REF] Zheng | Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens[END_REF][START_REF] Lai | A critical role of autophagy in plant resistance to necrotrophic fungal pathogens[END_REF][START_REF] Üstün | Bacteria Exploit Autophagy for Proteasome Degradation and Enhanced Virulence in Plants[END_REF]. In this bipartite system, the first line of resistance is call PAMP-triggered immunity (PTI) and it recognizes pathogen associated molecular patterns (PAMPs). The next level of defense, named effector-triggered immunity (ETI) is more specific and against pathogens that co-evolved with plants, which allowed them to override and suppress basal defense [START_REF] Wang | ATG2, an autophagy-related protein, negatively affects powdery mildew resistance and mildew-induced cell death in Arabidopsis[END_REF]Zhang et al., 2020). Moreover, ETI is often associated with host PCD [START_REF] Lenz | Autophagy controls plant basal immunity in a pathogenic lifestyle-dependent manner[END_REF]. Phytopathogens can be divided into two categories: necrotrophic and biotrophic. Their infection strategies are totally different, necrotroph organisms kill host to obtain nutrients from dead tissue whereas biotroph pathogens need a living host to proliferate inside [START_REF] Lenz | Autophagy controls plant basal immunity in a pathogenic lifestyle-dependent manner[END_REF]. These two strategies explain the duality of autophagic activity in the infected host, leading in one case in a beneficial impact and in the other in a detrimental effect. Necrotrophic pathogens include particularly mildew like Golovinomyces cichoracearum or Phytophthora infestans and necrotrophic fungi such as Botrytis cinerea or Alternaria brassicicola. These pathogens rely on a range of virulent factors and hydrolytic enzymes to promote host cell death [START_REF] Lai | A critical role of autophagy in plant resistance to necrotrophic fungal pathogens[END_REF].

Among necrotrophs, Wang and colleagues reported that autophagy mutant atg2-2 has better resistance against powdery mildew G. cichoracearum that wild-type plants, but the mutant loses its resistance when key actors of salicylic acid (SA) pathway are mutated [START_REF] Wang | ATG2, an autophagy-related protein, negatively affects powdery mildew resistance and mildew-induced cell death in Arabidopsis[END_REF]. The majority of plant's activity is regulated by signaling hormones such as SA, jasmonic acid (JA) or ethylene. SA pathway is known to plays a critical role in defense responses against biotrophic pathogens [START_REF] Zheng | Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens[END_REF]. SA pathway is also independent of potential negative role of autophagy [START_REF] Wang | ATG2, an autophagy-related protein, negatively affects powdery mildew resistance and mildew-induced cell death in Arabidopsis[END_REF]. Study of the response of Arabidopsis autophagy defective mutants atg5, atg7 and atg18 facing fungal pathogens seems to indicate that autophagy plays a positive role in plant resistance to these organisms [START_REF] Lai | A critical role of autophagy in plant resistance to necrotrophic fungal pathogens[END_REF]. Zhang et al., showed that blocking autophagy by knockout of ATG genes causes hypersensitive responses (HR) to necrotrophic fungi (Zhang et al., 2020). Furthermore, phosphorylation of ATG18a suppresses AP formation which results in reduced autophagy activity and decrease plant resistance against Botrytis cinerea. Conversely, overexpressing a hyper-autophagy inducing mutant of ATG18a -where the five phosphorylation sites (Thr241, Ser328, Ser344, Ser361 and Thr387) are mutated to alanine-enhances resistance against this pathogen (Zhang et al., 2020). Note that certain kind of necrotrophic pathogens can hijack autophagy to their advantage. For example, the effector PexRD54 from the potato blight pathogen Phytophthora infestans binds the autophagy protein ATG8CL to stimulate AP formation in order to eliminate defense-related compounds. Thus, pathogens can activate autophagy for their own benefit [START_REF] Dagdas | An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor[END_REF].

Among the biotrophic pathogens, the most studied example is Pseudomonas syringae pv tomato (Pst). Üstün et al., identified both pro-and antibacterial functions of autophagy upon infection of Arabidopsis thaliana with Pst. This biotrophic pathogen manipulates and modulates autophagy machinery via various effectors [START_REF] Lal | Phytopathogen Effectors Use Multiple Mechanisms to Manipulate Plant Autophagy[END_REF]. On the one hand, Pst can activate autophagy with the effector HrpZ1 to enhance autophagy through the modulation of ATG8 cleavage by ATG4. On the other hand, Pst can also negatively regulate autophagy with the effectors HopF3 and AvrPtoB by targeting the ATG8 and ATG1 module of the autophagy machinery, respectively [START_REF] Lal | Phytopathogen Effectors Use Multiple Mechanisms to Manipulate Plant Autophagy[END_REF].

Plants can also use autophagy as mean of defense to viruses. Autophagy deficient Arabidopsis thaliana mutants atg5 and atg7 and the cargo receptor mutant nbr1 exhibited stronger symptoms to Cauliflower Mosaic virus (CaMV) infection compare to WT [START_REF] Hafrén | Selective autophagy limits cauliflower mosaic virus infection by NBR1-mediated targeting of viral capsid protein and particles[END_REF]. These elements suggest a protective role of autophagy against this virus. The authors have identified that P4, a major structural protein of viral capsid and assembled P4viral-capsides are selectively targeted by NBR1 to mediate their degradation and restrict CaMV infection [START_REF] Hafrén | Selective autophagy limits cauliflower mosaic virus infection by NBR1-mediated targeting of viral capsid protein and particles[END_REF]. Similarly, To fight against RNA virus, plants use autophagy to target and degrade the RNA-dependent RNA polymerase. Li and colleagues have shown that infection by the Turnip mosaic virus (TuMV) activates autophagy in N. benthamiana [START_REF] Li | Beclin1 restricts RNA virus infection in plants through suppression and degradation of the viral polymerase[END_REF]. The protein BECLIN1/ATG6 was found to interact specifically to NIb, the only RNAdependent RNA polymerase of TuMV. BECLIN1/ATG6 was found to interact with ATG8a to trigger Nib degradation by autophagy and inhibit viral proliferation of TuMV [START_REF] Li | Beclin1 restricts RNA virus infection in plants through suppression and degradation of the viral polymerase[END_REF]. In N. benthamiana, the regulator of gene silencing calmodulin-like protein (rgs-CaM) interact with the two viral effectors, HC-pro and 2b, involved in the suppression of the plant RNA silencing machinery which mediates defense against various viruses. Autophagy was found to mediate the degradation of the rgs-CaM complex together with the two viral effectors [START_REF] Nakahara | Tobacco calmodulin-like protein provides secondary defense by binding to and directing degradation of virus RNA silencing suppressors[END_REF].

While plants can use autophagy for defense against viruses, the co-evolution of those pathogens and plants led to the establishment of counter-defense strategies by viruses, notably by suppressing autophagy. This is the strategy used by the Barley stripe mosaic virus (BSMV), a RNA virus [START_REF] Yang | Barley stripe mosaic virus γb Protein Subverts Autophagy to Promote Viral Infection by Disrupting the ATG7-ATG8 Interaction[END_REF]. The viral genome encodes the γb protein which functions as a viral suppressor of RNA silencing and a modulator of host defences (Zhang et al., 2017). Suppression of autophagy by BSMV occurs via its γb protein which physically interact with ATG7. The γb/ATG7 interaction directly competes with the ATG7/ATG8 interaction that is essential for ATG8 lipidation and AP formation. Thus, by disrupting the interaction between ATG7 and ATG8, γb inhibits autophagy and promotes viral infection.

Together, these results illustrate how BSMV could interfere with host autophagy antiviral defence to infection.

All of the stresses that we have presented above can also result in the production of ROS that will act as a defense-signaling molecule for the plant and allow the establishment of a defense pathway including 26S proteasome and/or autophagy. Xiang et al., showed that autophagy is induced in wild-type Arabidopsis thaliana plants after transfer of seedlings to MS medium containing either hydrogen peroxide or MV (Xiong et al., 2007). In addition, the RNAi AtATG18a mutant exhibits hypersensitivity to oxidative stress (Xiong et al., 2007;Xiong et al., 2007). Likewise, the atg5, atg7 and nbr1 mutants also present hypersensitivity to oxidative stress after treatment with MV [START_REF] Zhou | NBR1-Mediated Selective Autophagy Targets Insoluble Ubiquitinated Protein Aggregates in Plant Stress Responses[END_REF] whereas the overexpression of ATG5 and ATG7 leads to enhance resistance to MV (Minina et al., 2018). Recently, both PCD and reactive oxygen species (ROS) level were reported to be regulated by autophagy during the compatible plant and virus interaction [START_REF] Zhou | Autophagy contributes to regulate the ROS levels and PCD progress in TMV-infected tomatoes[END_REF]. Moreover, treatment of these plants with autophagy inhibitor 3-MA increase PCD systemically (not at infection site) but not ROS level. This suggests that ROS act as signalling molecules and are produced in excess upon infection; in these conditions, autophagy is induced to eliminate the excess of ROS thus protecting cells, where ROS are not needed to trigger PCD [START_REF] Zhou | Autophagy contributes to regulate the ROS levels and PCD progress in TMV-infected tomatoes[END_REF]. Recently, Chen and colleagues have also shown that autophagy is activated in Arabidopsis thaliana following hypoxic stress by submergence via the accumulation of ROS [START_REF] Chen | Autophagy contributes to regulation of the hypoxia response during submergence in Arabidopsis thaliana[END_REF].

II-3. How autophagy mediates plant adaptation to stresses?

Plants are subjected to multiple environmental stresses during their life cycle, causing them to constantly adapt to their living environment. Autophagy is an essential quality control pathway for maintaining homeostasis and therefore survival. As we saw in the previous section, mutants with less or no autophagy are more sensitive to a wide range of stresses while autophagy-overexpressing plants are more resistant to environmental stresses suggesting a mostly pro-resistance role of autophagy in the majority of conditions studied. How does autophagy mediate plant resistance to such a variety of stresses?

Protein degradation

Autophagy selectively degrades specific proteins to modulate targeted plant metabolic pathways or intracellular processes. For example, it was shown that autophagy participates in the degradation of TRYPTOPHAN-RICH SENSORY PROTEIN (TSPO) [START_REF] Vanhee | The Arabidopsis Multistress Regulator TSPO Is a Heme Binding Membrane Protein and a Potential Scavenger of Porphyrins via an Autophagy-Dependent Degradation Mechanism[END_REF].

Vanhee and colleagues have shown that the degradation of the TSPO protein was inhibited in the atg5 mutant [START_REF] Vanhee | The Arabidopsis Multistress Regulator TSPO Is a Heme Binding Membrane Protein and a Potential Scavenger of Porphyrins via an Autophagy-Dependent Degradation Mechanism[END_REF]. Furthermore, the expression of TSPO is induced in vegetative tissues during osmotic stresses (mannitol and salt) as well as during treatment with abscisic acid (ABA) [START_REF] Guillaumot | The Arabidopsis TSPO-related protein is a stress and abscisic acid-regulated, endoplasmic reticulum-Golgi-localized membrane protein[END_REF] and magnesium deficiency [START_REF] Hermans | Early transcriptomic changes induced by magnesium deficiency in Arabidopsis thaliana reveal the alteration of circadian clock gene expression in roots and the triggering of abscisic acid-responsive genes[END_REF]. Hachez et al., also reported that TSPO interacts with aquaporin PLASMA MEMBRANE INTRINSIC PROTEIN 2;7 (PIP2;7) within membrane compartments of the early secretory pathway and downregulates its abundance in the cell [START_REF] Hachez | The Arabidopsis abiotic stress-induced TSPO-related protein reduces cellsurface expression of the aquaporin PIP2;7 through protein-protein interactions and autophagic degradation[END_REF]. In particular, the authors propose that the heterocomplex is delivered in the vacuole by autophagy, thereby reducing the abundance of PIP2;7 at the PM and protecting the cell from water deficit [START_REF] Hachez | The Arabidopsis abiotic stress-induced TSPO-related protein reduces cellsurface expression of the aquaporin PIP2;7 through protein-protein interactions and autophagic degradation[END_REF]. Furthermore, PIP2;7 was shown to be also a negative regulator of drought stress in the barrelclover Medicago truncatula (Li et al., 2020). Finally, Jurkiewicz and colleagues have also demonstrated that TSPO played a role in storage lipid homeostasis in A. thaliana seeds and seedlings [START_REF] Jurkiewicz | The multistress-induced Translocator protein (TSPO) differentially modulates storage lipids metabolism in seeds and seedlings[END_REF].

Another example of the function of autophagy in protein degradation to modulate intracellular processes is to counter the antiviral defence mechanism: some viruses have developed various strategies including suppressors that inhibit the virus-induced RNA silencing process in plant cells [START_REF] Agius | RNA Silencing and Antiviral Defense in Plants[END_REF]. We previously mention how viruses can inhibit autophagy to promote their replication. However, how autophagy interacts with plant virus is not as simple as it seems. Hijacking autophagy by viruses to use it as their own advantage is also part of the strategies they developed. In this way, upon Turnip mosaic virus infection, autophagy target the NIb through BECLIN1/ATG6 as a way to limit viral spread but this virus also trigger an selective autophagy pathways in a unfolded protein response (UPR) dependent manner to promote virus infection (Li et al., 2020). A small integral membrane protein, the 6K2, remodels the ER to form viral replication vesicles suitable for the virus replication complex [START_REF] Jiang | The Vesicle-Forming 6K2 Protein of Turnip Mosaic Virus Interacts with the COPII Coatomer Sec24a for Viral Systemic Infection[END_REF]. This protein induces the INOSITOL-REQUIRING PROTEIN-1/BASIC-LEUCINE ZIPPER TRANSCRIPTION FACTOR60 (IRE1/bZIP60) pathway, a major branch of the UPR in N. benthamiana (Li et al., 2020). In turn, this pathway upregulated the cargo NBR1 that interact with the viral polymerase Nib. This complex interacts with ATG8f and colocalize with 6K2 at the viral replication complex at the tonoplast.

Interestingly, overexpressing ATG8f or NBR1 facilitates TuMV infection while their silencing has the opposite effect, suggesting the importance of autophagy for viral replication. The TuMV activates and manipulates autophagy in an UPR-dependent pathway to target the viral replication complex to the tonoplast in order to promote viral replication. Similarly, the TuMV use autophagy as its own advantage also by triggering the degradation of SUPPRESOR OF GENE SILENCING3 (SGS3) and its interacting partner RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), two essential components of the RNA silencing pathway for antiviral immunity. In their study, Cheng and colleagues showed that the VIRAL PROTEIN GENOME-LINKED (VPg) played a role in this machinery by its interaction to SGS3 to mediate its degradation by autophagy [START_REF] Cheng | The Potyvirus Silencing Suppressor Protein VPg Mediates Degradation of SGS3 via Ubiquitination and Autophagy Pathways[END_REF]. Another example of autophagy-mediated regulation and adaptation to stress by means of protein degradation is the control of the level of the protein ARGONAUTE1 (AGO1) in the context of viral infection.

AGO1 is a component of the RNA-induced silencing multi-protein complex (RISC) associating with small interfering RNA (siRNA) thus allowing gene silencing in plants. Derrien and colleagues showed that the viral suppressor of RNA silencing P0 protein encode by the Polerovirus triggers the degradation of AGO1 by autophagy [START_REF] Derrien | Degradation of the antiviral component ARGONAUTE1 by the autophagy pathway[END_REF] thus modulating gene silencing and allowing viral replication.

Organelle degradation and selective autophagy

Selective autophagy is an additional solution to modulate the adaptation of plants to environmental stresses and to degrade unwanted, damaged or supernumerary organelles.

This process is carried out by the intermediary of receptor or adapter proteins binding to the cargo to be eliminated but also to the ATG8 protein via ATG8-interacting motifs termed AIMs (consensus sequence: F/W/Y-X-X-L/I/V) [START_REF] Kalvari | iLIR: A web resource for prediction of Atg8-family interacting proteins[END_REF]Xie et al., 2016). First of all, chlorophagy, i.e., the selective degradation of a chloroplast, is a peculiarity of photosynthetic organisms compared to mammals and yeasts. The recycling of chloroplast proteins during senescence is an important event for the maintenance of homeostasis of the plant. This is made possible in particular by the transfer of proteins, such as the RIBULOSE-1,5-BIPHOSPHATE CARBOXYLASE-OXYGENASE (RuBisCo), from the stroma via spherical double membrane structures called Rubisco containing bodies (RCBs) using autophagy pathway [START_REF] Wada | Autophagy plays a role in chloroplast degradation during senescence in individually darkened leaves[END_REF][START_REF] Ishida | Mobilization of Rubisco and Stroma-Localized Fluorescent Proteins of Chloroplasts to the Vacuole by an <em>ATG</em> Gene-Dependent Autophagic Process[END_REF][START_REF] Izumi | The Autophagic Degradation of Chloroplasts via Rubisco-Containing Bodies Is Specifically Linked to Leaf Carbon Status But Not Nitrogen Status in Arabidopsis[END_REF]. Wang and colleagues have also found a second kind of structure called small starch granule-like structures (SSGLs) during the degradation of starch in leaves [START_REF] Wang | Autophagy contributes to leaf starch degradation[END_REF]. In particular, this team showed that starch accumulated in Nicotiana benthamiana seedlings treated with the 3-MA autophagy inhibitor or in several VIGS-ATG mutants [START_REF] Wang | Autophagy contributes to leaf starch degradation[END_REF]. In addition, Michaeli et al.,have also detected vesicles associated with plastids, termed ATI1-PS bodies, accumulating during carbon deficiency, salt stress as well as during senescence (Michaeli et al., 2014). RCBs, ATI-PS and SSGLs are grouped under the name of macrochlorophagy while degradation of chloroplast by invagination of chloroplast contents is termed microchlorophagy [START_REF] Zhuang | Chloroplast Degradation: Multiple Routes Into the Vacuole[END_REF]. Recently, Izumi et al., also showed that the combined action of low temperature (10 °C) and high luminous intensity of visible light but also by UV-B treatment could induce chlorophagy of entire chloroplasts (Izumi et al., 2017).

In addition to chlorophagy, mitophagy is a process of mitochondria degradation also demonstrated in plants. Li and colleagues have showed this process in the atg11-1 mutants which present an accelerated chlorosis phenotype as well as a decrease in the mitochondrial proteins turnover in comparison with the WT following a dark-induced senescence (Li et al., 2014). Furthermore, using confocal microscopy, the authors notably demonstrated the colocalization of a fluorescent mitochondrial marker (Mito-YFP) with the marker of autophagic membranes mCherry-ATG8a in AB but also the co-localization of the mitochondrial marker MitoTracker Green FM with mCherry-ATG11 in Arabidopsis thaliana (Li et al., 2014). However, the understanding of how mitophagy works in Arabidopsis thaliana and the involvement of ATG11 is not yet understood. Recently, Dündar and colleagues have also shown that treatment with a high dose of UV-B leads to the inactivation and fragmentation of mitochondria as well as their degradation by mitophagy [START_REF] Dündar | Autophagy-deficient Arabidopsis mutant atg5, which shows ultraviolet-B sensitivity, cannot remove ultraviolet-B-induced fragmented mitochondria[END_REF].

Unlike mammalian and yeast models, ribophagy is a mechanism not yet demonstrated in plant organisms [START_REF] Kazibwe | The Ins and Outs of Autophagic Ribosome Turnover[END_REF]. However, several studies regarding the nonspecific endoribonuclease of the RNase T2 family (RNS2) seem to highlight the existence of rRNA turnover by plant vacuoles [START_REF] Hillwig | RNS2, a conserved member of the RNase T2 family, is necessary for ribosomal RNA decay in plants[END_REF][START_REF] Floyd | Localization of RNS2 ribonuclease to the vacuole is required for its role in cellular homeostasis[END_REF][START_REF] Floyd | Evidence for autophagy-dependent pathways of rRNA turnover in Arabidopsis[END_REF]. Hillwig and colleagues first demonstrated that rns2-2 mutant have a longer half-life 28S rRNA [START_REF] Hillwig | RNS2, a conserved member of the RNase T2 family, is necessary for ribosomal RNA decay in plants[END_REF] but also exhibited a constitutive autophagy with an higher number of AP than wildtype under normal growth [START_REF] Floyd | Evidence for autophagy-dependent pathways of rRNA turnover in Arabidopsis[END_REF][START_REF] Hillwig | RNS2, a conserved member of the RNase T2 family, is necessary for ribosomal RNA decay in plants[END_REF]. Moreover, Floyd et al., have also shown that rRNA accumulates in vacuoles from rns2-2 and rns2-2 atg9-4 mutants but not in the rns2-2 atg5-1 mutant suggesting different functions of ATG9 and ATG5 in the trafficking and turnover of rRNA in the vacuole [START_REF] Floyd | Evidence for autophagy-dependent pathways of rRNA turnover in Arabidopsis[END_REF]. The same team also observed that the localization of RNS2 to the vacuole via a C-terminal targeting signal is essential for the maintenance of homeostasis and the degradation of rRNA in Arabidopsis thaliana [START_REF] Floyd | Localization of RNS2 ribonuclease to the vacuole is required for its role in cellular homeostasis[END_REF].

Similarly, selective autophagy can suppress RNA virus infections. The RNA silencing suppressor protein p3 encoded by the Rice stripe virus (RSV) was shown to be targeted by a novel protein called P3 INTERACTING PROTEIN (P3IP) in N. benthamiana [START_REF] Jiang | The plant protein NbP3IP directs degradation of Rice stripe virus p3 silencing suppressor protein to limit virus infection through interaction with the autophagy-related protein NbATG8[END_REF]. Further experiments showed that in plants where autophagy is inhibited through 3-MA, p3 protein levels are higher. Moreover, P3IP interacts with ATG8f. A P3IP mutant that is unable to bind to ATG8f did not inhibit the RNA silencing function of p3, suggesting that P3IP mediates p3 degradation by interacting with ATG8f. Meanwhile in ATG5 or ATG7 silenced plants the RSV infection was promoted. On the contrary, when autophagy induced by the silencing of GAPC it suppresses RSV infection. Altogether these results demonstrate that autophagy could act as an antiviral defense against the RSV.

Pexophagy is also a selective degradation system for degradation of peroxisomes in plants. Like ribophagy, the mechanism of pexophagy is not yet clearly understood in photosynthetic organisms. Using BiFC, Xie et al., have demonstrated that peroxisomal PEROXIN proteins (PEX6 and PEX10) interacted in planta with ATG8 via their AIM motifs (Xie et al., 2016).

Apart from autophagy, there are other control machines that allow cell quality to be maintained, such as the ubiquitin-26S proteasome system (UPS). This system takes part in the degradation of misfolded, denatured or obsolete proteins after being tagged by multiple ubiquitins allowing their recognition [START_REF] Marshall | Dynamic Regulation of the 26S Proteasome: From Synthesis to Degradation[END_REF]. Marshall and colleagues have shown that there is a selective autophagy process, called proteaphagy, allowing the elimination of non-functional 26S proteasome [START_REF] Marshall | Autophagic Degradation of the 26S Proteasome Is Mediated by the Dual ATG8/Ubiquitin Receptor RPN10 in Arabidopsis[END_REF]. The authors showed that this process could take place either by nitrogen deficiency or by the effect of MG132, a 26S proteasome inhibitor [START_REF] Marshall | Autophagic Degradation of the 26S Proteasome Is Mediated by the Dual ATG8/Ubiquitin Receptor RPN10 in Arabidopsis[END_REF]. In this process, the ubiquitin-binding proteasome subunit REGULATORY PARTICLE NON-ATPASE SUBUNIT10 (RPN10) plays a role as a specific receptor for proteaphagy [START_REF] Marshall | Autophagic Degradation of the 26S Proteasome Is Mediated by the Dual ATG8/Ubiquitin Receptor RPN10 in Arabidopsis[END_REF]. Üstün and colleagues have also shown that Pseudomonas syringae pv tomato can activate autophagy with the type III effector (T3E) and stimulate autophagic removal of proteasomes via proteaphagy to promote bacterial proliferation (Üstün et al., 2018). Other kinds of selective autophagy are also proposed in plants such as aggrephagy [START_REF] Jung | Arabidopsis cargo receptor NBR1 mediates selective autophagy of defective proteins[END_REF], reticulophagy [START_REF] Wu | ATI1 (ATG8-interacting protein 1) and ATI2 define a plant starvation-induced reticulophagy pathway and serve as MSBP1/MAPR5 cargo receptors[END_REF] and nucleophagy (Li et al., 2020) but there is also few evidence at the time of writing this review.

Autophagy mediates nitrogen recycling and lipid metabolism

By mediating the degradation of selected or random cargo (proteins, organelles, etc.), autophagy promotes the recycling of nitrogen before or during senescence. The connection between autophagy and nitrogen remobilization/recycling have been extensively studied in recent years in Arabidopsis thaliana [START_REF] Guiboileau | Physiological and metabolic consequences of autophagy deficiency for the management of nitrogen and protein resources in Arabidopsis leaves depending on nitrate availability[END_REF]Guiboileau et al., 2012), Zea mays [START_REF] Li | Autophagic recycling plays a central role in maize nitrogen remobilization[END_REF] and Oryza sativa [START_REF] Wada | Autophagy supports biomass production and nitrogen use efficiency at the vegetative stage in rice[END_REF] models. Guiboileau and colleagues have notably shown that the atg mutants presented an altered C/N ratio as well as a greater accumulation of ammonium, amino acid and proteins in their rosette before senescence suggesting that autophagy controls the C/N ratio and the protein contents in A. thaliana [START_REF] Guiboileau | Physiological and metabolic consequences of autophagy deficiency for the management of nitrogen and protein resources in Arabidopsis leaves depending on nitrate availability[END_REF]. Senescence is a process finely regulated by the plant and in particular by autophagy via a tight balance between the conservation of cell longetivity and the degradation of inefficient and ageing photosynthesis organs for example [START_REF] Havé | Nitrogen remobilization during leaf senescence: lessons from Arabidopsis to crops[END_REF].

Physiological studies of atg mutants reveal early senescence phenotype [START_REF] Breeze | High-Resolution Temporal Profiling of Transcripts during Arabidopsis Leaf Senescence Reveals a Distinct Chronology of Processes and Regulation[END_REF]Doelling et al., 2002;[START_REF] Hanaoka | Leaf Senescence and Starvation-Induced Chlorosis Are Accelerated by the Disruption of an Arabidopsis Autophagy Gene[END_REF]Li et al., 2014;[START_REF] Wada | Autophagy plays a role in chloroplast degradation during senescence in individually darkened leaves[END_REF]Xiong, Contento, and Bassham 2005) as well as an accumulation of SA responsible for this phenotype (Yoshimoto et al., 2009).

Finally, although this was not the focus of our review, it would seem that the autophagic pathway is also intrinsically connected with lipid metabolism. For example, disrupting autophagy impedes the lipid turnover that results from the degradation of organelles membranes in mature and senescing leaves (Fan et al., 2019). Also, autophagy atg5 mutants were seen to display ER-stress independently of the growth conditions and lipidomic analysis showed that these mutants accumulated high levels of ER-synthesized phospholipids as well as PM-localized sphingolipids, indicating an implication of autophagy in the turnover of lipids from both membranes [START_REF] Havé Marien | Proteomic and lipidomic analyses of the Arabidopsis atg5 autophagy mutant reveal major changes in endoplasmic reticu-lum and peroxisome metabolisms and in lipid composition[END_REF]. Moreover, proteomic and lipidomic analysis performed on maize leaves from atg12 mutants upon nitrogen starvation showed changes in lipids and secondary metabolites accumulation compared to WT plants, indicating that autophagy mutants are affected in lipid homeostasis [START_REF] Mcloughlin | Maize multi-omics reveal roles for autophagic recycling in proteome remodelling and lipid turnover[END_REF]. All of these results indicate that autophagy plays roles in the maintenance of lipid homeostasis and thus further illustrate how autophagy can redirect plant metabolism in order to face adverse environmental conditions.

Conclusion

In conclusion, throughout this review we have described how lipids are not only structural building blocks for autophagic membranes, but rather, are fundamental key players mediating several steps in the autophagosome biogenesis process as well as defining critical membrane features such as curvature or electrostatics. Further, we have seen how a wide range of environmental stresses can induce autophagy, and how plants adapt to these stresses by mediating the degradation of damaged intracellular materials (from proteins up to entire organelle) via the autophagic pathway. How lipids are mobilized/delivered to the autophagy and how these pathways are regulated to support increasing rates of AP formation upon environmental stresses remain a critical aspect of plant physiology that future research should address. Plants, being sessile organisms, are frequently confronted to diverse and sometimes harsh environmental conditions. Yet, plants are capable of withstanding these conditions thanks to a wide range of intracellular mechanisms that allow them to tightly orchestrate adaptive responses to stresses. One fundamental mechanism required for plant survival is the proper management of damaged cellular contents that tend to accumulate in these conditions [START_REF] Hall | Cellular mechanisms for heavy metal detoxification and tolerance[END_REF][START_REF] Huang | Mechanisms of ROS regulation of plant development and stress responses[END_REF][START_REF] Liu | Endoplasmic reticulum protein quality control and its relationship to environmental stress responses in plants[END_REF]. Waste management is assured in plants by two main catabolic pathways: (i) the ubiquitin-26S proteasome system, which involves covalent binding of ubiquitin moieties to selected target proteins, for their recognition and degradation by the 26S proteasome [START_REF] Vierstra | The ubiquitin-26S proteasome system at the nexus of plant biology[END_REF] and (ii) autophagy.

II-SCIENTIFIC CONTEXT AND OBJECTIVES

Autophagy is an evolutionary conserved mechanism in which parts of the cytoplasm are delivered to the lytic vacuole for degradation; the macromolecules resulting from this degradation are re-exported to the cytoplasm, providing energy for the cell as well building blocks for required anabolic reactions (Marshall & Vierstra, 2018). The type of cargo that can be degraded by autophagy ranges from proteins and protein aggregates [START_REF] Svenning | Plant NBR1 is a selective autophagy substrate and a functional hybrid of the mammalian autophagic adapters NBR1 and p62/SQSTM1[END_REF][START_REF] Zhou | Role and regulation of autophagy in heat stress responses of tomato plants[END_REF] to entire organelles [START_REF] Izumi | The Autophagic Degradation of Chloroplasts via Rubisco-Containing Bodies Is Specifically Linked to Leaf Carbon Status But Not Nitrogen Status in Arabidopsis[END_REF]Broda et al., 2018;[START_REF] Shibata | Highly oxidized peroxisomes are selectively degraded via autophagy in Arabidopsis[END_REF]. Autophagy is carried by the so-called autophagy machinery, well conserved across organisms and composed of a core of around 18 ATG (AuTophaGy related) proteins. ATG genes were first discovered via a genetic screen in yeast [START_REF] Harding | Isolation and characterization of yeast mutants in the cytoplasm to vacuole protein targeting pathway[END_REF][START_REF] Thumm | Isolation of autophagocytosis mutants of Saccharomyces cerevisiae[END_REF][START_REF] Tsukada | Isolation and characterization of autophagy-defective mutants of[END_REF] which over the years, has led to the identification of their orthologues in other organisms (Feng et al., 2014). The sequential and coordinated actions of ATG proteins govern the formation of the central structure of autophagy: the autophagosome (AP). APs are specialized double membrane vesicles which targets and sequesters the degradation cargoes and traffics if to the vacuole/lysosome (Figure 5). Unlike other endomembrane vesicles, APs do not bud from a preexisting compartment; instead, they are formed de novo in response to autophagy-inducing conditions. AP formation begins with the relocalization of the ATG machinery from the cytosol to a lipido/proteic core called the Pre Autophagosomal Structure (PAS) which nature and structure remains enigmatic in plants. At the PAS, ATG proteins coordinate the de novo nucleation of an initial membrane structure called the phagophore, by mechanisms which are still not well understood [START_REF] Hurley | Mechanisms of Autophagy Initiation[END_REF]. Morphologically, the phagophore is characterized by its cup-shape with a highly curved rim that is subject to tight regulation to avoid premature closure of the structure. Upon nucleation, the phagophore expands by addition of lipids, while keeping its structure to ensure cargo engulfment, until the rim fuses, thus forming a complete AP (Melia et al., 2020). Finally, the AP traffics to the vacuole where the outer membrane fuses with the tonoplast, releasing a single membrane vesicle and its containing cargo, an intermediate structure called the autophagic body (AB) into the vacuolar lumen for degradation (Marshall & Vierstra, 2018).

BOX 1 -LIPID CLASSES IN PLANT CELLS

Membranes in plant cells are composed mainly of three class of lipids: glycerolipids (mainly phospholipids), sterols and sphingolipids (Fig. 6).

o Glycerophospholipids contain a polar head as well as an hydrophobic portion composed of a DAG molecule which contains saturated or cis-unsaturated fatty acids chains of variable lengths. Among them, the glycerophospholipidsphosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidic acid (PA)-represent the major structural lipids in membranes. o Sphingolipids consist of a long chain base (LCB) with an amidated fatty acid molecule, and a polar head attached to the alcohol residue of the LCB. Plant cells display a greater LCB diversity than animals and yeast, and thus, there are more than 500 molecular species of sphingolipids in plant cells (Pata et al, 2010). The LCB of sphingolipids allows them to form taller and narrower cylinders than glycerolipids for example, and because of this they are able to pack more tightly and create solid 'gel' like phases in membranes, which can be fluidized by sterols. o Sterols are the major nonpolar lipids of cell membranes, they are formed by a cyclopentaperhydrophenantren moiety made up of four rigid rings, hydroxylated at position 3 (Fig. 6). While animals and yeast include only one type of sterol in their membranes (cholesterol and ergosterol respectively), membranes of plant cells contain multiple types of plant-specific sterols, called phytosterols. Phytosterols are classified by the number and the position of double bonds in the cycle, and by the nature (branched or linear, saturated or unsaturated) of their lateral chain.

Except for complex sphingolipids, which are synthesized in the trans-golgi network, most lipids are assembled in the endoplasmic reticulum and transported through the secretory pathway to other organelles (Van Meer et al., 2008). Membrane remodeling events are numerous during autophagy, with the de novo formation of a membrane structure, its shaping, its expansion, as well as multiple membrane fusion events. How most of these events are occurring (i.e., molecular actors and mechanism) remain poorly understood especially in plants where autophagy research has lagged behind that of yeast or mammalian models. In fact, most studies have focused on the function of proteins, and in particular ATG proteins in the formation of APs but even though autophagy is a membrane-based process, far less attention has been given to the importance of lipids as potential actors of the autophagy machinery.

Lipids are essential components of biological membranes; first, because they are building blocks of membranes with thus important structural inputs on membrane morphology, and second because they play important role by mediating the recruitment of membrane associated peripheral proteins. In plant cells, three lipid classes exist: glycerolipids (phospholipids, phosphoinositides, and galactolipids), sphingolipids, and sterols (Figure 6).

These classes differ from each other by their physical and biochemical properties, and because of these, each class of lipids has different impacts on membrane properties such as its fluidity, curvature, and overall architecture thus controlling fusion or fission events. Attributes such as the size of the polar head, or the length and insaturation degree of their acyl chains define the geometry of lipids which affect the curvature of the membrane where they accumulate (Bigay & Antonny, 2012), Figure 7). These different lipid geometries can also affect lipid density within membranes with cylinder-shaped lipids creating densely packed regions, whereas lipids that differ from the canonical cylindrical shape can create loosely packed regions within the membrane ("i.e. lipid packing defects", Bigay & Antonny, 2012). Membranes with lipid packing defects are known to be preferred by cytosolic proteins with hydrophobic terminal residues who take advantage in these packing defects for insertion (Bigay & Antonny, 2012). Other lipids, like anionic phospholipids, bear electrostatic charges on their polar heads, thus creating particular electrostatic field in the membranes where they are present (Figure 7). These particular electrostatic fields are functionally important as they allow the recruitment of proteins through electrostatic interactions. All of these properties (membrane curvature, lipid density, electrostatics) contribute to defining membrane identity (Bigay & Antonny, 2012). Each organelle has its own "membrane identity", dictated by the nature and the relative quantity of the lipids and proteins composing it. The membrane identity of each organelles has a direct impact on their ultrastructure and their activity, as it mediates the recruitment of proteins to these membranes assuring multiple biological functions [START_REF] Noack | Functions of Anionic Lipids in Plants[END_REF].

In that context, lipids have the potential to be fundamental component of each and blocks for the pagophore expansion, (4) shaping and reshaping autophagy structures from a coalescence of vesicles to a cup-shaped double membrane to a complete double membrane vesicle through controlling membrane fluidity, curvature and (5) instructing the multiple membrane fusion events required for AP formation and delivery to the vacuole.

Increasing research in yeast and mammalian models recognizes the importance of lipids in autophagy. In fact, since the beginning of my PhD in 2017, exciting and groundbreaking studies on the relation between lipids and autophagy have unraveled molecular mechanism behind critical steps of AP formation that had remained elusive for over 20 years (Judith et al., 2019;[START_REF] Maeda | The autophagic membrane tether ATG2A transfers lipids between membranes[END_REF][START_REF] Matoba | Atg9 is a lipid scramblase that mediates autophagosomal membrane expansion[END_REF][START_REF] Osawa | Atg2 mediates direct lipid transfer between membranes for autophagosome formation[END_REF]Schütter et al., 2020;[START_REF] Valverde | ATG2 transports lipids to promote autophagosome biogenesis[END_REF]. In plants, almost virtually nothing is known about the nature and functional contribution of lipids in autophagy. Notably, although some aspects of autophagy are well conserved among organisms, fundamental differences in the machinery and morphology exist which impose a dedicated look at the mechanisms of plant autophagy.

In fact, PI3P was shown to be essential but its function remains largely uncharacterized.

Similarly, the conjugation of ATG8 to a lipid is mandatory for AP formation, yet the nature of this lipid and its impact on the phagophore ultrastructure stays elusive.

In that context, the aims of my PhD work were to identify lipids and lipid-related proteins involved in autophagy in Arabidopsis, in order to characterize their function as well as their impact in the autophagic process and by this, start unraveling how these key lipids contribute to autophagy and therefore, to plants adaptation to environmental stress.

III-1 Identifying lipids required for autophagy: a lipid inhibitor screen

In this part of my PhD work, we wanted to identify lipids that are critical for autophagy. To this end, we performed a screen in which we tested the effects of blocking the synthesis of distinct lipids on autophagy activity. We reasoned that if a lipid is essential for autophagy, inhibiting its synthesis will result in a decrease in autophagy activity. Throughout this part, lipid synthesis was inhibited using pharmacological treatments directed against lipid synthesizing enzymes (Figure 8). We decided to use lipid inhibitors rather than mutants of lipid synthesis because:

(1) obtaining and crossing these mutants with autophagy markers would be very time consuming; (2) mutants defective in the synthesis of lipids often display severe phenotypes due to the fact that multiple pathways are usually affected [START_REF] Mou | Deficiency in Fatty Acid Synthase Leads to Premature Cell Death and Dramatic Alterations in Plant Morphology[END_REF]. In contrast, the use of short-time treatments should allow to minimize possible pleiotropic effects. We thus went through the literature and identified a range of lipids inhibitors already described/characterized in plant cells that we used accordingly to target all classes of lipids: phospholipids, sterols and sphingolipids.

All experiments performed in this screen were performed on seven day old 35S::GFP-ATG8a (Thompson et al., 2005) seedlings. Following seven day of culture in sucrosecontaining full MS plates, we transferred seedlings into a nutrient rich full MS liquid medium, or to a liquid MS medium lacking nitrogen and carbon (-NC) in order to induce autophagy.

Inhibitor treatments were applied concomitantly with the transfer to liquid media, for a duration of 3 hours (unless indicated otherwise in the figures). At the end of the treatments, we dissected roots from the seedlings and extracted root proteins (See experimental procedures).

We then measured the impact of lipid inhibitors on autophagy by quantitatively measuring autophagy flux using the well-established GFP-ATG8 degradation assay (Shin et al., 2014, Figure 9,A). ATG8 is a central protein in autophagy which has the particularity of being present in both the inner and outer membranes of the AP. Because of this, unlike many other ATG proteins which are recycled from the AP upon completion of the vesicle and prior to its the fusion with the vacuole, ATG8 is degraded within the vacuole in addition to all autophagic cargo. Thus, by using plants that express a version of ATG8 tagged with GFP (GFP-ATG8a), GFP-ATG8 is incorporated into autophagic structures and delivered to the vacuole. Inside the vacuole, ATG8 is rapidly degraded resulting in the release of free GFP that, because of its resistance to vacuolar hydrolases, accumulates over time. In Figure 9B, levels of GFP-ATG8a and GFP moieties were detected and measured by immunoblotting using an anti-GFP antibody. When autophagy is induced by nutrient starvation, we observe an accumulation of the free GFP band compared to that of untreated plants (Figure 9, B); in the same conditions, when autophagy is blocked, for example by using Wortmannin (a PI3K inhibitor; (Shin et al., 2014)), free GFP fails to accumulate. Thus, measuring the ratio of GFP/GFP-ATG8a illustrates the rate of ATG8 degradation and more generally reflects the rate of autophagic flux, thus allowing to quantitatively measure autophagic activity.

To explore the role of lipids in plant autophagy, we compared the GFP/GFP-ATG8 ratio of control plants (DMSO, which was set to 100%) to that of plants treated with lipid inhibitors.

As a negative control we used Wortmannin, a known inhibitor of autophagy (Shin et al., 2014) (Figure 9,B). Results of the screen are presented and discussed below.

III-1.1 Sphingolipids

In our screen we tested the importance of sphingolipids in plant autophagy. For this, we used two different inhibitors acting at different stages of sphingolipid metabolism (Figure 10, A-B).

(1) We inhibited the ceramide synthase, a central enzyme in sphingolipid metabolism, using Fumonisin B1 (Fb1) at 1 µM [START_REF] Qin | Sphingosine kinase AtSPHK1 functions in fumonisin B1-triggered cell death in Arabidopsis[END_REF]. In these conditions we did not observe any significant defects in the ratio of GFP/GFP-ATG8 compared to control conditions (DMSO)

suggesting that autophagy flux is not affected (Figure 10,C).

(2) We inhibited the GlucosylCeramide Synthase (GCS) which catalyzes the first step for Glycosyl Inositol Phospho Ceramides (GIPCs) synthesis, a family of unique plant sphingolipids [START_REF] Gronnier | GIPC : Glycosyl Inositol Phospho Ceramides , the major sphingolipids on earth[END_REF].

To block GCS activity we used PDMP (D,L-threo-1-pheny-2decanoylamin-3-morpholinopropanol) at 50 µM as previously described [START_REF] Melser | Links between lipid homeostasis, organelle morphodynamics and protein trafficking in eukaryotic and plant secretory pathways[END_REF]. In these conditions the autophagic flux was significantly decreased of about 20%, indicating a possible contribution of GCS to the autophagic process (Figure 10 alteration of the autophagic flux. In mammals, altering the sphingolipid metabolism by Fumonisin B1 treatment lead to an impairment in the autophagic activity notably by affecting the lipidation of ATG8 [START_REF] Matarrese | Evidence for the involvement of GD3 ganglioside in autophagosome formation and maturation[END_REF]. Although we did not check the lipidation of ATG8 in our experiments, the lack of impact on the autophagic flux suggests that notable differences can be seen between plants and mammals.

When inhibiting the GCS, we observed a decrease in the autophagic flux of around 20%, which suggests that the synthesis of glucosylceramides is required for the autophagic process. The GCS enzyme has been shown to be critical for Golgi morphology (Melser et al., 2010) and for protein transport through the secretory pathway in plant cells [START_REF] Melser | Links between lipid homeostasis, organelle morphodynamics and protein trafficking in eukaryotic and plant secretory pathways[END_REF]. Thus, the impact of the autophagic flux we observed could be resulting from dysregulation in these pathways in which GCS has been shown to be important. Moreover, glucosylceramides are the entry point for GIPCs synthesis. GIPCs are known to contribute to the formation of lipid nanodomains within the PM [START_REF] Gronnier | Structural basis for plant plasma membrane protein dynamics and organization into functional nanodomains[END_REF][START_REF] Cacas | Revisiting plant plasma membrane lipids in tobacco: A focus on sphingolipids[END_REF].

In mammals, PM nanodomains have been shown to interact with core-initiation proteins of autophagy and required for AP formation [START_REF] Matarrese | Evidence for the involvement of GD3 ganglioside in autophagosome formation and maturation[END_REF][START_REF] Garofalo | Evidence for the involvement of lipid rafts localized at the ERmitochondria associated membranes in autophagosome formation[END_REF]. Thus, if plant nanodomains are also functionally important for AP formation, inhibiting the GCS enzymes could lead to a dysregulation in the formation of these domains and thus have an impact on autophagy. In addition, our results show that KCS inhibition leads to a sharp increase in the autophagic flux. This suggests a higher turnover of autophagic structures in these conditions, ie that the structures are more rapidly degraded in these conditions. Autophagy can be activated as a response to stress, and the accumulation of C16-C18 acyl-CoAs resulting from the Metazachlor treatment could be sensed as one. However, this does not seem to be the case as we observed no significant increase of the autophagic flux when we performed the metazachlor treatment in nutrient rich conditions (compared to the untreated plants in rich conditions; Figure 10,E). This suggests that in autophagy inducing conditions, the KCS activity is in some way implicated in the regulation of the process; and that in KCS inhibiting conditions, the autophagic pathway is even more active. This suggests that the KCS activity plays a role as a negative regulator for autophagy in our conditions. Inhibition of the KCS enzyme leads to a decrease in the length of fatty acids. The length of the fatty acids present in sphingolipids has been implicated in the clustering of proteins and defects have led to alteration of vesicular pathways in the trans-golgi-network [START_REF] Wattelet-Boyer | Enrichment of hydroxylated C24-and C26-acyl-chain sphingolipids mediates PIN2 apical sorting at trans-Golgi network subdomains[END_REF]. In this manner, altering the length of fatty acids could also have an impact in autophagy as autophagy is tightly connected with secretory pathways in other organisms (Zhuang et al., 2015). Sphingolipids are known regulators for different steps in the AP biogenesis process in [START_REF] Taniguchi | Regulation of autophagy and its associated cell death by "sphingolipid rheostat": Reciprocal role of ceramide and sphingosine 1-phosphate in the mammalian target of rapamycin pathway[END_REF]. In plants, our results suggest that not all sphingolipids affect autophagy in the same manner, indicating a particular function for each of them.

III-1.2 Sterols

In order to deplete plants from sterols, we selected two different inhibitors: Fenpropimorph (fen) and Lovastatin (lova), which act at different steps along the biosynthetic pathway (Figure 11,A). Both have been well characterized and are efficient in Arabidopsis roots [START_REF] Grison | Specific Membrane Lipid Composition Is Important for Plasmodesmata Function in Arabidopsis[END_REF]. In contrast to other inhibitors, and according to previous publications, these treatments were performed by transferring seedlings for 48 h to Fenpropimorph or lovastatin-containing plates prior to shift to nutrient deprived (-NC) liquid MS, also supplemented with the corresponding lipid synthesis inhibitor, for 3 hours.

(1) We inhibited the HMG-CoA reductase, using Lovastatin at 1 µM [START_REF] Boutté | Fluorescent in situ visualization of sterols in Arabidopsis roots[END_REF][START_REF] Frescatada-Rosa | High lipid order of Arabidopsis cell-plate membranes mediated by sterol and DYNAMIN-RELATED PROTEIN1A function[END_REF][START_REF] Grison | Specific Membrane Lipid Composition Is Important for Plasmodesmata Function in Arabidopsis[END_REF]. In these conditions we did not observe any significant changes in the ratio of GFP/GFP-ATG8 compared to control conditions (DMSO) suggesting that autophagy flux is not affected. (2) We also inhibited the Cycloeucalenol cycloisomerase (CPI1), using Fenpropimorph at 165 µM. In these conditions we observed a drastic increase in the autophagic flux (Figure 11,B). It is to note that the autophagic flux was increased in these conditions even in nutrient rich conditions (Figure 11,B), unlike what we saw for example when we inhibited the KCS (Metazachlor treatment above).

Sterols are one of the major lipids found on membranes and are known to modulate fluidity [START_REF] Dufourc | Sterols and membrane dynamics[END_REF]. Local enrichment along with sphingolipids has been shown to induce a liquid-ordered phases, with both a high mobility and a high conformational order (Van Meer, 2008). Sterols have been implicated in membrane nano-domains formation as well as protein recruitment [START_REF] Gronnier | Structural basis for plant plasma membrane protein dynamics and organization into functional nanodomains[END_REF]. Sterols have also been shown to be enriched at plasmodesmata (PD), specialized subdomains of the plasma membrane that allow intracellular communication [START_REF] Grison | Specific Membrane Lipid Composition Is Important for Plasmodesmata Function in Arabidopsis[END_REF]. Also, sterols have been proposed as crucial signaling molecules, as their recognition by PM localized protein kinases is known to trigger intracellular pathways controlling root growth as well as development in A. thaliana [START_REF] Jaillais | Brassinosteroid signaling and BRI1 dynamics went underground[END_REF]. Thus, sterols seem to have an important role in membrane dynamic events, and as such, represent good candidates as possibly controlling membrane dynamic events involved in autophagy. Interestingly, it was found that an abnormal accumulation of sterols in endolysosomal membranes results in an impaired sorting of SNARE complexes that are required for the AP-lysosome fusion, thereby reducing the lysosomes ability to fuse with APs in mammalian cells [START_REF] Fraldi | Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders[END_REF].

Our results show that the two treatments did not cause the same effects. Inhibiting the HMG-CoA reductase did not affect the autophagy flux, suggesting that sterol synthesis is not required for the autophagic flux. However, inhibiting the Cycloeucalenol cycloisomerase (CPI1) with the Fenpropimorph chemical treatment caused a large increase in the autophagic flux, even in nutrient rich conditions. These diverging effects can be explained by the fact that both inhibitors alter the sterol composition differently: lovastatin affects sterols quantitatively, while Fenpropimorph affects sterols qualitatively, as it leads to the accumulation of two metabolic intermediates (cycloeucalenol and 24-Methylpollinastarol) with little effects on overall quantity of sterols (Figure 11, A and [START_REF] Frescatada-Rosa | High lipid order of Arabidopsis cell-plate membranes mediated by sterol and DYNAMIN-RELATED PROTEIN1A function[END_REF]). It is to note that Lovastatin treatment does not completely inhibit sterols synthesis, but decreases it by around 40% [START_REF] Frescatada-Rosa | High lipid order of Arabidopsis cell-plate membranes mediated by sterol and DYNAMIN-RELATED PROTEIN1A function[END_REF]. Thus, the residual sterols present in the cells could account for the lack of effects we observed in the autophagic flux and we cannot conclude that sterols are not required for autophagy. In the case of the Fenpropimorph treatment, the accumulation of these intermediates could be sensed as a stress if this compound was to become toxic if accumulated. In this case, it could explain the increase in the autophagic flux we observed.

Fenpropimorph treatment has been shown to induce the conversion of membrane lipids into triacylglycerol molecules in algae [START_REF] Kim | The small molecule fenpropimorph rapidly converts chloroplast membrane lipids to triacylglycerols in Chlamydomonas reinhardtii[END_REF]. Another explanation to the huge increase in the autophagic flux we observed could therefore be that in our conditions, treatment with Fenpropimorph also causes an abnormal TAG and lipid droplet formation causing the cell to induce lipophagy, a specific type of autophagy targeting lipid droplets that was recently show to occur in plant cells [START_REF] Barros | Multifaceted Roles of Plant Autophagy in Lipid and Energy Metabolism[END_REF][START_REF] Elander | Autophagy in turnover of lipid stores: trans-kingdom comparison[END_REF]Fan et al., 2019). Finally, Fenpropimorph treatment has been shown to affect cell division, where it lead to defects in stomatal asymmetric division [START_REF] Qian | Sterols are required for cell-fate commitment and maintenance of the stomatal lineage in Arabidopsis[END_REF]. Autophagic activity has been shown to be differentially regulated in each step of cell cycle, notably, AP formation was strongly suppressed during mitosis [START_REF] Hanamata | Cell cycle-dependence of autophagic activity and inhibition of autophagosome formation at m phase in tobacco by-2 cells[END_REF]. In that context, the large increase in autophagic flux observed upon Fenpropimorph treatment could also result from a dysregulation in the cell cycle and with thus subsequent effects on the autophagic pathway.

III-1.3 Anionic phospholipids

3-1 Phosphatidic acid (PA)

PA is anionic lipid precursor for the synthesis of many other lipids (Figure 12,A). Additionally, PA is an important second messenger implicated in many cellular responses [START_REF] Pokotylo | The phosphatidic acid paradox: Too many actions for one molecule class? Lessons from plants[END_REF]. In plants, PLD and PA are implicated in multiple biotic and abiotic stress responses such as cold, salt, heat and osmotic stress (Hong et al., 2016;Li et al., 2019).

(1) Inhibition of LPAAT activity

To inhibit de novo PA synthesis we aimed at targeting the lyso phosphatidic acid acyltransferase activity that converts lysoPA to PA. For this, we used Ci-976, an inhibitor that was first discovered to affect the secretory and endocytic membrane trafficking pathways in mammals [START_REF] Chambers | A unique lysophospholipid acyltransferase ( LPAT ) antagonist , CI-976 , affects secretory and endocytic membrane trafficking pathways[END_REF].This inhibitor has already has been used to prove the implication of LPAAT activity in intracellular vesicular transport in mammals, such as COPI trafficking [START_REF] Yang | COPI acts in both vesicular and tubular transport[END_REF], or ER-to-Golgi transport (J. [START_REF] Lin | Rab1A is required for assembly of classical swine fever virus particle[END_REF]; as well been implicated in the shaping of the Golgi structure and function [START_REF] Schmidt | Lysophosphatidic acid acyltransferase 3 regulates Golgi complex structure and function[END_REF]. Interestingly, in mammals, LPAAT activity was shown to contribute to the activation of a protein complex required for fission of several endomembranes [START_REF] Pagliuso | Golgi membrane fission requires the CtBP1-S/BARS-induced activation of lysophosphatidic acid acyltransferase δ[END_REF]. Taken together, LPAAT activity seems to be crucial for intracellular vesicular trafficking.

Although this has not been published yet, our colleagues V. Wattelet-Boyer and F.

Dittrich-Domergue in Patrick Moreau's team at the LBM showed that Ci-976 is also a potent inhibitor of LPAAT in plant cells (Figure 12,A). In particular, they showed that seedlings grown for five days on MS plates with 10 µM Ci-976, have a 50% decrease in the levels of de novo synthesized PA (Figure 12,B). To test the effect of Ci-976 on autophagy we tested two concentrations of this inhibitor. We observed a slight -yet not significant-decrease of the ratio GFP/GFP-ATG8 at 5 µM and a larger decrease at 10 µM (Figure 12,C). These experiments will have to be repeated to reach a firm conclusion, yet they suggest that inhibiting LPAAT may lead to autophagy defects in root cells. We have to note that this inhibitor is not that well described in plants, and could affect other lipids than PA by inhibiting not only LPAAT but also other lysophospholipids acyltransferases (LPCATs).

( (3) Inhibition of PA-Phosphatase (Propanolol, 50 µM) Propranolol is responsible for the inhibition of a phospholipase D and thus blocks PA production; as such, propanolol has been widely used in medical research [START_REF] Liu | The Role of Phospholipase D in Modulating the MTOR Signaling Pathway in Polycystic Kidney Disease[END_REF][START_REF] Sun | New strategy for in vitro activation of primordial follicles with mTOR and PI3K stimulators[END_REF]. In plants, propanolol has also been shown to modulate PA levels at a concentration of 50 µM [START_REF] Barbosa | Phospholipid composition and a polybasic motif determine D6 PROTEIN KINASE polar association with the plasma membrane and tropic responses[END_REF]. We thus chose this concentration for our experiments, which did not show any significant effect of this inhibitor on autophagic flux (Figure 12,E).

(4) Inhibition of Phosphoinositide-specific Phospholipase C (PI-PLC) (U-73122, 5 µM) Phospholipases C are enzymes responsible for phospholipid break down, producing a diacylglycerol (DAG) molecule and the head group specific to each phospholipid, two known signaling molecules. Two major types of phospholipases C exist in plants: one using phosphatidylcholine (PC) as a substrate (called PC-PLCs) and the other type being specific to phosphoinositides (PI-PLC). Their roles in plant signaling are wide and are known to regulate process such as tip growth, stomatal function and gravitropism [START_REF] Dowd | The Emerging Roles of Phospholipase C in Plant Growth and Development[END_REF]. In Arabidopsis, PI-PLC signaling has also been linked with either biotic or abiotic stress responses [START_REF] Munnik | PI-PLC: Phosphoinositide-Phospholipase C in Plant Signaling[END_REF]. We tested if the PI-PLC activity had any links with autophagy, by using the U-73122 specific PI-PLC inhibitor and its inactive homolog U-73343 (Figure 8).

Upon U-73122 treatment in nutrient starvation, we could observe a significant decrease in the autophagic flux compared to the control plants (Figure 12, F), however the same decrease is observed with the U-73343 inactive homolog, indicating that the decrease is not specific to a PI-PLC inhibition but is rather a side effect of the molecules for which we have no explanation at this time.

Our results showed that by inhibiting different enzymes leading to the same product, we obtained different results in the autophagic flux. This could be explained by the fact that in plant cells, different pools of PA exist, and are implicated in distinct cellular processes. The different pools of PA result from two distinct PA-producing enzymes: DAG Kinase (DGK) or Phospholipase D (PLD) [START_REF] Pleskot | Turnover of phosphatidic acid through distinct signaling pathways affects multiple aspects of pollen tube growth in tobacco[END_REF]. PA seems to be localized mainly at the PM [START_REF] Simon | A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants[END_REF]. Interestingly, PA also seems to be present in the ER and in chloroplasts, although to a minor extent than in the PM, highlighting the different pools of PA within the cell [START_REF] Simon | A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants[END_REF]. Thus, one can imagine that the PA required for autophagy is exclusively produced by one of these biosynthetic pathways leading to PA production. For example, our results suggest that LPAAT-produced PA may be required for autophagy while PLD produced PA is not (Figure 12 C,E). The R-59022 inhibitor has been tested in neuronal cells at 5 µM [START_REF] Takita | Diacylglycerol kinase inhibitor R59022-induced autophagy and apoptosis in the neuronal cell line NG108-15[END_REF] and was shown to induce autophagy in neuronal cell lines, although the signaling pathways involved in this activation were not elucidated in this study [START_REF] Takita | Diacylglycerol kinase inhibitor R59022-induced autophagy and apoptosis in the neuronal cell line NG108-15[END_REF]. In another study in mammalian cells, R-59022 (20 µM) treatment did not affect the autophagic flux (Holland et al., 2016). In our case, the R59022 treatment at 50 µM induced a large increase in the autophagic flux, while using this inhibitor at the lower concentration of 12,5 µM showed no effect. This would mean that in both mammals and plants, the effects seen upon R59022 treatment are dose dependent. The increase we observed in the 50 µM conditions suggests that the DAGK could be a negative regulator of autophagy in plant cells.

DAGKs have been implicated in regulating plant development [START_REF] Gómez-Merino | Arabidopsis AtDGK7, the smallest member of plant diacylglycerol kinases (DGKs), displays unique biochemical features and saturates at low substrate concentration: The DGK inhibitor R59022 differentially affects AtDGK2 and AtDGK7 activity in vitro and alte[END_REF][START_REF] Pleskot | Turnover of phosphatidic acid through distinct signaling pathways affects multiple aspects of pollen tube growth in tobacco[END_REF]. Thus, DAGKs could be responsible of regulating autophagy as to allow proper plant development in conditions where autophagy is not required.

Taken together, these results suggest that PA is important for plant autophagy, although its exact function has yet to be identified. As an important signaling molecule, PA could be responsible of activating/repressing autophagy induction pathways.

3-2 Inhibition of PIP kinases

Phosphoinositides (PIs) are a minor class of lipids, yet they control many signaling pathways.

PIs are known to control organelle biology by regulating vesicular trafficking, but they also modulate other aspects such as lipid distribution and metabolism thanks to their close relationship with lipid transfer proteins [START_REF] Balla | Phosphoinositides: Tiny lipids with giant impact on cell regulation[END_REF]. In plants, PIs are unevenly distributed in different compartments and help define their specific membrane identity required for proper functioning of these organelles [START_REF] Simon | A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants[END_REF]. We thus inhibited the synthesis of different PIP kinases to assay if this had an impact on the autophagic flux in our conditions (Figure 13).

(1) We inhibited the PI3Kinase enzyme using Wortmannin at 10 µM as a negative control. PI3P is already known to control autophagy in yeast and mammalian cells. In Arabidopsis, PI3P is also important for autophagy. The Arabidopsis homolog of the PI3K complex was recently characterized and shown to regulate autophagy (Liu et al., 2020). Using the Wortmannin inhibitor, we observed an expected significant decrease in the autophagic flux (Figure 13,B).

(2) To test the implication of PI(3,5)P2 in autophagy we used YM-201636, an inhibitor which was already been shown to inhibit PI(3,5)P2 production without affecting the concentration of any other phosphoinositides [START_REF] Hirano | Phosphatidylinositol 3-phosphate 5-kinase, FAB1/PIKfyve kinase mediates endosome maturation to establish endosome-cortical microtubule interaction in Arabidopsis[END_REF]. As seen in Figure 13 C, treatment with YM-201636 did not cause any significant defect in the ratio of GFP/GFP-ATG8a

suggesting that PI(3,5)P2 is not involved in autophagy in these conditions. (3) We inhibited the PI4Kinase using the phenylarsine oxide (PAO) inhibitor to test the importance of PI4P in plant autophagy and showed a drastic block in autophagy activity (Figure 14). These results will be presented in more details in the next chapter.

The results we obtained by inhibiting PIP kinases indicate that, as in other organisms, phosphoinositides play important roles in the regulation of autophagy in plants. Inhibiting the PI3Kinase resulted in a decrease in the autophagic flux (Figure 13,B). This inhibitor was already shown to decrease the number of autophagic structures which is congruent with our observations (Shin et al., 2014). This result also reassured us as to validate the experimental conditions that we used for performing this lipid inhibitor screen. PI(3,5)P2 is an anionic phosphoinositide with multiple functions in plant cells. Notably it seems to play essential roles in pollen development [START_REF] Whitley | Arabidopsis FAB1/PIKfyve proteins are essential for development of viable pollen[END_REF], as well as controlling multiple membrane trafficking events [START_REF] Hirano | Inhibition of phosphatidylinositol 3,5bisphosphate production has pleiotropic effects on various membrane trafficking routes in Arabidopsis[END_REF]. PI(3,5)P2 also controls many membrane trafficking event in mammals and yeast [START_REF] Balla | Phosphoinositides: Tiny lipids with giant impact on cell regulation[END_REF] and it has been found to be crucial for the endocytic pathway, intracellular signaling, and adaptation to stress [START_REF] Hasegawa | PI5P and PI(3,5)P2 : Minor, but Essential Phosphoinositides[END_REF]. PI(3,5)P2 is present on autophagic structures in mammals; and its presence is highly regulated by phosphatases to allow proper fusion with the lysosome (Hasegawa et al., 2016). Our results

show that PI(3,5)P2 is not required for autophagic flux in our experimental conditions suggesting fundamental differences in plant compared to mammalian autophagy. PI(3,5)P2 is a very minor lipid in plants, where it represents only 0.02% of total phospholipids in Arabidopsis [START_REF] Hirano | Phosphatidylinositol 3-phosphate 5-kinase, FAB1/PIKfyve kinase mediates endosome maturation to establish endosome-cortical microtubule interaction in Arabidopsis[END_REF], but it plays essential roles in endosome maturation [START_REF] Hirano | Phosphatidylinositol 3-phosphate 5-kinase, FAB1/PIKfyve kinase mediates endosome maturation to establish endosome-cortical microtubule interaction in Arabidopsis[END_REF].

The FAB1 homolog in Arabidopsis, as well as its product PI(3,5)P2, localize mostly to the plasma membrane [START_REF] Hirano | PtdIns(3,5)P2 mediates root hair shank hardening in Arabidopsis[END_REF]. Although PI(3,5)P2 synthesis did not affect the autophagic flux in our conditions, we cannot exclude that PI(3,5)P2 might be required for other types of autophagy or in other plant tissues or developmental stages. ATG18 bears a PI(3,5)P2

binding motif (in addition to its PI3P binding motif) in both yeast and in mammals, which ensures its proper localization and function in autophagy in these organisms [START_REF] Dove | Svp1p defines a family of phosphatidylinositol 3,5-bisphosphate effectors[END_REF][START_REF] Baskaran | Two-site recognition of phosphatidylinositol 3-phosphate by PROPPINs in autophagy[END_REF]. In plants, ATG18a has been reported as being implicated in autophagy (Xiong et al., 2005), and, to localize to autophagic structures (Zhuang et al., 2017), but direct binding of ATG18 to PI(3,5)P2 has not been reported. Our results indicate that PI(3,5)P2 is not required for autophagic flux in our experimental conditions; thus, it would be interesting to check if, as in mammals, ATG18 is indeed binding PI(3,5)P2 or if in plants it binds another lipid.

III-1.4 Conclusion and perspectives

From the results that we obtained from this screen, we observed that distinct treatments differentially affected autophagy. Some lipid inhibitors increased considerably the autophagic flux, other treatments had a negative impact on the autophagy flux, and some had no impact.

Therefore, the inhibition of different lipids (from distinct lipid classes) affects the autophagic flux differently. This suggests that all lipids do not have the same impact in autophagy, rather, they seem to have specific functions and roles in the process. Overall, these results support that lipids are more than mere building blocks for the production of AP membranes and rather, that key lipids have specific functions in the autophagy pathway. Inhibition of some lipids decreased the autophagic flux, which could mean that their synthesis is required for (1) the formation (size/number) or proper structure of autophagic compartments or (2) for their fusion with the vacuole. Further experiments using confocal microscopy could be helpful for answering these questions. If some lipids are required for the formation or the proper shaping of autophagic structures, treated plants would display a reduced number (or misshapen) of autophagic structures compared to untreated plants. Conversely, to assay if they are important for the fusion with the vacuole, we could perform experiments using Concanamycin A 1µM (an inhibitor or vacuolar hydrolases), which results in the accumulation of ABs inside of the vacuole. In these conditions, if a given lipid is indeed important for the fusion with the vacuole, treated plants would display a decrease in the number of ABs within the vacuole compared to untreated plants.

On the other hand, inhibition of the synthesis of other lipids resulted in a dramatic increase in the autophagic flux, which could result from: (1) the plant senses this inhibition as a stress and induces autophagy as a mechanism of defense (it could be the case if our treatment produces pleiotropic effects or leads to the accumulation of toxic metabolite intermediates or damaged organelles), or (2) the lipid/enzyme we inhibited plays a role as a negative regulator of autophagy, and thus in its absence (or of its activity) the process is activated. Multiple types of mechanisms can negatively regulate autophagy. For example, the nutrient sensing master regulator mammalian Target Of Rapamycin (mTOR) prevents autophagy activation by phosphorylation of key components of the autophagic machinery when the nutritive conditions are optimal [START_REF] Neufeld | TOR-dependent control of autophagy: Biting the hand that feeds[END_REF]. Interestingly, the lipid synthesizing enzyme phospholipase D (PLD), through a signaling cascade implicating its product PA, is capable of inhibiting autophagy via mTOR activation [START_REF] Armi | The Phospholipase D1 Pathway Modulates Macroautophagy[END_REF][START_REF] Zoncu | MTOR: From growth signal integration to cancer, diabetes and ageing[END_REF]. In this manner, lipid-synthesizing enzymes that produce second messengers (such as PA) can negatively regulate autophagy. Another way to inhibit the autophagic pathway is by transcriptional repression of ATG genes. Multiple transcription factors have been identified as being regulators of autophagy gene expression in mammals (Di [START_REF] Malta | Transcriptional regulation of autophagy: Mechanisms and diseases[END_REF]. Among these transcriptional factors, ZINC FINGER WITH KRAB AND SCAN DOMAINS 3 (ZKSCAN3)

was characterized as a transcriptional suppressor of autophagy in nutrient rich conditions [START_REF] Chauhan | ZKSCAN3 (ZNF306) is a Master Transcriptional Repressor of[END_REF][START_REF] Pan | The role of ZKSCAN3 in the transcriptional regulation of autophagy[END_REF]. Although this remains to be proven, it could be that by inhibiting the synthesis of a specific lipid we might have indirectly activated one of the transcriptional pathways leading to an autophagy activation (or vice-versa, we might have inhibited one of the transcriptional pathways known to repress autophagy).

Finally, some treatments did not show any impact on the autophagic flux, which suggests that, at least in the conditions we tested, their synthesis is not required for autophagy.

However, while we used these inhibitors as previously described in the literature, we did not systematically test their impact on the lipid composition in our conditions and thus we cannot exclude that the inhibition was not efficient. Additionally, we cannot exclude that these lipids may be important in other conditions such as longer periods of autophagy, other types of autophagy (selective) or in other tissues or developmental stages. More generally in the discussion of the results/effects that we observed here (or the absence of thereof) we should keep in mind that (1) we induced autophagy by nutrient starvation (nitrogen and carbon); other conditions induce other types of selective autophagy which may be dependent on different types of lipids (2) we extracted proteins exclusively from roots; thus we cannot exclude that these lipids are required for other types of autophagy or in other organs and at different developmental stages (3) we performed our screen using the GFP-ATG8 processing assay; nine ATG8 isoforms have been identified by sequence homology in the Arabidopsis genome (Kellner et al., 2017); for simplicity, in our inhibitor screening we only used one isoform, ATG8a but other populations of APs, marked by other ATG8 isoforms may react differently, (4) we induced autophagy for a relatively short period of time: lipids may be available from other sources (reservoirs in the ER or other organelles) but, after prolonged period of time, inhibition of their synthesis may be more limiting. Lastly, in our screen we obtained results that seemed to differ with what is occurring in mammalian cells (for example we were surprised that the inhibition of PI(3,5)P2 synthesis had no effect on the autophagic flux as this lipid seems to play important roles in the mechanism of fusion with the lysosome in mammals), which indicates that the function of lipids in autophagy displays major differences between organisms and that we should be very careful when extrapolating results. To sum up, this screen allowed us to identify interesting lipid candidates that seem to be important for plant autophagy, such as PA or glucosylceramides (or PI4P, see next chapter). Further studies directed towards their functional characterization will help us understand its role and by which mechanisms they contribute to autophagy in plants.

III-2 Phosphoinositol-4-phosphate controls autophagosome

INTRODUCTION

Macroautophagy (hereafter referred to as autophagy) is an evolutionary conserved mechanism essential for protein and organelle quality control. Autophagy is an intracellular degradation and recycling process during which intracellular material including proteins, proteins aggregates, organelles and/or invading pathogens are delivered to the lytic vacuole for degradation (Marshall & Vierstra, 2018). Autophagy exists at a basal level and is largely induced by a wide range of environmental stresses during which it plays essential in plant physiology by (1) clearing the cells from unwanted or toxic components thus ensuring quality control and (2) recycling organelles/molecules that can be re-used by the cell to support or reshape metabolism in periods of stress (Guiboileau et al., 2012). As such autophagy is a critical component of plant adaptation and plants lacking autophagy are unable to survive to various conditions such as nitrogen starvation, drought or pathogen attacks (Liu & Bassham, 2012).

Morphologically, autophagy starts with the de novo nucleation of an initial membrane structure called the phagophore which expands around autophagy cargo by addition of lipids and ultimately seals to form a double membrane vesicle called autophagosome (AP) (Soto-burgos et al., 2018). AP formation is critical in autophagy because it targets and engulfs the cargo to be degraded and traffics it to the vacuole. Upon fusion of the outer membrane of the AP with the tonoplast, the inner membrane and the cargo are released into the vacuolar lumen where they are rapidly degraded by hydrolases. The mechanism of AP formation has been a longstanding question in the field. The molecular mechanism governing autophagy is executed by a well-conserved machinery composed of a core of around 30 ATG genes (Feng et al., 2014;[START_REF] Galluzzi | Molecular definitions of autophagy and related processes[END_REF] first discovered in yeast, with many homologs now identified and characterized in plants (Marshall & Vierstra, 2018;Masclaux-daubresse et al., 2020). The core autophagic machinery is assembled into five protein complexes: (i) the initiation ATG1-ATG13 kinase complex; (ii) the phosphatydilinositol-3-kinase (PI3K) complex; (iii) the ATG9 complex, and finally both the (iv) ATG12-ATG5-ATG16 and (v) the ATG8-phophatidylethanolamine (PE) ubiquitin-like conjugation systems (Li & Vierstra, 2012;[START_REF] Liu | The vacuolar protein sorting-38 subunit of the arabidopsis phosphatidylinositol-3-kinase complex plays critical roles in autophagy, endosome sorting, and gravitropism[END_REF]Soto-burgos et al., 2018;[START_REF] Yoshimoto | Unveiling the Molecular Mechanisms of Plant Autophagy -From Autophagosomes to Vacuoles in Plants Special Focus Issue -Mini Review[END_REF]. Upon autophagy induction, ATG proteins are recruited from the cytosol to a lipido-proteic core named the pre-autophagosomal structure (PAS) which serves as the nucleation site for the phagophore. At the PAS, the ATG machinery regulates the assembly of the phagophore in a spatiotemporally coordinated manner and in a process that requires a flux of membrane lipids to assure proper expansion and maturation of the structure (Carlsson & Simonsen, 2015). In the nucleation step, the ATG1 complex allows the recruitment of the autophagy-specific PI3K which produces phosphatidylinositol 3-phosphate (PI3P) (Nascimbeni et al., 2017). The local enrichment of PI3P at the early autophagy structure is required for the recruitment of downstream ATG proteins including ATG18 as well as the ATG12-ATG5-ATG16 complex, which promotes the conjugation of ATG8 to PE thus allowing its incorporation into the autophagic membranes (Chung et al., 2010). ATG8 lipidation is a central and finely regulated step in autophagy as it allows the recruitment of ATG8 to the phagophore which (1) controls the recognition of cargo during selective autophagy and (2) supports the expansion of the phagophore membrane [START_REF] Bu | Multiple Functions of ATG8 Family Proteins in Plant Autophagy[END_REF]. In line with these facts, mutants defective for ATG5 or ATG4, another component of ATG8 lipidation, are unable to form mature APs (Yoshimoto et al., 2004;Thompson, 2005).

Despite extensive studies on ATG proteins, how APs are formed remains only partially understood, especially in plants. In particular, how the phagophore assembles stays elusive.

In yeast, the site of AP formation is in close proximity to the vacuole [START_REF] Hollenstein | Vac8 spatially confines autophagosome formation at the vacuole in S. Cerevisiae[END_REF], whereas in mammals early autophagic structures are connected to the endoplasmic reticulum (ER) [START_REF] Hayashi-Nishino | A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation[END_REF][START_REF] Ylä-Anttila | 3D tomography reveals connections between the phagophore and endoplasmic reticulum[END_REF]. Further high-resolution microscopy analysis of the ATG1/ULK1 initiation complex showed that this complex relocalizes to ERderived tubulovesicular compartments composed of ER-Golgi intermediate compartments (ERGIC) and ER exit sites (ERES) coalescing with ATG9-containing vesicles (Karanasios et al., 2016). Other ER-organelle membrane contact sites have been proposed as hubs for AP formation in mammals, such as ER-mitochondria contact sites (Hamasaki et al., 2013) or the ER-plasma membrane (Nascimbeni et al., 2017b). Taken together, these studies place the endoplasmic reticulum (ER) as the central platform for AP formation, with specific contributions of other organelles in different physiological conditions and cell types. In plants, how the phagophore is formed is less characterized, but evidence points to the ER as a critical platform for its nucleation and assembly. Notably, proteins required for early steps of AP formation such as the PI3P-and ATG8-binding SH3P2 (SH3 DOMAIN-CONTAINING PROTEIN 2) protein (Zhuang et al., 2013) and ATG5 were shown to localize on a cup shaped domain of the ER during AP formation (Le Bars et al., 2014). In addition to the ER, plasma membrane located proteins were also shown to be required for autophagy, indicating a possible contribution of ER/plasma membrane (PM) contacts for the formation of APs in Arabidopsis (P. Wang et al., 2019).

In addition to the protein machinery governing the formation of APs, lipids are also emerging key actors in the process. Lipids are not only structural building blocks of membranes; their physicochemical properties also determine crucial properties of membranes such as fluidity, curvature or electrostatics (Bigay & Antonny, 2012). These properties also define the identity and functions of membranes by mediating the recruitment of specific sets of peripheralmembrane proteins [START_REF] Noack | Functions of Anionic Lipids in Plants[END_REF]. In fact, research on this topic has progressed tremendously in yeast and mammals where recent studies have provided valuable information about the functions of lipids in autophagy highlighting novel aspects of AP formation (reviewed [START_REF] Gomez | Lipids in membrane dynamics during autophagy in plants[END_REF]Melia et al., 2020;[START_REF] Nishimura | Emerging roles of ATG proteins and membrane lipids in autophagosome formation[END_REF]. In particular, anionic lipids including PI3P, PI4P, PI(4,5)P2, PI(3,5)P2 or PA have been implicated in multiple steps of the autophagic process. The most prominent is PI3P, who was identified as being strongly accumulated in the site of AP formation (Axe et al., 2008, Zhuang et al., 2013). PI3P is required for the recruitment of downstream ATG proteins, called PI3P-effectors, that allow and ensure the progression of AP biogenesis (Proikas-Cezanne et al., 2015;Nascimbeni et al., 2017).

Another phosphoinositide required for the autophagic process is phosphatidylinositol-4phosphate (PI4P). In yeast, Atg9 vesicles trafficking require PI4K activity (Wang et al., 2012).

In mammals, the golgi-derived ATG9 vesicles delivers the class III PI4-Kinase β (PI4KIIIβ) to the phagophore nucleation sites, which was shown to be key for the formation of APs (Judith et al., 2019). In addition, mammalian PI4KIIα was found to interact with GABARAP (GAMMA-AMINOBUTYRIC ACID RECEPTOR-ASSOCIATED PROTEIN) proteins (ATG8 homologs in GFP/GFP-ATG8 ratio (%) mammals) which recruited them to APs for in situ PI4P production required for proper APlysosome fusion (Wang et al., 2015). Critically, the same lipid may control multiple steps along the pathway but lipid turn-over at the phagophore is also required to support proper autophagy (Nascimbeni et al., 2017a). The rapid interconversions between one phosphoinositide specie to another support the fast membrane remodeling events required during AP formation. This implies that their presence on autophagic structures is tightly regulated in time and space.

These findings indicate that lipids are indeed regulating many aspects of the autophagic process, notably by allowing the transient recruitment of membrane-associated proteins though electrostatic interactions (Nascimbeni et al., 2017a).

In plants, very little is still known about the nature and dynamics of lipids in the phagophore as well as their function during AP formation. The conjugation of ATG8 to a lipid moiety is indeed required for AP formation (Doelling et al., 2002;Yoshimoto et al., 2004), although solid evidence about the identity of this lipid is still lacking. The critical implication of PI3P in plant autophagy is well documented (Kang et al., 2018;Liu et al., 2020b) and multiple PI3P-effectors have been identified (Gao et al., 2015;Zhuang et al., 2013;Zhuang et al., 2017). However, its exact functions are not entirely understood. Similarly, although they have potential to regulate fundamental aspects of AP formation, the implication and functions of lipids other than PI3P in plant autophagy remains elusive.

In the present study, we show that pharmacologically or genetically inhibiting PI4Kinase activity blocks autophagy in plant cells. By combining biochemical and microscopy techniques, we investigated the function of PI4P in AP formation. Our findings suggest that PI4P is not required for the nucleation of the phagophore but is critical for its elongation and/or proper assembly notably by modulating the lipidation of ATG8. Our analyses further suggest that PI4P is present on autophagy structures and thus proposes a model in which PI4P present at the PM might be incorporated or at least be required for the formation of autophagic structures in Arabidopsis.

Furthermore, the PI4P required for autophagy in plant cells is likely to be produced by the PMlocalized PI4 Kinase (PI4Kα1) as a reduced expression of this gene hampered autophagic activity.

RESULTS

PAO, an inhibitor of PI4K activity, prevents autophagic flux

To identify which and how lipids affect the autophagy process in plants, we performed a lipid inhibitor screen. Briefly, we treated Arabidopsis seedlings with a range of inhibitors known to Graphics represent the signal intensity of the probe within the drawn area (red line), created using the "plot profile" feature (Fiji software). Scale bar = 20 µm affect the synthesis of specific lipids and measured the impact of these inhibitors on autophagy activity upon nutrient starvation conditions. This screen notably revealed that the PI4K inhibitor PAO (phenylarsine oxide) strictly blocks autophagy flux in roots of Arabidopsis in these conditions (Figure 14 A-B). Specifically, plants were treated with PAO at 30 µM, as previously described [START_REF] Barbosa | Phospholipid composition and a polybasic motif determine D6 PROTEIN KINASE polar association with the plasma membrane and tropic responses[END_REF][START_REF] Platre | A Combinatorial Lipid Code Shapes the Electrostatic Landscape of Plant Endomembranes[END_REF][START_REF] Simon | A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants[END_REF], in nutrient starvation conditions, and autophagy flux was measured using the GFP-ATG8 assay in comparison to control plants treated with DMSO. During autophagy, ATG8 is recruited to the membrane of the phagophore, the precursor to the AP and a portion of the protein is delivered in the vacuole after fusion of the AP with the tonoplast. In the vacuolar lumen, ATG8 proteins fused to the GFP (GFP-ATG8) are rapidly degraded while the more stable GFP moiety accumulates.

Therefore, the ratio between GFP-ATG8 and free-GFP reflects the rates of autophagy cargo delivery (Shin et al., 2014). In control conditions, plants transferred from a nutrient-rich medium (+NC) to a nutrient-depleted medium (MS medium deprived in carbon and nitrogen, -NC)

showed an accumulation of free GFP overtime reflecting an increase in autophagic flux in these conditions (Figure 14, A-B, DMSO). In contrast, when plants were treated with PAO, we observed a large decrease in the accumulation of free GFP compared to control conditions after 1.5 or 3 hours of nutrient starvation suggesting a block in autophagy autophagy activity in these conditions (Figure 14, A-B, PAO). To validate that PAO treatment was effectively inhibiting the synthesis of PI4P in our conditions, we performed confocal microscopy analysis on P7Y plants, which express a fluorescent probe (mCitrine-PH OSBP ) designed from a PI4Pbinding region of the Oxysterol-binding protein (OSBP) and used as a bona-fide PI4P reporter in plants [START_REF] Michaeli | ATG8-INTERACTING PROTEIN1 Is Involved in Autophagy-Dependent Vesicular Trafficking of Plastid Proteins to the Vacuole[END_REF][START_REF] Simon | A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants[END_REF]. Under normal untreated conditions, the fluorescence of the PI4P reporter strictly localizes at the PM (Figure S14, B, DMSO).

Consistent with the litterature [START_REF] Simon | A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants[END_REF] we found that, upon PAO treatment, as PI4P is no longer being sytnhesized, the fluorescent probe largely excedes the lipid available for binding, resulting in diffuse fluorescence of the probe throughout the cytosol (Figure S14, B, PAO). These results allow us to validate that our PAO treatment is efficiently inhibiting the synthesis of PI4P. Although PAO has been conventionally used to inhibit PI4K activity at concentration of 30 µM for 1 hour or 60 µM for 30 min [START_REF] Barbosa | Phospholipid composition and a polybasic motif determine D6 PROTEIN KINASE polar association with the plasma membrane and tropic responses[END_REF][START_REF] Platre | A Combinatorial Lipid Code Shapes the Electrostatic Landscape of Plant Endomembranes[END_REF][START_REF] Simon | A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants[END_REF], a recent paper reported that it acts as a potent PI4K inhibitor at lower concentrations (8 µM) [START_REF] Nagashima | Multiple quality control mechanisms in the ER and TGN determine subcellular dynamics and salt-stress tolerance function of Korrigan1[END_REF]. Our results recapitulated these data, showing a redistribution of P7Y fluorescence from the PM to the cytosol when seedlings were shifted from control -NC medium to a -NC medium supplemented with PAO at 7.5 µM or 15 µM (Figure S14,A). Further, our results show that at these lower concentrations, the inhibition of PI4K by PAO coincided with a block of autophagy flux, similar to what we observed for a treatment at 30 µM, (Figure 14, C-D). Together these results show that PAO is an inhibitor of autophagy induction upon nutrient starvation in root cells. TOR is a conserved master regulator of autophagy (Liu & Bassham, 2010;[START_REF] Rexin | TOR signalling in plants[END_REF]Soto-Burgos & Bassham, 2017). In plants, down-regulation of TOR in Arabidopsis RNAi lines lead to constitutive autophagy and decreased growth (Liu and Basham, 2010). To investigate if the synthesis of PI4P was required for TOR-dependent autophagic induction, we used the TOR inhibitor AZD8055 (Chresta et al, 2010;[START_REF] Pu | TOR-Dependent and -Independent Pathways Regulate Autophagy in Arabidopsis thaliana[END_REF] in combination with PAO and then performed the GFP-ATG8a degradation assay. As expected, we observed a massive accumulation of free GFP and an almost disapearrance of GFP-ATG8 upon treatment with 1 µM AZD treatment, indicative of a large increase in autophagy flux when TOR activity is inhibited (Figure 14, E-F). In contrast, when plants were treated with both AZD and PAO, the degradation of GFP-ATG8 to free GFP was largely stalled in a dose dependent manner (Figure 14, E-F) indicating that PAO blocks autophagy flux in these conditions and suggesting that PI4K activity is required for TOR-induced autophagy activation. Altogether, our results

show that PAO is a potent autophagy inhibitor and suggest that PI4P synthesis is critical for autophagy in root cells.

Downregulating PI4Ka, rather than PI4Kb, decrease autophagic degradation

Our results suggest that PI4P synthesis is required for autophagy. PI4P is synthesized by phosphatidylinositol-4-kinases (PI4-Kinases); in Arabidopsis, twelve genes encode for putative PI4-Kinases, subdivided in three classes 8 PI4Kγ, 2 PI4Kβ and 2 PI4Kα. Among those, three PI4K class γ were shown to be protein kinases [START_REF] Galvão | Characterization of a new family of protein kinases from Arabidopsis containing phosphoinositide 3/4-kinase and ubiquitin-like domains[END_REF][START_REF] Mueller-Roeber | Inositol phospholipid metabolism in Arabidopsis. Characterized and putative isoforms of inositol phospholipid kinase and phosphoinositide-specific phospholipase C[END_REF] and PI4Kα2 is considered to be a pseudogene as there are no transcripts of this gene in expressed sequence tag libraries. At this time, only PI4Kβ1, PI4Kβ2 and PI4Kα1 have a characterized phosphatidylinositol-4-kinase activity, i.e. are bona fide PI4P producing enzymes [START_REF] Okazaki | Phosphatidylinositol 4-phosphate negatively regulates chloroplast division in arabidopsis[END_REF][START_REF] Stevenson | A phosphatidylinositol 4-kinase pleckstrin homology domain that binds phosphatidylinositol 4-monophosphate[END_REF][START_REF] Xue | A Plant 126-kDa Phosphatidylinositol 4-Kinase with a Novel Repeat Structure[END_REF]. PI4Kβ1 and PI4Kβ2 both localize to the trans-golgi network/early endosomes (TGN/EE), and have been implicated in vesicular sorting events, endocytosis, and lateral root growth [START_REF] Kang | Electron Tomography of RabA4b-and PI-4Kβ1-Labeled Trans Golgi Network Compartments in Arabidopsis[END_REF][START_REF] Lin | A dual role for cell plate-associated PI4Kβ in endocytosis and phragmoplast dynamics during plant somatic cytokinesis[END_REF][START_REF] Preuss | A role for the RabA4b effector protein PI-4Kβ1 in polarized expansion of root hair cells in Arabidopsis thaliana[END_REF][START_REF] Rubilar-Hernández | PI4KIIIβ activity regulates lateral root formation driven by endocytic trafficking to the vacuole[END_REF]. In contrast, PI4Kα1 was found to localize to the PM, and a knockdown mutation decreased the amount of PI4P in chloroplasts [START_REF] Okazaki | Phosphatidylinositol 4-phosphate negatively regulates chloroplast division in arabidopsis[END_REF]; additionally, PI4Kα1 has also been observed to localize to perinuclear membranes [START_REF] Stevenson-Paulik | Differential Regulation of Two Arabidopsis Type III Phosphatidylinositol 4-Kinase Isoforms. A Regulatory Role for the Pleckstrin Homology Domain[END_REF]. To genetically confirm the result obtained using PAO as well as exploring the subcellular origin of PI4P for autophagy, we compared the relative contribution of PIKα1 and PI4Kβ in autophagy activity using reverse genetics. The pi4kβ1 pi4kβ2 double KO mutant was used as previously described [START_REF] Kang | Electron Tomography of RabA4b-and PI-4Kβ1-Labeled Trans Golgi Network Compartments in Arabidopsis[END_REF]Lin et al., 2019, Figure S15,A). However, no KO line for PI4Ka was available as homozygous tDNA-insertion mutants in PI4kα1 are embryo lethal [START_REF] Delage | Arabidopsis type-III phosphatidylinositol 4-kinases β1 and β2 are upstream of the phospholipase C pathway triggered by cold exposure[END_REF]. We thus constructed an artificial is massively degraded upon autophagy induction by nutrient starvation; addition of concanamycin A, which de-acidifies the vacuole resulting in a reduction of vacuolar degradation (Shin et al., 2014), partially inhibits NBR1 degradation. In contrast, the protein highly accumulates in the atg5-1 mutant (in which autophagy is blocked) and shows a minor reduction in level upon autophagy induction. In the same conditions, the pi4kβ1 pi4kβ2 double mutant showed no difference in regard to the level of NBR1 compared with the wild type (preliminary results, Figure 15, A-B). In contrast, the ami:PI4Kα1 showed a significant accumulation of the protein compared to the WT upon autophagy induction by nutrient starvation (Figure 15, A-B). Note that mRNA analyses showed no impact of knocking out PI4Kβ or knocking down PI4Kα1 in NBR1 expression across conditions (preliminary results, Figure 15, C), supporting that the differences in NBR1 protein levels are due to modified rates of protein degradation rather than differences in expression. These results suggest that PI4Kα1, but not PI4Kβ, is required to support proper autophagy degradation. Autophagy has been consistently shown to promote plant survival upon nutrient limitation [START_REF] Chen | Autophagy and Nutrients Management in Plants[END_REF].

To further assess the implication of PI4Kα1 in autophagy, we thus compared the phenotype of amiRNA:PI4Kα1 plants to that of WT plants as well as of the atg5-1 mutant grown in +NC conditions for seven days and then shifted to -NC conditions for an additional seven days. As segregation. Using these lines, we plan on quantitatively assess rates of autophagy flux using the GFP-ATG8 assay as well as looking at autophagosome formation.

Pi4K activity is essential for AP formation

Our results show that autophagic flux is blocked following the inhibition of PI4K; we thus decided to explore the role of PI4P in autophagy, i.e., the step(s) of the pathway at which this lipid may function. To this end, we used confocal microscopy to visualize autophagy structures in vivo in the roots of Arabidopsis seedlings upon PAO treatment in comparison to control conditions. The GFP-ATG8 protein decorates different autophagic structures, from the expanding phagophore, up to mature APs, and is thus widely used as a marker for autophagic structures [START_REF] Jiménez-Nopala | Autophagy mediates hydrotropic response in Arabidopsis thaliana roots[END_REF]Shin et al., 2014;Thompson et al., 2005). Plants expressing a GFP-ATG8a construct were analyzed in rich conditions as well as after 30 minutes of nutrient starvation +/-PAO. We chose to use a treatment of PAO at 60 µM for 30 minutes as it was previously shown to sharply disrupt PI4P synthesis (Figure S15B) without affecting the pool of PI(4,5)P2 [START_REF] Simon | A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants[END_REF]. As seen in (Figure 16, A), in rich conditions, GFP-ATG8a mostly localizes in the cytosol. Upon 30 minutes of autophagy induction by nutrient starvation, GFP-ATG8a localized to multiple punctate structures throughout the cytoplasm, which correspond to either phagophores or APs. These structures do exist in nutrient rich conditions, as there is a basal level of autophagy occurring at any given time in cells. Upon nutrient starvation (-NC), the number of GFP-ATG8a punctas significantly increased which reflects an increase in AP formation (Figure 16, A-B), compare +NC and -NC, DMSO conditions). In comparison, when plants were treated with PAO, very little GFP-ATG8 punctae were detected and GFP-ATG8a was mostly found in the cytosol (Figure 16,B)

suggesting that AP formation was prevented. Similarly, treatment with PAO greatly affected the number of GFP-ATG8a puncta upon autophagy induction by AZD8055 compared to control conditions (Figure S16,B), suggesting that PI4K activity is critical for TOR-dependent AP formation.

The Arabidopsis genome contains nine genes encoding ATG8 isoforms (Avin-Wittenberg et al, 2012), and specific ATG8 isoforms decorate specific population of APs depending on the stress used for autophagy induction [START_REF] Zess | N-terminal β-strand underpins biochemical specialization of an ATG8 isoform[END_REF][START_REF] Bu | Multiple Functions of ATG8 Family Proteins in Plant Autophagy[END_REF]. It has been hypothesized that ATG8 specialization contributes to the differentiation of selective autophagy pathways (i.e. different ATG8s isoforms interact with specific cargo receptors) [START_REF] Zess | N-terminal β-strand underpins biochemical specialization of an ATG8 isoform[END_REF]. To further explore the importance of PI4P synthesis for AP formation, we analyzed the impact of PAO on the subcellular localization of additional ATG8 isoforms, namely ATG8e and blocks the formation of GFP-ATG8e or GFP-ATG8f punctae upon nutrient starvation conditions (Figure 16, A-B). Together, these analyses show that PAO inhibits the formation of ATG8labeled structures in multiple autophagy inducing conditions suggesting that PI4K activity is essential for AP formation. Additionally, this activity seems to be necessary for the formation of different populations of APs based on the analyses of three isoforms of ATG8.

PI4K activity controls ATG8 lipidation

A prerequisite for the formation of APs, is the recruitment of the central protein ATG8 to the phagophore membrane (Xie et al., 2008) which requires the covalent lipidation of ATG8 to the lipid PE. Our results show an absence of ATG8 puncta structures in PAO treated conditions

showing that AP formation is prevented and raising the question of ATG8 recruitment to the phagophore in these conditions. To address this question, we tested the effect of PAO on ATG8 lipidation. For this purpose, we performed cell fractionation by using high-speed ultracentrifugation to separate soluble proteins (S100) from membrane-bound proteins (P100) in WT plants in either rich conditions (R) or in nutrient starvation conditions with PAO (P) or DMSO as control (D) (Figure 17, A-B). Immunoblot analyses using an endogenous anti-ATG8

antibody revealed that the quantity of ATG8 was similar in total lysates or in the soluble fractions across treatments (Figure 17, A-B) suggesting that PAO has no effect on the production of the protein. However, the level of the ATG8-PE band was significantly reduced in the membrane fraction upon PAO treatment (Figure 17, A-B, ATG8-PE) suggesting that the lipidation of ATG8 is prevented in these conditions. Note that the anti-ATG8 antibody that was used for these analyses recognizes all ATG8 isoforms, we thus cannot discriminate if -and which-only specific isoforms of ATG8 are affected. Yet, our previous results showed that PAO blocks the formation of different types of APs (labelled with ATG8a, ATG8e or ATG8f)

suggesting that it could block the lipidation of multiple ATG8 isoforms.

We further assessed ATG8 lipidation in PI4K mutants. Similar to what we found for NBR1 degradation, we observed that ATG8 lipidation was not affected in seedlings of the double pi4k 1 pi4k 2 mutant (Figure S17, A-B). In contrast, we found that the production and turnover of ATG8-PE was affected in the roots of the RNA:PI4K 1 i e af e 24 h f -estradiol treatment. In these conditions, in WT plants, we noted a slight increase in ATG8-PE after 0.5 h of nutrient starvation followed by a sharp reduction in the level of the protein after 3 h that was prevented by the addition of concanamycin A, and thus reflects the rate of ATG8-PE degradation by autophagy (preliminary results, Figure 17, C-D, see conversion index calculated as the level of ATG8-PE in conditions + concA/level of PE at 3h of -NC). In contrast,

we found that the level of ATG8-PE in the RNA:PI4K 1 line was mostly A. ATG8 and ATG8-PE levels in the microsomal fraction of WT, or seedlings. Immunoblot analyses were performed using an anti-ATG8 antibody. 8 day old plantlets were transferred from nutrient rich (+N) conditions to nutrient deprived (-N) or nutrient deprived conditions supplemented with concanamycin A 1µM (3+Ca) MS medium, for 0.5 or 3 hours. PIP2,7 was used as a loading control and normalization factor for (B). B. Quantification of the ATG8-PE levels in conditions presented in (A). Results represent the average of 3 independent experiments ± SD. C. ATG8 and ATG8-PE levels in the microsomal fraction of WT and seedlings.Immunoblot analyses were performed using an anti-ATG8 antibody. 7 day old plantlets were transferred to liquid nutrient transferred to liquid nutrient depleted medium to induce autophagy for 0.5 hours (0.5) and 3 hours (3) in control conditions or supplemented with concanamycin A (1µM, 3+Ca). PIP2,7 was used as a loading control and normalization factor for (D). D. Quantification of the ATG8-PE level in conditions presented in (C). The ATG8 formation index (right panel) represents the ratio of ATG8-PE in + Concanamycin A conditions (3+Ca) divided by the level ATG8-PE in nutrient deprived conditions (3) which reflects the net rate of ATG8-PE formation after 3 hours of autophagy induction. unchanged upon nutrient starvation either after 0,5 or 3 h and only showed a moderate increase upon addition of concanamycin A (preliminary results, Figure 17, C-D) which translated as a large reduction in the ATG8-PE conversion index compared to WT (preliminary results, Figure 17,D). These results suggest that both the formation and degradation of ATG8-PE are hampered in the amiRNA:PI4Kα1 line which further support a role for this kinase in the autophagy pathway.

PI4P synthesis is critical for the proper assembly/elongation of the phagophore

Our results propose that the formation of the mature autophagy structure, the autophagosome, is prevented in the absence of PI4P; we next wondered if an earlier autophagic structure could still be formed in these conditions. To address the function of PI4P in the formation of the phagophore, we focused on proteins that are only present on phagophores but dissociate from completed autophagosomes. Further, because our results suggest that ATG8 lipidation is prevented upon PAO treatment, we chose to focus on proteins which recruitment to the phagophore precedes ATG8 lipidation at this membrane. In other organisms, both the initiation ATG1 complex as well as ATG9 proteins act upstream and are required for the lipidation of ATG8 (Koyama-Honda et al., 2013) . In plants however, both atg13 (a member of the initiation complex) and atg9 mutants do not display a default in the lipidation of ATG8, indicating that in Arabidopsis these proteins are dispensable for this step (Suttangkakul et al., 2011;Zhuang et al., 2017). Thus, we first focused on ATG5, an essential component of the ATG8 lipidation complex (Thompson et al., 2005). ATG5 is part of the E3-like ATG12-ATG5 complex, and acts with the E1-like ATG7 and the E2-like ATG3 proteins to mediate the conjugation of Atg8 to the headgroup of PE (Sawa-Makarska et al., 2020). ATG5 is covalently conjugated to ATG12 and that is required for ATG8 lipidation. Both ATG5 and ATG12 are soluble proteins which reside in the cytosol at steady state. Upon autophagy induction, they are rapidly recruited to the membrane of the phagophore/PAS prior to the recruitment and lipidation of ATG8 at this membrane. In fact, dynamics studies showed that ATG5-ATG12 conjugation precedes that of ATG8-PE during phagophore formation in Arabidopsis (LeBars et al., 2014) and the atg5-1 mutant shows no lipidation of ATG8 (Thompson et al., 2005; Figure 17, atg5-1).

We reasoned that if the absence of PI4P synthesis blocks the overall autophagy pathway -with no nucleation of the PAS/early phagophore-the ATG12-ATG5 conjugate proteins would remain in the cytosol and thus be detected in the soluble fraction and depleted in the membrane fraction upon cell fractionation, similar to what we found for ATG8 (Figure 17). To test this hypothesis, we extracted proteins from roots of ATG5-mcherry and performed cell fractionation as described above for ATG8. Immunoblot analyses showed that both ATG5- mcherry and the ATG12-ATG5-mCherry conjugate display the same levels in all conditions tested, indicating that PAO treatment did not affect ATG5-mcherry protein production nor its conjugation with ATG12 (Figure 18, A-B). However, we observed that both ATG5-mCherry and ATG12-ATG5-mCherry accumulated at the membrane fraction from +NC to -NC treatment in PAO conditions compared to control DMSO-treated plants (Figure 18, A-B). The observed accumulation of ATG5 in the membrane fraction show that PI4P is not required for ATG5 or ATG12-ATG5 membrane association. ATG5 is known to accumulate in cup-shaped structures in close proximity to the ER that is considered to be the early autophagic structure (Phagophore / PAS) (Le Bars et al., 2014). In that context, our results suggest that early events of autophagy are still occurring in the absence of PI4K activity with notably the nucleation of an early phagophore structure. This hypothesis was further supported by our analyses of two additional ATG proteins: ATG4 and ATG18. ATG4 is a cysteine protease which cleaves the N-terminal Glycine residue of ATG8, a prerequisite for ATG8 lipidation (Woo et al., 2014;Yoshimoto et al., 2004). ATG18 is a PI3P-binding protein which was recently suggested to tether the PAS to the ER, in complex with ATG2, to initiate membrane expansion during autophagosome formation in yeast (Kotani et al., 2018). In Arabidopsis, ATG18a has also been implicated in autophagosome formation although its exact function is less characterized (Liu et al., 2009;Xiong et al., 2005). Similar to other ATG proteins, ATG18a displays a cytosolic localization in nutrient rich conditions, but relocalizes to the phagophore upon autophagy inducing conditions (Zhuang et al., 2017). Similar to what we found for ATG5, cell fractionation analyses of RFP-ATG4a and YFP-ATG18a showed that both proteins (1) still associate with membranes in autophagy inducing conditions when PI4K activity is inhibited by PAO, (2) actually accumulate in the membrane fraction in these conditions (Figure 18, C-F). These results suggest that the absence of PI4P does not prevent the recruitment of early ATG proteins to the PAS/early phagophore where they accumulate because autophagosome formation is blocked.

To visualize where the ATG5, ATG4 and ATG18 localize within the cell when PI4K activity is inhibited, we performed confocal microscopy analyses using the same conditions (lines, autophagy induction and PAO treatment) than for cell fractionation analyses. As previously described, ATG5 and ATG18 were mostly found in the cytosol in rich conditions and formed puncta representing phagophore upon autophagy inducing conditions (Figure 18, G-H; -NC, DMSO; Le Bars et al., 2014;Zhuang et al., 2017). RFP-ATG4 presented a similar punctate signal in nutrient starvation conditions; likely representing autophagy-related structures as we found that RFP-ATG4a and RFP-ATG4b co-localizes with GFP-ATG8a in transgenic lines coexpressing the two constructs (Figure S18, A-C). Upon PAO treatment, we observed a drastic decrease in the number of puncta for all proteins -i.e., ATG5-mCherry, YFP-ATG18, RFP-ATG4a and RFP-ATG4b-which, instead, showed a diffuse signal throughout the cytosol (Figure 18, G-H, Figure S18,D). This result may seem contradictory with the membraneassociated accumulation of the proteins revealed biochemically; yet given the function of ATG8 in phagophore expansion (Xie et al., 2008) and the fact that PAO inhibits ATG8 lipidation, we propose that, in the absence of PI4P, phagophore expansion gets stalled very early causing the accumulation of premature membrane structures that are too small to be resolved by confocal microscopy. Similar observations were made in the atg13 mutant where, although ATG8 is lipidated and accumulates at the membrane, autophagosome formation is blocked and the ATG8 signal looks diffuse in the cytosol (Suttangkakul et al., 2011). Altogether our data suggest that PI4P does not prevent the nucleation of the PAS/early phagophore but functions in early stages of autophagosome formation by supporting the proper structuration/expansion of the phagophore, notably through direct or indirect promotion of ATG8 lipidation.

PI4P is detected on autophagic structures.

PI4P is a major anionic phospholipid in plant cells where it acts as a docking site to recruit specific set of proteins via electrostatic interactions [START_REF] Simon | A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants[END_REF]. Our results show that PI4K activity is required for proper autophagy upon nutrient starvation in root cells. To test whether PI4P is directly present in the autophagic membranes we explored the localization of a set of PI4P-specific probes in regard to that of markers of autophagy structures in various conditions. Multiple lines probes for PI4P exist (expressing different PI4P-binding domains);

we thus crossed plants expressing mcherry-ATG8f with previously described lines expressing either P7Y (mCitrine-1PH OSBP ), P21Y (mCitrine-2xPH FAPP1 ) or the mutated version of FAPP1

(mCitrine-1xPH FAPP1-E50A-H54A ) [START_REF] Platre | A Combinatorial Lipid Code Shapes the Electrostatic Landscape of Plant Endomembranes[END_REF][START_REF] Simon | A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants[END_REF] to create individual homozygous lines.

We performed two types of experiments and compared the signal of PI4P probes to that of autophagic structures. First, we induced autophagy using nutrient starvation for 2 or 5 hours in control conditions (-NC+DMSO). In addition, to increase the number of autophagic structures and address the potential dynamics of PI4P throughout autophagy structures, plants placed in nutrient starvation were treated with 1µM concanamycin A, an inhibitor of hydrolar vacuolases which results in the accumulation of autophagic bodies inside the vacuole (Shin et al., 2014 and Figure 19,A). In nutrient starvation conditions, we observed PI4P probes mainly at the plasma membrane (PM) as well as on endomembrane compartments, with 2PH FAPP1 and 1xPH FAPP1-E50A-H54A , corresponding to the TGN (Figure 19, A; Figure S19, and data not shown)

which is in accordance with previous publications [START_REF] Simon | A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants[END_REF]. In these conditions we observed none to very few punctate PI4P signal colocalizing with ATG8-labeled structures (Figure 19, A, Figure S19). In particular, the P7Y (mCitrine-1PH OSBP ) x mcherry-ATG8f line did not display colocalization in punctate structures while the P21Y (mCitrine-2PH FAPP1 ) x mcherry-ATG8f line displayed occasional but rare co-localization events in both conditions (with less than 0,20% PI4P puncta showing a mChery-ATG8 signal, Figure S19,A). The probe corresponding to the mutated version of FAPP1 (mCitrine-1PH FAPP1-E50A-H54A ) showed an overall stronger signal as well as more numerous colocalization events with mCherry-ATG8f

(Figure 19, A-B). Although upon 5 hours of autophagy induction, only around 1% of autophagic structures displayed a PI4P signal when 4% of PI4P punctate structures displayed an ATG8fmcherry signal (Figure 19, A-B, -NC).

While ATG8 structures showing labeling for PI4P probes were rarely observed, we noticed that a significant portion of ATG8 puncta was located at, or at least close, to the plasma membrane.

Given that PI4P mostly accumulates at the PM in plant cells (Figure S14, Figure 19, Figure S19 and [START_REF] Simon | A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants[END_REF]) and that we found that the PM-localized PI4Kα1 producing enzyme is required for autophagy (Figure S17, C-D), we reasoned that this organelle could act as a reservoir of PI4P for autophagosome biogenesis. We thus analyzed the localization of ATG8 puncta in relation to the PI4P signal at the plasma membrane at high resolution using the Zeiss Airyscan technology which led to the identification of three types of ATG8 population (Figure 19, C-D). A vast majority (68 %) of ATG8 puncta were located in the cytosol, most likely labelling APs en-route to the vacuole; 23 % of them were at close proximity of the plasma membrane and 9 % were located on the PI4P signal at the plasma membrane. It is worth mentioning that puncta located at the plasma membrane did not show the same pattern as mature autophagosomes found in the cytoplasm (i.e spherical ring like structures) but rather presented a solid structure that followed the shape of the plasma membrane (Video supplemental full volume of a puncta at plasma membrane). These results suggest that a substantial amount of autophagic structures reside either in close proximity to the PI4Penriched plasma membrane, or fully accolated to it (Figure 19, C-D). Moreover, the fact that autophagic structures accolated to the plasma membrane do not display a conventional dotlike structure but rather a flattened shape that resembles the linear signal of the plasma membrane (Figure 19, C-D) suggest that these structures are in fact constrained to it, rather than only in close proximity due to the reduced cytoplasm found in plant cells because of their massive vacuole.

Finally, when plants were treated with concanamycin A, we observed the accumulation of PI4P signal as dots inside the vacuole which strongly colocalized with ATG8 (80%) suggesting that PI4P labels autophagic bodies (Figure 19, A, -NC + Ca). This suggest that PI4P accumulates on late mature autophagy structures.

DISCUSSION

PI4P controls autophagosome formation in plant cells

Autophagosome formation is a highly dynamic process that requires fine tuning of several membrane remodeling steps to ensure proper functioning of the autophagic process. This process relies on the concerted action of ATG proteins, yet these are not the only actors involved in this process. Lipids, aside from being structural membrane components are also known to modulate many membrane trafficking events in eukaryotic cells [START_REF] Noack | Functions of Anionic Lipids in Plants[END_REF]. In that context, we speculated that lipids play critical roles in the formation of autophagic vesicles. Here, our results show that PI4K activity is crucial for autophagy upon nutrient starvation in Arabidopsis. Inhibition of PI4K activity leads to a large block in the autophagic flux (Figure 14, A-F) and a complete block in autophagosome formation (Figure 16, A-B; Figure S16, A-B). Our data indicate that in the absence of PI4K activity, the lipidation of ATG8 is hampered, indicating that the protein is no longer able to be inserted into autophagic structures (Figure 17, A-B). However, inhibition of the PI4Kinase activity did not alter the membrane association of earlier ATG proteins (Figure 18, A-F). As these early ATG markers reside in the cytosol at steady state, their recruitment to membranes in the absence of PI4P suggests that this lipid is not critical for the earliest steps in PAS formation and/or nucleation. However, the absence of ATG-labeled puncta observed by fluorescent microscopy in these conditions (Figure 18, A-F) support the idea that PI4P is critical for the proper structuration and expansion of the phagophore, likely by, directly or indirectly, promoting ATG8 lipidation. Furthermore, a significant amount of autophagosomes were seen to localize on or in close proximity with the plasma membrane (Figure 19, C-D) and PI4P was detected on late autophagy structures in the vacuole (Figure 19,A)

Membrane sources for autophagosome formation in plants

Previous work on plant autophagy has shown that the ER is a critical platform for autophagosome formation (Le Bars et al., 2014, Zhuang et al., 2014;Zhaung et al., 2017). A recent study suggest that the plasma membrane could also participate in autophagy; in particular, the relocalization of PM-localized proteins to ER-PM contacts is required to support autophagosome biogenesis at these sites (Wang et al., 2019). In other organisms, both the ER as well as the plasma membrane and PM-derived compartments have been reported as contributing to the formation of autophagic structures [START_REF] Karanasios | Dynamic association of the ULK1 complex with omegasomes during autophagy induction[END_REF]Puri et al., 2013;[START_REF] Ravikumar | Plasma membrane contributes to the formation of pre-autophagosomal structures[END_REF]. Notably, contacts between the ER and the PM have been shown to be critical for autophagosome formation, by promoting localized PI3P synthesis at these contact sites (Nascimbeni et al., 2017b). Here we found that PI4P, a hallmark of the PM in plant cell [START_REF] Simon | A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants[END_REF] is critically engaged in AP formation in plants. In mammals the trans-golgi-network (TGN) and the TNG-localized PI4 kinase have been proposed as a source for PI4P required for autophagy [START_REF] De Tito | The Golgi as an Assembly Line to the Autophagosome[END_REF]. In plants, two major pools of PI4P have been reported, the major one present at the plasma membrane, and another consequent pool residing at the TGN [START_REF] Simon | A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants[END_REF]. Our preliminary results show that the TGN-residing PI4Kβ1 and PI4Kβ2 [START_REF] Kang | Electron Tomography of RabA4b-and PI-4Kβ1-Labeled Trans Golgi Network Compartments in Arabidopsis[END_REF] are not required for ATG8 lipidation or NBR1 degradation (Figure S17, A-B). These data suggest that, in contrast to yeast and mammal models, these proteins are not involved in autophagy in plants, or that their absence can be, at least partially, compensated by alternative proteins, including PI4Kα1. In contrast to PI4Kβ1 and PI4Kβ2, down-regulating PI4Kα1, the PM-residing PI4K [START_REF] Okazaki | Phosphatidylinositol 4-phosphate negatively regulates chloroplast division in arabidopsis[END_REF], reduced autophagy flux suggesting that it is required for that process (Figure S17, C-D). Further, we found that a substantial amount of autophagic structures localized in close proximity to the PM (Figure 19, C-D). We could also observe the presence of very small structures at the plasma membrane that could be representative of phagophores emerging from the PM (Figure 19, C-D). Interestingly, some ATG8-labeled compartments displayed a flattened shape that seemed to follow the shape of the plasma membrane and thus suggests that these autophagic structures could be constrained to it (Figure 19, C-D). In that context, our results suggest a functional contribution of the PM that could provide the PI4P required for autophagosome formation. As mentioned above, the ER represents a critical platform for autophagosome formation in plants (Le Bars et al., 2014) and ER-PM contact sites have been proposed to contribute to autophagosome formation in mammals (Nascimbeni et al., 2017b). PI4P has been shown to contribute to the formation of ER-PM contact sites (EPCS) in plants, by recruiting tether proteins that allow the establishment of such EPCS [START_REF] Brault | Multiple C2 domains and transmembrane region proteins ( MCTP s) tether membranes at plasmodesmata[END_REF][START_REF] Pérez-Sancho | The arabidopsis synaptotagmin1 is enriched in endoplasmic reticulum-plasma membrane contact sites and confers cellular resistance to mechanical stresses[END_REF]. Hence, a potential function for PI4P in autophagosome formation might be to contribute to the establishment of EPCS required for this process. Additionally, or alternatively, PI4P could function directly in the phagophore membrane. It is to note that PI4Ka1 has been reported to localize to compartments other than the PM, including the nuclear membrane in protoplasts of Arabidopsis cells [START_REF] Stevenson-Paulik | Differential Regulation of Two Arabidopsis Type III Phosphatidylinositol 4-Kinase Isoforms. A Regulatory Role for the Pleckstrin Homology Domain[END_REF]. Thus, we cannot exclude a possible implication of PI4P from other pools than that at the PM, and further studies should explore the potential localization of PI4Kα1 directly at autophagy structures.

Presence of PI4P on autophagy structures.

Our results, suggest that autophagic structures emerge from sites close to the PI4P-enriched plasma membrane (Figure 19, C-D). Moreover, we have found that PI4P is critical for the early stages of autophagosome formation such as the lipidation of ATG8 (Figure 16, A-B). However, our data concerning the presence of PI4P on autophagic structures is rather ambiguous. We observed few co-localization events between phagophores/autophagosomes and PI4P biosensors in the cytosol (Figure 19, A-B, -NC). However, we did observe a high colocalization between autophagic structures and PI4P biosensors in conditions in which we blocked autophagic degradation inside the vacuole (Figure 19, A-B, -NC + cA). This indicates a presence of PI4P on autophagic bodies (i.e. the single-membrane autophagic structure delivered in the vacuole). These observations could be explained by different reasons. First, PI4P might be mediating autophagosome formation by contributing to PM-associated events (such as the establishment of EPCS) required for that process; in that situation, the close proximity/contact of the phagophore with the PM may be sufficient to access the pool of PI4P required for AP formation. Second, if the concentration of PI4P on autophagic structures is low, PI4P-probes may not be recruited to these membranes, but rather titrated by highlyaccumulating compartments such as the plasma membrane or the TGN [START_REF] Simon | A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants[END_REF].

Third, a general bias for the use of lipid-probes is the fact that these can only bind 'inactive' pools of lipids, ie, lipids that are not already bound to a protein.

As reported before for other organelles, PI4P mostly act as a docking site for the recruitment of peripheral membrane proteins; thus, the active pool of PI4P at the phagophore would likely bind specific proteins and may not be available to recruit PI4P-probes, especially if the quantity is low. Fourth, PI4P can be rapidly interconverted to other phosphoinositide species (PI, PI(4,5)P2) and thus its presence in autophagic structures may only be transient, which could explain why we observed only few colocalization events in the cytosol. Supporting the hypothesis of high dynamic turn-over of PI4P at autophagy structures, PI4P has been detected on autophagy structures in other organisms where it was proposed to play distinct roles at different steps of the process, notably in early steps by mediating the trafficking of ATG9 vesicles (Judith et al., 2019;Wang et al., 2012), but also in the final steps by mediating the fusion of autophagosomes with the lytic compartments (Kurokawa et al., 2019;Wang et al., 2015). Furthermore, an abnormal accumulation of PI4P in autophagosome membranes due to a deficiency of the SAC1 phosphatase (which de-phosphorylates PI4P) abrogated autophagosome-vacuole fusion caused by a failure to recruit the SNARE complexes implicated in the fusion step (Zhang et al., 2020). Using nanoscale particle identification, PI4P was found to be present on the luminal leaflet of the autophagosome membrane in yeast (Kurokawa et al., 2019), whereas in mammals, PI4P was found exclusively in the cytoplasmic leaflet of both inner and outer membrane of the autophagosomes [START_REF] Kurokawa | Phosphatidylinositol 4-phosphate on Rab7-positive autophagosomes revealed by the freeze-fracture replica labeling[END_REF]. The different distributions of PI4P in both organisms supports the role that PI4P mediates multiple steps of autophagic pathway. Hence, the fact that we observed a high number of autophagic structures

PI4P ATG4/ATG5/ ATG18 ATG8 PE Cargo Plasma Membrane (PI4Kα1) Conjugation Expansion ER PI4K inhibition PI4P- binding protein

Figure 20. Model of autophagosome formation in plants

Upon autophagy induction early ATG proteins, including ATG5, ATG18 and ATG4 are recruited from the cytosol to the PAS, the lipoproteic core from which the phagophore emerges. The PAS is located on specific subdomains of the ER, presumably in contact with the PM. Upon phagophore nucleation, the ATG8 is lipidated and inserted to the membrane. Inhibiting PI4K activity blocks the lipidation of ATG8, preventing phagophore expansion and AP formation. We speculate that PI4P, at sites of the PM close or in contact with the PAS, promotes the recruitment of (a) key protein(s) acting upstream of ATG8 lipidation in the machinery of AP formation.

displaying a PI4P signal within the vacuole might be reminiscent of an accumulation of PI4P in the maturation steps of autophagosome formation to allow their subsequent fusion with the vacuole. Future studies in plant cells to finely characterize the PI4P distribution within phagophores as well as autophagosomes would provide helpful insights concerning the presence and dynamics of this lipid during the different steps of autophagosome formation and thus help characterize the functions of this lipid in the autophagic process.

Molecular function of PI4P in plant autophagy

Accumulation of PI4P and its combination with other anionic lipids contributes to the identity and activity of membranes by mediating the selective recruitment of specific sets of proteins through electrostatic interactions [START_REF] Noack | Functions of Anionic Lipids in Plants[END_REF]. Here our results show that the absence of PI4P causes a block in ATG8 lipidation and prevent autophagosome formation.

Yet, at least a portion of early ATG proteins is still being recruited to membranes suggesting that early stages of phagophore nucleation are still occurring (Figure 18, A-H). In that context, whether PI4P is directly present in autophagic membranes or in PM-ER contact sites from where phagophores emerge, we propose that PI4P act as a docking site for the recruitment of a key protein, working upstream of the lipidation of ATG8 to support the expansion of the phagophore (Figure 20).

As mentioned above, PI4P has been already involved in autophagy in other organisms. In saccharomyces cerevisiae, PI4P promotes the anterograde traffic of ATG9 from the Golgi complex and/or post-Golgi compartments (TGN) towards the PAS, this process is required for the recruitment of other essential ATG proteins (Wang et al., 2012). Both of the PI4K of yeast: Pik1p and Stt4p have been implicated in autophagy (Kurokawa et al., 2019). However, both seem to control different steps of autophagy: mutation of Pik1p led to a reduced number of autophagosomes in the cytosolic compared to the WT, while Stt4p mutants did not display any difference. Stt4p was indeed implicated in producing PI4P required for fusion with the vacuole (Kurokawa et al., 2019). Similarly, it was shown that the mammalian phophatidylinositol kinase-4 IIIβ is delivered to the initiation membrane site via ATG9-postitive vesicles. Production of PI4P by this kinase is critical for the early steps of autophagosome formation in mammals presumably by controlling the recruitment of ATG13, a member of the initiation complex, to the phagophore/isolation membrane (Judith et al., 2019). In mammals, PI4P also participates in latter stages of autophagy, notably by promoting autophagosome fusion with lysosomes (Wang et al., 2015).

In Arabidopsis, our results show that a total block in PI4P synthesis prevents early steps of autophagosome formation although our results suggest fundamental differences concerning the role and/or effector(s) of PI4P in the pathway compared to other organims. (1) Our data shows that blocking PI4P synthesis hampers ATG8 lipidation (Figure 17) yet plant KO mutants for the initiation complex (atg13) are not impaired in this process (Suttangkakul et al., 2011). This suggests that, in contrast to what was proposed in mammals, PI4P does not function by mediating the recruitment of ATG13. (2) Our results show that the absence of PI4P hampers the formation of autophagosome. As stated above, PI4P has been implicated in the trafficking of ATG9 vesicles in other organisms which is required for the expansion of the autophagic structures. In Arabidopsis, atg9 mutants are also affected in autophagy but, in contrast to other models, ATG8 lipidation is not prevented and atg9 mutants display abnormally large and tubular ATG8-labeled structures (Zhuang et al., 2017). This indicates that ATG9 in plants is dispensable for the expansion of autophagic structures, as well as for the lipidation of ATG8 and illustrates the major differences in phenotypes caused by the absence of ATG9 compared to the lack of PI4P reported in the present work. Further, in addition to the PI4P-regulation of ATG9 trafficking reported in yeast and mammals; ATG9 vesicles were shown to deliver TGNresident PI4KIIIβ to the site of phagophore expansion to promote AP formation (Judith et al., 2019). Here, our preliminary results show that mutants lacking the TGN-resident PI4Kβ are not affected in autophagy. Taken together, this suggest that, in Arabidopsis, PI4P functions prior to -and independent from-the implication of the ATG9 complex. Although we cannot exclude that PI4P also mediates ATG9-dependent mechanisms that were not uncover in this study where the sharp block in PI4P synthesis blocked autophagy at a very early stage. Additional studies, out of the scope of this work, focusing on the identification and functional characterization of PI4P-binding proteins found at autophagy structures should unravel the PI4P effectors involved in plant autophagy.

In sum, this study identified a critical component in autophagy in Arabidopsis: the lipid phosphatydilinositol-4-phosphate (PI4P). Our results show that PI4K activity is critical for autophagosome formation and ATG8 lipidation and that its absence causes a block in early stages of autophagy. Further, we found that autophagy structures form closely to the PM and that the PM-associated PI4Ka1 is required for proper autophagy flux suggesting an important contribution of the PM to plant autophagy. We speculate that PI4P mediates the recruitment of a yet-unidentified PI4P-binding protein(s) that work(s) upstream of ATG8 lipidation to support proper assembly/expansion of the phagophore (Figure 20). Altogether, our study provides fundamental insights into the mechanism of plant autophagy and acknowledges the critical roles that lipids play in that process. Autophagy is an important mechanism for plants adaptation to stressful environments; thus, having a better understanding of this process could contribute to deal with the adverse environmental changes that are expected to occur in the future. efficiency, we compared the expression of PI4Ka1 in each line compared to wild type upon treatment with 10µM β-oestradiol in various conditions using RT-qPCR. From over 30 lines tested, only one line, corresponding to amiRNA sequence 2 (see Table S2) showed a significant reduction in PI4Ka1 mRNA and was therefore selected for further experiments.

Growth conditions, autophagy induction and chemical treatments

Seeds were vernalized in water and darkness, at 4 °C, for 24 to 48 hours. Seeds were next surface sterilized in 10% bleach for 20 min, and sown on Murashige and Skoog (MS) agar medium plates (0.8% plant agar, 1% sucrose and 2.5 mM morpholinoethanesulfonic acid (Sigma), pH 5.7). Seeds were sown on a piece of mesh to facilitate root dissection from leaves.

Seedlings were grown vertically for 7 days, at 21 °C, under long-day conditions (16h-light/ 8hdark photoperiod, 300 µE m² s -1 ). To induce autophagy, seedlings were starved for nutrients by transfer from MS plates into liquid MS -NC medium (Murashige and Skoog liquid medium depleted for nitrogen and carbon: Murashige and Skoog micronutrient salts (Sigma, M0529), 3 mM CaCl2, 1.5mM MgSO4, 5 mM KCl, 1.25 mM KH2PO4, 0.5% (w/v) mannitol, 3 mM 2-(Nmorpholino)ethanesulphonic acid, pH 5.7), and incubated in darkness (wrapped in aluminium foil) for different times, as indicated in the figures. For rich conditions, seedlings were sampled directly from the MS plates or incubated in liquid Murashige and Skoog (MS) medium (1% sucrose and 2.5 mM morpholinoethanesulfonic acid (Sigma), pH 5.7 under normal light conditions. Chemical treatments were applied concomitantly with autophagy induction, as indicated in figures, as followed. To inhibit PI4kinase activity, seedlings were treated with PAO (Phenylarsine oxide, Sigma P3075) as previously described [START_REF] Simon | A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants[END_REF][START_REF] Vermeer | Imaging phosphatidylinositol 4-phosphate dynamics in living plant cells[END_REF] with concentration ranging from 7.5 µM, 15 µM , 30 µM or 60 µM and for different times as indicated in the figures. For TOR inhibition, AZD8055 1 µM (MedChemExpress) [START_REF] Pu | TOR-Dependent and -Independent Pathways Regulate Autophagy in Arabidopsis thaliana[END_REF] was used in liquid MS medium and for different times as indicated in figures.

Concanamycin A 1 µM (Sigma-Aldrich) was used as previously described (Shin, 2014) as a vacuole acidification inhibitor to accumulate autophagic bodies in the vacuole. For all chemical treatments, DMSO (dymethylsulfoxide, Sigma) was used as vector control (untreated plants) in the same conditions (volume, time) than the treated plants. To induce the expression of the artificial micro-RNA against PI4Kα1 (AT1G49340), seedlings were incubated for 24h with 10 µM β-estradiol (Sigma Aldrich). To test the impact of dowregulating PI4Kα1 (AT1G49340) on plant response to nutrient starvation (Figure 15 D), seedlings were grown for 6 days on full MS plates with sucrose prior to being transferred for 24h on full MS plates with sucrose supplemented with 10 µM β-estradiol to induce the expression of the miRNA. Seedlings were then transferred to a minimal media lacking sucrose and nitrogen (medium was prepared as in Gaude et al., 2007, without sucrose) supplemented with 10 µM β-estradiol for 8 days. Day 0 describes the day of transfer to -NC conditions; a picture was taken at D0 and D8. Then, seedlings were transferred back to full MS medium with sucrose for an additional 10 days; a picture was taken at D18.

Microscopy analysis

All observation were performed in on root epidermal cells of the elongation to early differentiation zone, employing 7-day-old seedlings mounted in culture medium with chemical treatments as indicated in the figures or in the following. Roots were kept under the microscope for no longer than 10 minutes in order to avoid secondary effects due to prolonged treatment times. Confocal images were acquired with a ZEISS LSM880 confocal system. For mCitrine, an Argon laser 488-nm was used for excitation, a dual-band dichroic mirror (488/561) was used for detection and the mCitrine signal was detected using band-pass filters of 409-696nm.

Laser excitation lines for the different fluorophores were 488 nm for GFP or mCitrine and 561 nm for tagRFP. Fluorescence emissions were detected at 409-696nm for mCitrine, 490-597 nm for GFP and 580-650 nm for tagRFP. In multi-labeling acquisitions, detection was in sequential line-scanning mode with a line average of 4. An oil-corrected × 63 objective, numerical aperture=1.4 was used. Confocal microscope images were processed using the Zen Black Software (Zeiss) for intensity optimization. High resolution Airyscan imaging of GFP and mCherry were performed using the ZEISS LSM880 confocal microscope equipped with a Plan-Apochromat 63×/1.4NA oil objective and a super-resolution AiryScan module with a band-pass filter 495-550nm and a long pass filter 570 nm.

Number of puncta, probe localization, cell number and surface areas were quantified using Zen Black Software (Zeiss) and Image J. Fluorescent puncta (ATG8, ATG18, ATG5, ATG4, PI4P probes) were counted manually on a minimum of 12 images per condition and per independent experiment (representing over 80 cells total in at least 3 independent roots). At least 3 independent experiments were performed for each analysis and statistical significance was assessed using Prism software. The p-value is indicated as following: *p-value<0.05; **p-value<0.01; ***p-value<0.001.

GFP-ATG8 assay

For the GFP-ATG8 assay, total proteins were extracted from dissected roots exclusively. Roots were frozen in liquid nitrogen, disrupted using a TissueLyser (Qiagen), homogenized in 2X Laemmli buffer (20 µL/mg of fresh weight) and clarified by two consecutive centrifugations at 3000 rpm for 1 min and 10 000 rpm for 10 min, respectively. Proteins were denaturated at 55 °C for 15 min. After migration on 12% SDS-PAGE (TGX Stain-Free FastCast Acrylamide kit, BioRad), equal protein quantity per lane was verified by stain-free activation of the loaded gel.

To assess the level of ATG8 lipidation, total lysates (TL), soluble (S100) and membrane fractions (P100) were prepared as described above. Equal amounts of proteins were loaded onto 13% SDS-PAGE gel containing urea (40% Acrylamide, 6M Urea). ATG8 and ATG8-PE were detected using an anti-ATG8 antibody (Agrisera, AS142811, 1/1000). Extracts from WT plants were compared to that of the atg5-1 mutant to clearly identify the ATG8-PE band, absent from the atg5-1 mutant, enabled to lipidate ATG8. ATG8 and ATG8-PE levels were quantified using PIP2.7 for the membrane fraction and UGP signals for the cytosolic fraction for normalization.

ATG8 lipidation was analyzed in dissected roots of 7 day old seedlings in experiments using PAO (Fig. 4) or in the ami:PI4Kα1 line (Figure S17). To assess the level of ATG8 lipidation in the pi4kβ1 pi4kβ2 mutant (Figure S17), whole seedlings of 8 days were used (because of reduced rates of growth in the mutant compared to WT which led to a reduced amount of available material).

To assess NBR1 levels, 7-day old seedlings were transferred from full MS plates into either rich MS liquid medium (+N), -NC liquid medium (-NC) or -NC liquid medium supplemented with 1µM ConcanamycinA for 16 hours for pi4kβ1 pi4kβ2 lines compared to WT. To test the impact of PI4Kα1 on NBR1 degradation, 6-day old seedlings of the ami:PI4Kα1 line, WT and atg5-1 mutant, were first transferred to full MS plates supplemented with 10µM β-estradiol for 24h prior to being tranfered to liquid +N, -NC, or -NC+concA for 16 hours, each supplemented with 10 µM β-estradiol. Upon treatments, proteins from total lysates were prepared was described above, loaded onto 10% SDS-PAGE and analysed by immunoblot using an anti-NBR1 antibody (Agrisera, AS142811, 1/5000) and a peroxidase-coupled Goat anti-Rabbit antibody as described above. For each sample, NBR1 transcripts were analysed in parallel by RT-qPCR as described in the next section.

RT-qPCR analysis

Total RNA was extracted using the RNeasy mini kit (Qiagen). To eliminate genomic DNA contamination, an additional DNase treatment was performed according to the RNeasy kit instruction with the RNase-free DNase set (Qiagen). One microgram of total RNA was reversetranscribed into cDNA in a 20 μL reaction mixture using the Superscript II reverse transcriptase enzyme (Invitrogen). cDNA levels were then analyzed using the iQ™ Sybr Green supermix (BioRad) on the iQ iCycler thermocycler (BioRad) with the gene-specific primers listed in Table S1. The thermocycling program consisted of one hold at 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. After completion of these cycles, melting-curve data were then collected to verify PCR specificity, contamination and the absence of primer dimers. As mentioned previously, autophagy relies on many membrane-remodeling events required for AP formation, notably to allow structure expansion but also to maintain the highly curved edges that characterize growing autophagic structures. In order to characterize the role of lipids in these events, we aimed at identifying lipid-related proteins (metabolic enzymes, lipid transfer proteins, etc.) that may be involved in autophagy. Prior to the beginning of my PhD work, A.

F GGGGACAAGTTTGTACAAAAAAGCAGGCTCTATCTTTGCTTGCTTGAAATTCGC R GGGGACCACTTTGTACAAGAAAGCTGGGTATCAAGCAACGGTAAGAGATCC F AGCATTTTCCCAGGCACCACAAC R TGCGACTTCTCCACCTCTCCAC F GAGGACCCAGACCGGAAGG R gacaaacacgacgaggatgc F CCGAGCAGCATGAAGATTAAG R CATACTCTGCCTTAGAGATCCACA F GTGTTGTAACGGCTTGAgca R tgtttgcatctttggtacgg F GGGGACAAGTTTGTACAAAAAAGCAGGCTCTATGAAGGCTTTATGTGATAGATTTGTTC R GGGGACCACTTTGTACAAGAAAGCTGGGTATCAGAGCATTTGCCAGTCATCTTCACG F AGGACGTAACCAGAGGGGTAG R CGTTGTGACCCGTCATTAATC F AAACCTCCTTATCTTCCGCTG R ATGAACGAAATTGGGTTCTCC tDNA F TGGTTCACGTAGTGGGCCATCG ATG4a AT2G44140
Bernard performed in silico analyses to compare the expression profiles of lipid-related genes (Lipid Gene Ontology) with that of ATG genes in various tissues, developmental stages and stress conditions associated with autophagy induction (nitrogen starvation, heat, dark and osmotic stress) using data from micro-arrays and RNA-seq experiments available at Arabidopsis databases (BAR, TAIR, Genevestigator). Gene co-expression analyses has been widely used for predicting gene functions because genes within modules of a co-expression network may be involved in common biological processes and exhibit similar biological functions. We thus reasoned that genes showing patterns of expression similar to that of ATG genes might be involved in autophagy.

These analyses notably identified two genes, AT4G19860 and AT3G03310 which were found in the first 100 genes co-expressed with at least 5-7 ATG genes upon abiotic stresses. AT4G19860 was found to co-express with ATG13a, ATG9, ATG18a,h and ATG8c,e,i. The expression of AT3G03310 also showed similarity with that of ATG13a, ATG9, ATG18a but not with ATG8s. The protein encoded by the AT4G19860 gene is annotated as a lecithin:cholesterol acyltransferase (LCAT). However, it was characterized as having a PLA2

activity by heterologous expression in yeast [START_REF] Zkscan3 Chen | Identification and characterization of an LCAT-like Arabidopsis thaliana gene encoding a novel phospholipase A[END_REF]. More precisely, PLA2 showed optimal activity at acidic pH (5.0), and had a head group preference for PC (G. [START_REF] Zkscan3 Chen | Identification and characterization of an LCAT-like Arabidopsis thaliana gene encoding a novel phospholipase A[END_REF]. Moreover, immunoprecipitation experiments performed in N. benthamiana identified the protein encoded by AT4G19860 as a potential physical interactor of ATG8 (Y. Dagdas, GMI Vienna, personal communication). The protein encoded by the AT3G03310 gene is also annotated as a lecithin:cholesterol acyltransferase (LCAT). However, it was described as having a phospholipase A1 activity, with an optimal activity at pH 6-6.5 and a preference for PC as a substrate [START_REF] Noiriel | Expression in yeast of a novel phospholipase A1 cDNA from Arabidopsis thaliana[END_REF]. Interestingly, both of these proteins display amino acid sequences atypical for PLAs: they were first annotated as LCAT, and do not share high similarities with other PLAs of Arabidopsis [START_REF] Noiriel | Expression in yeast of a novel phospholipase A1 cDNA from Arabidopsis thaliana[END_REF][START_REF] Zkscan3 Chen | Identification and characterization of an LCAT-like Arabidopsis thaliana gene encoding a novel phospholipase A[END_REF]) (Figure 21,A). Furthermore, these proteins display a 52% amino acid sequence identity indicating a potential redundancy of these proteins (Figure 21,B). From these results we hypothesized that AT4G19860 and AT3G03310 might be implicated in plant autophagy.

III-3.2 Phospholipases in biological membranes

The variety of membrane lipids' structure and chemical properties (conferred by the nature of their polar head and acyl chains) as well as their accumulation and distribution define the specific biophysical features of biological membranes. These notably include membrane electrostatics, packing, thickness or curvature, as well as its fluidity and plasticity which contributes to fusion or fission events (Bigay & Antonny, 2012). In addition to this, lipids actively contribute to membrane activity notably by mediating the recruitment of proteins to specific membrane domains based on protein-lipid interactions (Jouhet, 2013). In particular, polar heads greatly differ from one lipid class to another, and this is directly reflected in the geometry and organization of the membranes they are present in. When present in membranes, cylindrical-shaped lipids promote the formation of flat lipid bilayers, conical lipids promote positively curved membranes, and inversed-cone lipids promote negative curvatures to membranes (Figure 22, A-B) (Bigay & Antonny, 2012). Membrane curvature is thus determined by asymmetric lipid composition of both bilayers, as well as physical forces that can be exerted by membrane-bound proteins. Thus, proteins modifying lipid geometry can have a great impact on the membrane curvature of membranes. Phospholipase A activity produces lyso-phospholipids and free fatty acids. Lysophospholipids, the final products of the phospholipase, are known to induce positive curvatures when they accumulate in membranes [START_REF] Furt | Lipids of the plant plasma membrane[END_REF]. In this manner, PLs are important actors in membrane remodeling and thus actively contribute to the maintenance of membrane lipid homeostasis and stability [START_REF] Takáč | Recent advances in the cellular and developmental biology of phospholipases in plants[END_REF]. For example, PLAs were shown to control various plant cellular and developmental processes, including cell elongation, organization of the endomembrane system of pollen grains as well as protein trafficking by regulating critical membrane properties. Yet little is still known about the functions of PLAs in Arabidopsis and notably about AT4G19860 and AT3G03310 which belong to an 'atypical' class of PLAs (Figure 21,A)

In that overall context, we postulated that the phospholipases PLA1 and PLA2 encoded by AT3G03310 and AT4G19860, respectively, might participate in autophagy by mediating lipid/membrane remodeling during AP formation. In the following text, AT4G19860 will be referred to as PLA2 while AT3G03310 will be referred to as PLA1. We speculated that if PLA1 or PLA2 were implicated in the autophagic process, they would localize to autophagic structures. To test this hypothesis, we created C-terminal fluorescenttagged versions of these proteins under the control of their endogenous promoter or the UBQ10 promoter. For PLA2, we created versions tagged with 2 different fluorochromes:

III

tagBFP and tagRFP. For PLA1, we created a version tagged with tagRFP. We then generated a set of stable transgenic lines expressing PLA1-tagged or PLA2-tagged either in WT background or in plants expressing GFP-ATG8a.

To address if PLA1 or PLA2 is recruited to autophagic structures, we used these sets of transgenic lines to perform confocal microscopy on seedlings in nutrient deprived conditions (-NC) to induce autophagy. As previously shown, GFP-ATG8a signal is found in the cytosol as well as on punctae scattered in the cell which correspond to autophagic structures (phagophores, APs) (Figure 23). PLA1-tagRFP displayed a cytosolic signal, and also seems to be recruited to punctae-like structures (Figure 23), but these structures where distinct than that of GFP-ATG8a, suggesting that these are not GFP-ATG8a-labelled autophagic structures.

In the same manner, we performed confocal microscopy on GFP-ATG8a x UBQ10p:PLA2-tagRFP plantlets in nutrient rich (+NC) as well as nutrient deprived conditions (-NC) to induce autophagy. We favored the use of this line, as the signal using PLA2-RFP under its endogenous promoter was too low to be detected. In both conditions, the two proteins (ATG8a and PLA2)

were observed on punctae within the cytosol at which they co-localized (Figure 24, A, +NC, -NC) suggesting that PLA2 is present on autophagy structures. Further, when plants were placed in nutrient deprived conditions (-NC) supplemented with Concanamycin A (1 µM), a treatment that inhibits the vacuolar ATPase, de-acidifiying the vacuole and inactivating vacuolar proteases, resulting in the accumulation of undegraded autophagic structures (Yoshimoto et al., 2004), we observed a drastic increase in the number of GFP-ATG8a and PLA2-RFP punctate structures inside the vacuole (Figure 24, A, -NC + cA). PLA2-RFP puncta co-localized with the GFP-ATG8a signal in the vacuole indicating that PLA2 is present on ABs and suggesting that like ATG8, PLA2 remains present in autophagic structures until their delivery to the vacuole. In fact, while we observed that both GFP-ATG8a and PLA2-RFP were highly present in the cytosol in +NC conditions (Figure 24, A, +NC), when autophagy was induced for 3 hours (-NC) we observed a shift of the bulk of the PLA2-RFP signal from the cytosol to the vacuolar lumen (Figure 24, A, -NC). Note that similar results were recapitulated with a line expressing PLA2-tagBFP under the control of the UBQ10 promoter in the wild-type background. In this line, upon transfer to nutrient deprived conditions (-NC) supplemented with concanamycin A (1 µM), PLA2 displayed a vacuolar signal and was also seen in ring-like structures within the vacuole indicating its presence on ABs (Figure 24,B).

APs biogenesis is a process that depends on the activity of Pi3 Kinases (Shin et al., 2014). In order to investigate whether PLA2-associated dot-like structures are genuine autophagy structures, we tested if their formation also depended on the activity of PI3K. We placed plants in nutrient deprived conditions (-NC) to induce autophagy and supplemented with Wortmannin (30 µM), a well characterized PI3K inhibitor (Shin et al., 2014). In these conditions, no APs are formed (Figure 24, A, Wm), as it has previously been reported (Shin et al., 2014). In addition, we observed very little to no PLA2 puncta in the cytosol, supporting the idea that PLA2 indeed localizes on autophagic structures. Further, the vacuolar localization of PLA2 was completely abolished by the addition of Wortmannin and, instead, the PLA2 signal was found exclusively in the cytosol. This suggest that the vacuolar localization of PLA2 is mediated by autophagy.

Together, these data show that PLA2, but not PLA1, localizes to both the cytosol and APs all the way into the vacuole; upon autophagy induction, PLA2 rapidly and almost entirely re-localizes from the cytosol to the vacuolar lumen and this seems to be dependent on autophagy as inhibiting AP formation prevents this re-localization. These data therefore suggest that PLA2 may either be degraded or 'transported' to the vacuole by autophagy.

3-2 Expression profile of PLA2

Our results suggest that PLA2 is present on autophagic structures and is rapidly transported into the vacuole upon autophagy inducing conditions. Autophagy mediates the degradation of cytosolic material in response to various stresses. In that context, and given the localization of PLA2 in the vacuole, it could be postulated that PLA2 is targeted by autophagy to be degraded upon nutrient starvation. However, this seems counter-intuitive with the fact that microarray data showed PLA2 co-expressed with ATG genes. We thus wanted to double check these large-scale data by analyzing the pattern of expression of PLA2 alongside that of ATG genes.

In plants, ATG genes expression is induced in nutrient deprivation conditions as well as in senescing organs (Yang & Bassham, 2015). In order to characterize the expression of PLA2 compared to that of ATG genes, we placed wild type plants (WT) plants in liquid nutrient rich (+NC) or nutrient starvation conditions (-NC) to induce autophagy and performed RT-qPCR analysis with samples collected at 3 h, 6 h or 16 h after the beginning of the treatment.

We compared AT4G19860 gene expression to that of ATG8 (a, c, e and i), ATG9 and ATG18

(a and h). Results from these experiments showed that the expression of most ATG genes was induced in nutrient starvation (-) conditions when compared to nutrient rich conditions (+) (Figure 25,A). Furthermore, AT4G19860 gene expression was also induced in nutrient starvation conditions compared to nutrient rich conditions (Figure 25,A). Furthermore, results from the Master 2 internship of J. Castets in the team, showed that an up-regulation of PLA2 expression in leaves during plant aging (Figure 25,B). This indicates that, similarly to that of ATG genes, the expression of PLA2 is upregulated in experimental or developmental conditions where autophagy is induced. These results corroborate our in silico analyses and favors the hypothesis that PLA2 is implicated in the progression of autophagy rather than being solely a cargo for autophagy degradation.

3-3 Function of PLA2 in autophagy and plant physiology

In order to test the implication of PLA2 in autophagy, we generated a toolbox composed of genetic lines KO or KD for PLA2.

First, we identified and ordered two tDNA insertional lines available for AT4G19860:

Salk_147672 and Salk_061713 (Figure 26,A). Additionally, we collected a tDNA insertional line for AT3G03310 (PLA1): SALK_025488. Genotyping of Salk_147672 confirmed the presence of the tDNA insertion within the AT4G19860 coding sequence (Figure 26,B).

Furthermore, RT-semi-qPCR analysis performed on Salk_147672 showed no expression of the full length AT4G19860 gene (Figure 26,C). These results indicate that Salk_147672 (hereafter called pla2-1) is indeed a knock-out line for AT4G19860. Interestingly, pla2-1 plants do not display any visible growth or developmental defects in regular conditions (data not shown). RT-semi-qPCR analysis of the other tDNA insertion line, Salk_061713, showed no differences in the gene expression of AT4G19860 compared to WT plants indicating that this line is not a knockout line, and was therefore not pursued further (data not shown).

Second, in order to avoid possible pleiotropic effects due to the implication of PLA2 in other cellular functions that autophagy, or a potential functional redundancy by an alternative mechanism that might compensate for the knockout of PLA2, we designed artificial micro RNA (miRNA) sequence directed against AT4G19860 and cloned it under the control of a β-estradiol inducible promoter [START_REF] Curtis | A Gateway Cloning Vector Set for High-Throughput Functional Analysis of Genes in Planta[END_REF]. These amiRNA::AT4G19860 constructs are a powerful tool as they allow to modulate gene extinction and would thus be utilized to diminish gene expression only and precisely before autophagy induction.

In order to study the implication of AT4G19860 in autophagy, we crossed the pla2-1 line with plants expressing the autophagy marker 35S::GFP-ATG8a (Thompson et al., 2005).

In the same manner, we transformed our amiRNA::AT4G19860 construct into 35S::GFP-ATG8a expressing lines. Using RT-qPCR analyses, we showed that, in these lines, the expression of AT4G19860 decreases by around 90% after 3 h of β-estradiol treatment, indicating that they are indeed functional knockdown lines for AT4G19860 (Figure 26,D).

These transgenic lines will be used in the future to assay the impacts of AT4G19860 knock-out or knock-down on plant autophagy by (1) look if these plants display an impact on their autophagic flux by performing the GFP-ATG8a assay, (2) if they display any differences in the size, number or shape of autophagic structures compared to WT plants using confocal microscopy. Moreover, we will measure the impact of KO for PLA2 on the plant lipid composition and assay how these plants respond to environmental stress to know if plant physiology is affected in these mutant backgrounds.

III-3.4 Conclusions and perspectives

In this part of the thesis, we identified two candidate genes which we found co-regulated with ATG genes in the Arabidopsis databases: AT4G19860 (PLA2) and AT3G0331 (PLA1).

Furthermore, immunoprecipitation experiments performed in N. Benthamiana by our collaborators suggested that PLA2 physically interacts with ATG8a central protein for autophagy (Y Dagdas lab, personal communication). We speculated that if the proteins encoded by these genes were implicated in plant autophagy, these proteins could be recruited to autophagic structures in autophagy inducing conditions. Confocal microscopy analysis showed that PLA1 did not co-localize with autophagic structures (Figure 23). We thus did not continue to further investigate its implication in plant autophagy. However, PLA1 was observed at dot-like structures, indicating a possible implication in another pathway of the endomembrane system (localization to other endomembrane organelles such as endosomes or TGN vesicles). Contrary to PLA1, PLA2 co-localizes with autophagic structures (Figure 24,A). Interestingly, PLA2 co-localized with autophagic structures in both nutrient rich as well as nutrient starvation conditions, indicating a possible implication in basal autophagy as well as nutrient starvation-induced autophagy. Furthermore, PLA2 also displayed a large relocalization from the cytosol to the vacuolar lumen upon nutrient starvation (Figure 24, A, -NC). Our results also showed that PLA2 co-localized with autophagic structures within the vacuolar lumen upon concanamycin A (1 µM) treatment, indicating that, unlike most ATG proteins which are only transiently recruited to autophagic structures (Koyama-Honda et al., 2013), PLA2 is escorted to the vacuole by APs. Finally, we observed that upon PI3K inhibition by Wortmannin, APs failed to form as expected (Figure 24, A, Wm). Interestingly, PLA2 vacuolar localization was also lost in these conditions (Figure 24, A, Wm). Taken together, our results indicate that PLA2 is recruited to APs in both nutrient rich and nutrient starvation conditions, as well as displaying a vacuolar localization.

What could be the function of PLA2 upon autophagy, what is the physiological relevance of its interaction with the autophagy machinery?

Based on our localization results, it could be suggested that PLA2 is degraded by autophagy upon nutrient starvation. The interaction between PLA2 and ATG8 could mediate the recruitment and engulfment of the protein inside APs for degradation. This mechanism has been previously shown for other proteins to selectively lower their level upon specific signaling and thus modify plant metabolism to mediate plant-stress adaptation (see section I-2, implication of autophagy in protein degradation). Therefore, one hypothesis is that autophagy help clear the cell from PLA2 to limit its activity upon nutrient starvation or other stresses.

Yet, this hypothesis does not fit well with our data, in particular, we found that the expression of PLA2 is in fact upregulated upon autophagy-inducing conditions which seems opposite to the cell wanting to have less of the protein. Therefore, another hypothesis for the function of PLA2 in relation to autophagy could be that PLA2 participate in the machinery of autophagy by controlling lipid remodeling at the AP membrane. Initially, we thought that, by producing lysophospholipids, PLA2 might support the membrane curvature of the phagophore. This hypothesis will be tested using transgenic lines (KO and KD) looking at number size, shape of autophagy structures using confocal microscopy and Correlative Light Electron Microscopy (CLEM) analyses. Another interesting hypothesis that we currently favor is that PLA2 may be involved in the turn-over of autophagy membranes inside the vacuole. Several lines of evidence support this hypothesis: (1) The fact that this protein was characterized as being most active in acidic pH conditions (pH 5.0) when expressed in yeast [START_REF] Zkscan3 Chen | Identification and characterization of an LCAT-like Arabidopsis thaliana gene encoding a novel phospholipase A[END_REF];

(2) the similarity of PLA2 with the human lysosomal PLA2 which is involved in the turn-over of macro-molecules inside the lysosome [START_REF] Shayman | The Arabidopsis pi4kIIIβ1β2 double mutant is salicylic acid-overaccumulating: A new example of salicylic acid influence on plant stature[END_REF]; (3) our results showing a relocalization of the protein from the cytosol to the vacuole upon autophagy induction. The loss of PLA2 vacuolar signal upon Wortmannin treatment (Figure 24, A, Wm) suggests that PLA2 relies on autophagy in order to re-localize to the vacuole. One possibility is that PLA2 is recruited to autophagic structures as a mean to re-localize to the vacuole, where it is most active, to participate in the degradation/remobilization of the autophagic cargo within the vacuole, predominantly upon autophagy-induced conditions. Note that we only tested nutrient starvation but whether this occurs upon additional stresses need to be tested. In that context, PLA2 could be trafficked, together with its substrate (i.e., AP membranes), inside the vacuole to support a very fine-tuning of lipid turn-over during autophagy. Such trafficking resembles the cytoplasm-to-vacuole targeting (Cvt) pathway which in yeast delivers vacuolar proteases to their site of action [START_REF] Klionsky | Aminopeptidase I of Saccharomyces cerevisiae Is Localized to the Vacuole Independent of the Secretory Pathway[END_REF]. In plants, an ER to vacuole trafficking mediated by autophagy was proposed, notably to transport seed storage proteins (Michaeli et al., 2014).

(1) To test the putative role of PLA2 in the degradation of autophagic cargo inside the vacuole, we could assay whether the vacuolar degradation of ABs is hampered in the pla2-1 KO or enhanced in PLA2 overexpressing lines that we are currently generating. Notably, we could test autophagy degradation in three types of plants: GFP-ATG8a x wild type, GFP-ATG8a x pla2-1 (KO) and GFP-ATG8a x PLA2 OE (overexpressor). Additionally, we will use our KD line as well as a double mutant between PLA2 and PLA1. Although our preliminary analyses indicate that this protein does not co-localize with ATG8, it could compensate for the absence of PLA in the mutant pla2-1. To measure autophagic degradation, we could use the GFP-ATG8a degradation assay which reflects the autophagic flux, and thus autophagic degradation. If PLA2 is indeed important for autophagic degradation, we would observe a decrease I the autophagic flux in GFP-ATG8a x pla2-1 plants compared to GFP-ATG8a x wild type PLA2 plants. In this same manner, the autophagic flux of GFP-ATG8a x PLA2 OE plants would be enhanced compared to that of GFP-ATG8a x wild type PLA2, and harshly increased compared to GFP-ATG8a x pla2-1 plants. The team is currently working on the production of such lines in order to test these possibilities. Additionally, performing transmission electron microscopy (TEM) analyses in these lines will inform on the potential accumulation of AB in the vacuole of the pla2-1 plants. Furthermore, it would be interesting to determine the in vivo activity of PLA2: what kind of lipids does it use as substrate? When expressed in yeast, it was shown to preferentially use PC, but was able to also hydrolyze other phospholipids such as PA, PE and PS. Interestingly, PC is a major lipid in the composition of AP in yeast (Schütter et al., 2020). At this time, the lipid composition of AP membranes in plants is still unknown; testing the activity of PLA2 in planta could give us valuable information about the nature of the lipids composing and regulating AP formation in plants. In addition to our Arabidopsis transgenic lines, we therefore plan to transiently express PLA2 by agroinfiltrating N. benthamiana leaves and assay the impact of overexpressing PLA2 on the plants lipidome.

(2) To test the implication of the autophagy machinery in PLA2 trafficking to the vacuole, we could assess whether the vacuolar localization of PLA2 is lost in atg mutants. Further, our team is currently collaborating with the Dagdas lab to identify and mutate the residues involved in PLA2-ATG8 interaction (preliminary analyses identified a putative AIM motif). Further work will also involve complementation assays with mutated version of PLA2 unable to interact with ATG8 to test the functional relevance of PLA2-ATG8 interaction in PLA2 trafficking, autophagy and plant physiology.

In conclusion, this PLA2 protein seems indeed implicated in the autophagic process, and results suggest that it might be implicated in the degradation of autophagic cargo inside the vacuole. Further experiments to decipher its functions are in preparation and these will provide important answers concerning the exact role of this protein in plant autophagy. How ABs are degraded within the vacuole in plants is not well known, yet this is a critical step as it represents the primary purpose of autophagy: degradation and recycling of macromolecules to support basal metabolism.

IV -GENERAL

DISCUSSION AND PERSPECTIVES

results suggest that all lipids do not have the same impact on autophagy activity, rather, they seem to have specific roles and functions in the process. This highlights our initial hypothesis that lipids are not just mere building blocks for the formation of autophagic membranes but rather that some lipids have specific functions in the autophagy pathway. Further, results from this screen allowed us to unravel interesting lipids that seem to be important for plant autophagy such as PA or glucosylceramides as potential candidates for further functional analyses. Notably, we found very interesting that multiple treatments to inhibit the PA, resulted in different types of autophagic responses (Figure 27). This could be explained by the existence of multiple PA pools within the cell [START_REF] Simon | A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants[END_REF], probably each of them having distinct roles towards autophagy. The anionic phospholipid PA has been described as an important second messenger in eukaryotic organisms [START_REF] Pokotylo | The phosphatidic acid paradox: Too many actions for one molecule class? Lessons from plants[END_REF]. In plants, PA has been implicated in plants response to multiple abiotic stresses (Li et al., 2019;[START_REF] Mcloughlin | Identification of novel candidate phosphatidic acid-binding proteins involved in the salt-stress response of Arabidopsis thaliana roots[END_REF] and thus PA could play important roles in the activation of autophagy when plants face adverse environmental conditions.

Finally, although the autophagic process is conserved in eukaryotic organisms, important differences exist. For example, some ATG proteins display different functions in plants compared to their mammalian or yeast counterparts. It is the case of ATG9 which, in yeast and mammals, seems to play key roles in the initial steps of AP formation by providing membrane seeds for the initial membrane (Kakuta et al., 2012;Mari et al., 2010); while in plants, atg9 Arabidopsis mutants were seen to form abnormally large ATG8-labeled autophagic structures, indicating a role in the shaping of these structures and thus in later steps of the process (Zhuang et al., 2017). Furthermore, the nature of lipids differs greatly from one organism to another. For example, mammalian cells mainly produce one type of sterols (i.e., Cholesterol), while plants cells contain a great diversity of phytosterols. Adding to this, the lipid composition of organelles membranes differs greatly between organisms.

For example, the anionic phospholipid PI4P is mainly accumulated in the PM of plants [START_REF] Simon | A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants[END_REF], while in animals, PI4P is mainly accumulated at the TGN [START_REF] Hammond | A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi[END_REF]. This indicates that lipids have distinct functions in different organisms. In this manner, in our screen we obtained results suggesting fundamental differences in plant autophagy compared to that of other organisms such as mammalian autophagy. For instance, inhibition of PI(3,5)P2 biosynthesis did not affect the autophagic flux in our conditions, while in mammals this lipid seems to play important roles in the mechanism of AP fusion with lysosomes. Therefore, the function of peculiar lipids in autophagy is not identical in all organisms and thus one should be very prudent when extrapolating results.

Gene co-expression analyses of lipid-related genes lead to the identification of the AT4G19860 gene which pattern of expression resembled that of ATG genes. AT4G19860 encodes a protein with a phospholipase A2 activity (PLA2), with an optimal activity at pH=5.0 and a preference for PC as a substrate [START_REF] Zkscan3 Chen | Identification and characterization of an LCAT-like Arabidopsis thaliana gene encoding a novel phospholipase A[END_REF]. We hypothesized that PLA2 could be implicated in the remodeling of autophagic membranes during AP formation. Our results showed that the expression of PLA2 follows a pattern similar to that of ATG genes as we found it to be induced in nutrient starvation conditions as well as in senescing organs (Figure 25, A-B). Moreover, PLA2 was found to localize to ATG8-postitive dot-like structures in nutrient rich and in nutrient starvation conditions indicating that PLA2 is present in autophagic structures (Figure 24,A). PLA2 shows a vacuolar signal and we found that its vacuolar localization is likely dependent on the autophagic pathway as it was abolished in autophagyinhibition conditions (PI3K inhibition by Wortmannin) (Figure 24,A). Taken together, my results suggest that PLA2 is implicated in autophagy. The fact that PLA2 localized to autophagic structures and displayed a vacuolar localization in our experiments (Figure 24,A) in addition to the fact that it was characterized as having an optimal activity at an acidic pH (5.0) lead us to hypothesize that PLA2 might be utilizing the autophagic pathway to relocalize to the vacuole where it is most active. From our results, we speculate that PLA2 might be implicated in the degradation of membrane lipids of ABs within the vacuole, and as such, contribute to the ultimate goal of the autophagic pathway, which is to degrade cytoplasmic material for recycling. Alternatively, and/or additionally, PLA2 might be implicated in lipid remodeling during AP formation, by providing conical shaped phospholipids (Figure 28).

Further analysis to determine the number, size, and morphology of APs in the pla2 mutant background will address these hypotheses and provide valuable information about the function of this protein. PLA2 activity results in the formation of lysophospholipids (Figure 28).

It is important to note that in our lipid inhibitor screen, inhibiting lysophospholipid acyltransferase activity (LPCAT) (Ci-976 inhibitor), resulted in a slight reduction of the autophagic flux (Figure 27; Figure 12,C). Further experiments will allow to determine if this decrease is significant (not statistically significant with our current set of data, although a decrease is clearly observed). If these results confirm a decrease in the autophagic flux they would indicate that lysophosphatidyl acyltransferases (that use lysophosphatidic acid or other lysophospholipids as substrates) might be implicated in plant autophagy. Interestingly, a balance in the activity of PLs (which remove acyl chains from phospholipids forming lysophospholipids) and acyltransferases (which add a novel acyl chain to lysophospholipids generating a new phospholipid) is key to support lipid remodeling in membranes (Figure 28).

Phospholipid remodeling mediated by the combined action of PLA2 and LPCAT leads to modifications of phospholipid acyl chains and thus results in changes in lipid shape (Figure 28). As such, phospholipid remodeling enzymes participate in the shaping of the endomembrane system with implication in membrane deformation in either tubules or vesicles [START_REF] Ha | Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase y sorting in Saccharomyces cerevisiae[END_REF]. Numerous membrane remodeling events happening at the phagophore are critical for autophagosome formation: notably the nucleation of the initial structure, its shaping to a cup-like structure with a highly curved rim, expansion while keeping structure and function and finally the fusion of the phagophore rim-to a complete double membrane vesicle (Carlsson & Simonsen, 2015). All of these events imply fast and coordinated changes in the phagophore membrane structure during the progression of autophagy for which the actors are still largely unknown, particularly in plants. In that context, it is tempting to speculate that the balance between PLA2 and LPCAT could play a role in autophagy. Future work will aim at exploring these hypotheses.

The lipid inhibitor screen, as well as our co-expression analyses provided lipid and protein candidates potentially implicated in plant autophagy. Future experiments will confirm their implication as well as determine their role in the autophagic pathway. It is noteworthy that the function of these lipids and/or proteins in endomembrane related processes other (or additional to) than autophagy could indirectly result in modifications of the autophagic pathway. In order to directly identify and functionally explore lipids/lipid-related proteins that actually compose the autophagy membrane it would be crucial to characterize the molecular composition of autophagic structures. For this, the purification of autophagic membranes followed by lipidomic/proteomic analysis will provide valuable information and allow to identify players acting directly in autophagic structures.

Finally, our lipid inhibitor screen allowed us to identify PI4P as a critical lipid for plant autophagy. In its absence (in PI4K inhibiting conditions) we observed a severe block in autophagic flux (Figure 27, Figure 14). We thus investigated in more details how PI4P regulates autophagy in plant cells.

IV-2 PI4P is critical for plant autophagy

Our results suggest that PI4P synthesis is required for autophagy. Inhibition of PI4K activity led to a large block in the autophagic flux and to a complete block in AP formation. Moreover, our results indicate that PI4P is present in autophagic structures, indicating an important role of this lipid for autophagy in plant cells. These results raise multiple interesting questions in regards to the origin as well as the role of PI4P required for plant autophagy.

IV-2.1 Origin of PI4P required for autophagy?

AP biogenesis occurs de novo and is governed by the sequential recruitment of a group of dedicated proteins part of the core ATG machinery. Multiple membrane sources have been proposed to serve as seeds for the nucleation of the initial membrane in yeast or mammals.

Additionally, multiple organelles have been proposed to serve as a platform for the assembly of the phagophore and/or to provide membranes to support phagophore expansion. These membrane sources involve multiple organelles as well as membrane contacts between organelles notably the ER-Golgi intermediate compartment (ERGIC). Indeed, multiple type of vesicles deriving from these compartments such as the COPII vesicles have been observed

to coalesce with ATG9 vesicles (also post-Golgi) at the site of AP formation in mammals (Graef et al., 2013;Mari et al., 2010;Orsi et al., 2012;Yamamoto et al., 2012). Interestingly, the lipid PI4P has been linked with the trafficking of ATG9 vesicles in yeast and in mammalian cells. In yeast, the trafficking of Atg9 vesicles requires the activity of the PI4K Pik1p (Wang et al., 2012).

In mammals, ATG9 vesicles were found to deliver the PI4KIIIβ to the site of AP formation (Judith et al., 2019).

In Arabidopsis, the site of AP biogenesis seems to localize in close proximity to the ER.

The early ATG marker ATG5 was found in close proximity to specific cup-shaped subdomains of the ER (Le Bars et al., 2014). However, a possible contribution of the PM to the formation of AP has also been suggested. The TPLATE Complex (TPC) is a plant-specific internalization complex located at the PM which regulates clathrin mediated endocytosis [START_REF] Gadeyne | The TPLATE Adaptor Complex Drives Clathrin-Mediated Endocytosis in Plants[END_REF]. Two proteins part of the TPC, AtEH1/Pan1 and AtEH2/Pan1, were found to co-localize with AP and knocking down these genes lead to a reduced number of APs ( Wang et al., 2019).

VAP27-1 is a protein that, along with the actin cytoskeleton, is implicated in the formation of ER-PM contact sites (EPCS) [START_REF] Wang | The plant cytoskeleton, NET3C, and VAP27 mediate the link between the plasma membrane and endoplasmic reticulum[END_REF]. Interestingly, AtEH1/Pan1 and AtEH2/Pan1labelled APs were found to also co-localize with VAP27-1-positive regions, which correspond to EPCS (Wang et al., 2019). In mammals and in yeast, many lipid synthesizing enzymes accumulate in these EPCS, thus, one of the key functions of EPCS in these organisms is to regulate local lipid composition to allow multiple cellular functions like intracellular Ca2+ dynamics or notably, lipid traffic and homeostasis [START_REF] Lees | Regulator and substrate Dual roles for the ATG1-ATG13 kinase complex during autophagic recycling in Arabidopsis[END_REF]Saheki et al., 2013).

Interestingly, EPCS have been shown to recruit proteins that mediate phosphoinositide interconversion like the yeast Sac1 phosphatase [START_REF] Sohn | PI(4,5)P2 controls plasma membrane PI4P and PS levels via ORP5/8 recruitment to ER-PM contact sites[END_REF]. As mentioned before, phosphoinositides play key roles in AP formation. Thus, EPCS being a hub for rapid phosphoinositide interconversion represent a potential good candidate for autophagosome formation. Moreover, EPCS have been shown to regulate PC synthesis in yeast [START_REF] Tavassoli | Plasma membrane -Endoplasmic reticulum contact sites regulate phosphatidylcholine synthesis[END_REF]. Interestingly, PC synthesis on nascent APs has been recently reported in mammalian cells (Andrejeva et al., 2020).

The fact that EPCS accumulate lipid synthesizing enzymes and are thus capable of modifying its local lipid composition make it interesting structures for sustaining the multiple membrane dynamic events required in the formation of APs. Indeed, the ER-PM tethering extended synaptotagmins (E-Syts) proteins have been shown to be directly involved in the formation of APs in mammals. Notably, these E-Syts are implicated in the targeting of the PI3KC complex to the site of AP formation upon autophagy induction [START_REF] Molino | ER-driven membrane contact sites: Evolutionary conserved machineries for stress response and autophagy regulation?[END_REF], a known crucial step in the autophagosome biogenesis process (Nascimbeni et al., 2017b).

Although plant EPCS are not as well characterized as its mammalian or yeast counterparts, most of these lipid enzymes are conserved, and thus a possible accumulation of these type of enzymes in plant EPCS is possible. In Arabidopsis, three types of EPCS have been described, one for each of the tethering enzymes implicated in their formation: the S-EPCS, stabilized by synaptotagmins (SYTs), V-EPCS, maked by the vesicle-associated membrane protein 27 (VAP27), and plasmodesmata (PD)-EPCS, enriched in multiple C2 and transmembrane domain containing proteins (MCTPS) [START_REF] Noack | Functions of Anionic Lipids in Plants[END_REF]. Interestingly, SYTs and MCTPs have been shown to interact with PI4P [START_REF] Brault | Multiple C2 domains and transmembrane region proteins ( MCTP s) tether membranes at plasmodesmata[END_REF][START_REF] Pérez-Sancho | The arabidopsis synaptotagmin1 is enriched in endoplasmic reticulum-plasma membrane contact sites and confers cellular resistance to mechanical stresses[END_REF]. This indicates an active implication of PI4P in the establishment of plant EPCS by contributing to the stabilization of the different tethering proteins that establish such EPCS. EPCS therefore represent good potential candidates for participating in AP biogenesis in Arabidopsis. In that context, it is possible that the role of PI4P in plant autophagy is to support the establishment of such EPCS that would be required for phagophore assembly/expansion. Future studies addressing the localization of APs compared to that of plant EPCS marker proteins would allow to answer if such contact sites are indeed implicated in AP biogenesis.

Is there PI4P in the membrane of autophagosomes?

Our results indicate that (1) PI4K activity is required for the formation of APs (Figure 16),

PI4P is observed on ABs (Figure 19, A-B), (3) a significant amount of APs seem to be tightly constrained against the PM (Figure 19, A-B). This indicates that PI4P is either synthesized directly at autophagic membranes or that PI4P is transferred from a PI4P-containing compartment serving as membrane source for AP formation. Alternatively, PI4P may be critical for phagophore assembly without being present directly at the phagophore membrane but by supporting contacts between phagophore/ER/PM that would be required for phagophore expansion as mentioned above. This would explain why we rarely saw co-localization events of autophagic structures with PI4P in the cytoplasm (Figure 19, A-B). However, we did observe numerous co-localization between PI4P biosensors and autophagic structures within the vacuole (Concanamycin A conditions, Figure 19, A-B), indicating that PI4P is present in ABs.

In other organisms, PI4P has been implicated in the final steps of autophagy as to allow the fusion between APs and lysosomes (Wang et al., 2015;Zhang et al., 2020). APs have been shown to interact/fuse with the PI4P-containing endosomes in other organisms (Puri et al., 2013). It has been proposed that during the maturation of APs, fusion or interaction with other PI4P-containing endosomal compartments mediates the transfer of PI4P into APs required for their subsequent fusion with the vacuole. In plants, interactions between APs and endosomes have been reported (Gao et al., 2015), and thus the PI4P present in ABs we observed might be reminiscent of a transfer of PI4P from endosomes to APs in the late stages of the maturation step.

In fact, freeze-fracture electron microscopy techniques have confirmed the presence of PI4P in APs in yeast and mammalian organisms (Kurokawa et al., 2019[START_REF] Kurokawa | Phosphatidylinositol 4-phosphate on Rab7-positive autophagosomes revealed by the freeze-fracture replica labeling[END_REF]. Note that our observation of the absence of PI4P biosensor signal in cytosolic autophagic structures does not absolutely mean that there is no PI4P in these membranes. Lipid biosensors, have proved very valuable to visualize the subcellular localization of lipids in vivo, but they also introduce some bias. First, these biosensors compete with endogenous proteins for binding, which means that the signal observed at a membrane only reflects the pool of "free" lipids. Second, the lipid-binding domains with which they are composed can rely on coincidence binding, and thus, such probes might bind to the lipid only in the presence of a protein partner that alters its conformation to allow the interaction with the lipid. Additionally, if the concentration of PI4P in autophagic structures is rather low, it may not efficiently recruit PI4P-probes which are more likely to be titrated in PI4P-enriched compartments (notably the PM where it massively accumulates, or TNG, see below). To be sure about the presence of PI4P on autophagic membranes, one alternative would be to assay the presence of PI4P on purified phagophore/AP structures in lipidomic analysis. Notably, the novel LC-MS-MS methods in development in the lab will allow for a fine characterization of the phosphoinositide composition of autophagic membranes. Alternatively, the presence of PI4P in autophagic structures could be detected by use of electron microscopy techniques using next-generation labelling to specifically target PI4P. Our team is also currently developing such approaches to confirm the presence of PI4P in autophagic structures.

Is the plasma membrane providing for the PI4P required for plant autophagy?

PI4P localization in yeast, mammals and plants and their implication in autophagy.

The development of lipid biosensors has allowed to visualize the subcellular distributions of multiple anionic phospholipids, including PI4P in various organisms. In yeast for example, the main pool of PI4P is present at the TGN, with a minor pool localized at the PM [START_REF] Roy | Multiple pools of phosphatidylinositol 4-phosphate detected using the pleckstrin homology domain of Osh2p[END_REF]. In mammals, PI4P labels the PM, the Golgi/TGN, as well as Rab7-positive compartments that correspond to endosomes/lysosomes, and PI4P seems to be evenly distributed between these three compartments [START_REF] Hammond | A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi[END_REF]. In plants, PI4P labels the PM as well as the TGN/EE (early endosomes), with the major pool of PI4P being that of the PM [START_REF] Simon | A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants[END_REF].

In yeast and mammalian cells, both PM and TGN PI4P pools have been implicated in autophagy, although at different stages. First, the PI4P pool present at the TGN has been implicated in early stages of AP formation, notably the trafficking of ATG9 vesicles (Judith et al., 2019, Kurokawa et al., 2019). However, plant ATG9 does not seem to have the same function that their mammalian and yeast counterparts, as plant atg9 mutants are capable of forming large but misshapen autophagic structures (Zhuang et al., 2017), while atg9 mutants in yeast and mammals display a block in autophagy at the very early stages (Orsi et al., 2012;Yamamoto et al., 2012). In addition, in yeast and mammals, the pool of PI4P present at the PM has been associated with the AP fusion with the vacuole step (Kurokawa et al., 2019a).

How about in plants? To address this question, we focused on the PI4 Kinases of Arabidopsis thaliana.

Which PI4 kinase produces the pool of PI4P required for autophagy in Arabidopsis ?

PI4P is synthesized by phosphatidylinositol-4-kinases (PI4-Kinases); in Arabidopsis, twelve genes encode for putative PI4-Kinases exist but only three have been characterized as genuine PI4 kinases, i.e. are PI4P producing enzymes [START_REF] Okazaki | Phosphatidylinositol 4-phosphate negatively regulates chloroplast division in arabidopsis[END_REF][START_REF] Stevenson | A phosphatidylinositol 4-kinase pleckstrin homology domain that binds phosphatidylinositol 4-monophosphate[END_REF][START_REF] Xue | A Plant 126-kDa Phosphatidylinositol 4-Kinase with a Novel Repeat Structure[END_REF]. PI4Kβ1 and PI4Kβ2 both localize to the trans-golgi network/early endosomes (TGN/EE), and have been implicated in vesicular sorting events, endocytosis, and lateral root growth [START_REF] Kang | Electron Tomography of RabA4b-and PI-4Kβ1-Labeled Trans Golgi Network Compartments in Arabidopsis[END_REF][START_REF] Lin | A dual role for cell plate-associated PI4Kβ in endocytosis and phragmoplast dynamics during plant somatic cytokinesis[END_REF][START_REF] Preuss | A role for the RabA4b effector protein PI-4Kβ1 in polarized expansion of root hair cells in Arabidopsis thaliana[END_REF][START_REF] Rubilar-Hernández | PI4KIIIβ activity regulates lateral root formation driven by endocytic trafficking to the vacuole[END_REF]. In this manner, the PI4Kβ1 and PI4Kβ2 are thought to be responsible for producing the pool of PI4P present at the TGN/EE. In contrast, PI4Kα1 was found to localize to the PM, and a knockdown mutation decreased the amount of PI4P in chloroplasts, although the exact molecular mechanism linking PI4Kα1 and PI4P production at the chloroplast was not determined [START_REF] Okazaki | Phosphatidylinositol 4-phosphate negatively regulates chloroplast division in arabidopsis[END_REF]. In this manner, PI4Kα1 is thought to be responsible of establishing the PI4P pool present at the PM. In order to identify if and which PI4K is involved in autophagy in Arabidopsis, we performed reverse genetics to explore the impact of knockingout or knocking-down either the PI4Kβ or the PI4Kα. Our initial results suggest that PI4Kα1, but not PI4Kβ, is implicated in providing PI4P for plant autophagy. In fact, ATG8 lipidation was unaffected in pi4kβ1 pi4kβ2 lines (Figure S17, A-B) while, we observed a defect in the conversion of ATG8 to ATG8-PE in amiRNA:PI4Kα1 (Figure S17, C-D), indicating that this process is disturbed in these lines. The degradation of the NBR1 protein is another way to assay autophagy in plant cells (Minina et al., 2018). Our results showed that the rate of NBR1 degradation was affected in amiRNA:PI4Kα1 lines but not in pi4kβ1/pi4kβ2 lines (Figure 15, A-B). Finally, amiRNA:PI4Kα1 lines displayed marks of accelerated senescence compare to WT in nutrient starvation conditions (-NC), a phenotype reminiscent to that observed in atg mutants (Figure 15,D). Taken together, these data point to the fact that PI4Kα1, rather than PI4Kβ, is implicated in plant autophagy. To confirm these results, we crossed the double KO mutant pi4kβ1 pi4kβ2 (Jandaa et al., 2014) as well as amiRN:PI4Kα1 lines (this study) with GFP-ATG8a expressing plants. In the near future, the analyses of these transgenic lines will allow us to assay if (1) APs formation is hampered in the absence of PI4Kα and/or PI4Kβ and

(2) the autophagic flux is affected in the absence of these PI4K. GFP-ATG8a x amiRNA:PI4Kα1 lines are currently in segregation. Preliminary results from GFP-ATG8a x pi4kβ1 pi4kβ2 lines showed that in this mutant background, APs are still being formed (Figure 29). This supports the results obtained with NBR1 degradation and ATG8 lipidation assays (Figure 15, A-B) and further suggests that PI4Kβ are dispensable for autophagy under nutrient starvation conditions.

Altogether, these analyses place the PM localized PI4Kα as the main source of PI4P

for autophagy in Arabidopsis. This would mean that the pool of PI4P present at the TGN is probably not involved in plant autophagy, at least not at the initial steps of AP formation as it occurs in mammals and yeast. Furthermore, the absence of PI4P in other organisms results in an impaired trafficking of ATG9 vesicles, which leads to a defect in the lipidation of ATG8.

However, in plants, atg9 mutants do not display a defect in the lipidation of ATG8, indicating that, in plants the trafficking of ATG9 vesicles is not implicated in the lipidation of ATG8 (Zhuang et al., 2017) This suggests that the ATG8 lipidation defect we observed in the absence of PI4P (Figure 17, A-B) is due to a mechanism other than ATG9 vesicle trafficking, and hence, that PI4P mediates the lipidation of ATG8 through another mechanism. Our results suggest that it is PI4Kα1 that is implicated in plant autophagy, and the localization of PI4K at the PM [START_REF] Okazaki | Phosphatidylinositol 4-phosphate negatively regulates chloroplast division in arabidopsis[END_REF] suggests an implication of the pool of PI4P present in the plasma membrane on plant autophagy. This is further supported by high-resolution AiryScan confocal microscopy studies that showed that a significant amount of autophagosomes is observed as flat-shaped structures that seemed constrained to the plasma membrane (Figure 19,C). would provide for the PI4Kα-produced PI4P required for the formation of autophagic structures (Figure 30).

Alternatively, it is a possibility that PI4Kα is involved in autophagy to provide PI4P directly at the AP membrane. To test this hypothesis, we performed crosses between plants expressing GFP-ATG8a and individual plants expressing fluorescent versions of PI4Kα1 (as well as PI4Kβ). Plants are currently in segregation. With these plants we will perform confocal microscopy studies in order to test if any of these PI4K re-localizes to autophagic structures which would indicate a possible contribution of these PI4K in plant autophagy.

IV-2.2 Function of PI4P in autophagy in Arabidopsis

How is PI4P affecting autophagosome formation, direct or indirect effects?

Our results indicate that PI4K activity is required for AP formation (Figure 16, Figure S16).

Furthermore, PI4P seems to be present in autophagic structures (Figure 19). These findings indicate a role for PI4P in the formation of autophagic structures. However, PI4P plays important roles in other cell processes, and thus the effects we observed in autophagy could result from alterations in other cellular processes. For instance, PI4P is a key lipid contributing to the establishment of the "membrane identity" of different organelles from the endomembrane system which is essential for their proper trafficking [START_REF] Noack | Functions of Anionic Lipids in Plants[END_REF]. In this manner, PI4P localized at the TGN (produced by PI4Kβ1/ PI4Kβ2 as mentioned above) has been implicated in polar sorting of proteins required for proper root growth [START_REF] Kang | Electron Tomography of RabA4b-and PI-4Kβ1-Labeled Trans Golgi Network Compartments in Arabidopsis[END_REF][START_REF] Lin | A dual role for cell plate-associated PI4Kβ in endocytosis and phragmoplast dynamics during plant somatic cytokinesis[END_REF][START_REF] Preuss | A role for the RabA4b effector protein PI-4Kβ1 in polarized expansion of root hair cells in Arabidopsis thaliana[END_REF][START_REF] Rubilar-Hernández | PI4KIIIβ activity regulates lateral root formation driven by endocytic trafficking to the vacuole[END_REF]. Also, PI4P has been shown to play important roles in plants responses to biotic stress. For example, upon biotic stress, the ubiquitin-ligase PLANT-U-BOX ( PUB13) is responsible for the trafficking of the PM receptor FLAGELLIN-SENSING2 (FLS2) to initiate plant defense mechanisms. PUB13 trafficking of FLS2 from the PM towards the TGN was found to be dependent on an interaction with PI4P present at the TGN [START_REF] Antignani | Recruitment of PLANT U-BOX13 and the PI4Kβ1/β2 phosphatidylinositol-4 kinases by the small GTPase RabA4B plays important roles during salicylic acidmediated plant defense signaling in arabidopsis[END_REF]. In other organisms, autophagy has been characterized as being tightly linked with endomembrane vesicles trafficking. Notably, the Golgi-derived ATG9 vesicles, COPII vesicles and endosomes have been proposed as membrane sources for the initiation of autophagic vesicles (Graef et al., 2013;Mari et al., 2010;Shirahama-Noda et al., 2013;Tan et al., 2013). Thus, the impact of inhibiting PI4K activity on these intracellular processes could indirectly affect autophagy. The results we obtained here allowed us to specifically identify which step of the autophagosome biogenesis process is affected by the lack of PI4P. In this manner, even if the effects we observed are indirect, (meaning they affect endosomes of COPII vesicles trafficking rather than autophagy directly), they provide valuable information concerning the step of autophagosome biogenesis in which these trafficking events are required. More importantly, we showed that in our conditions, the activity of the TGN-localized PI4Kβ1/ PI4Kβ2 (and known to be important for vesicular sorting)

was dispensable for autophagosome formation (Figure 29) as well as ATG8 lipidation (Figure 17). This suggests that pathways leading to (1) vesicular trafficking or (2) autophagy, are at least somewhat specific to each mechanism and thus, limits the possibility that the effects we observed are indirect. This in addition to the fact that we observed PI4P on autophagic bodies suggest that PI4P is indeed incorporated into these structures and thus has a direct impact on plant autophagy (Figure 19,A). Thus, we propose that PI4P plays a role as a structural lipid present in autophagosomes.

To address how PI4P directly impacts the phagophore structure or function, we aimed at modulating PI4P levels specifically at the phagophore. In order to alter the PI4P content on early autophagic structures, we fused the phosphatidylinositol-4-phosphate Sac1p yeast phosphatase to the ATG5 protein and to a mTagBFP2 fluorochrome. Sac1p is a phosphatase which localizes to the PM in yeast where it converts PI4P to PI3P [START_REF] Hammond | PI4P And PI(4,5)P2 Are Essential But Independent Lipid Determinants Of Membrane Identity[END_REF]. In plant cells, an engineered construct consisting of the catalytic site of this phosphatase fused to a PM-anchoring peptide was successfully addressed to the PM where it decreased the overall level of PI4P without affecting the levels of PS or PI(4,5)P2 [START_REF] Gronnier | Structural basis for plant plasma membrane protein dynamics and organization into functional nanodomains[END_REF][START_REF] Simon | A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants[END_REF]. To modulate the content of PI4P specifically at the phagophore we generated a similar construct, where the Sac1 catalytic site -or a mutated inactive version called Sac1 dead , was fused to the ATG5 protein to serve as a guide for targeting to autophagic structures (Figure 31). We transformed our constructs into the atg5-1 knock out mutant (Thompson et al., 2005;Yoshimoto et al., 2009) genetic background and recently selected homozygous single insertion lines. In the near future, these lines will be used to first verify that the constructs are effectively targeted to go to the phagophore by ( 1) assessing the complementation of the atg5-1 phenotype (looking at the restoration of ATG8 lipidation), (2) formation of punctate structure upon autophagy inducing conditions, and (3) their colocalization with autophagy markers (after transformation into mcherry-ATG8 expressing plants for example). Second, and granted that the constructs were specifically addressed to autophagy structures, we will use these transgenic lines to test the direct implication of PI4P in the phagophore membrane by comparing differences between Sac1 WT and Sac1 dead in autophagy activity. We notably expect that Sac1 WT expressing constructs will show a block in autophagy similar to the one caused by PAO with a decreased ATG8 lipidation, a lack of phagophore elongation and an accumulation of ATG5 in the membrane fraction. In comparison, Sac1 dead expressing constructs should display a normal ATG8 lipidation, normal phagophore elongation, and no accumulation of ATG5 in the membrane fraction. With these constructs we will be able to directly target the PI4P pool present at the phagophore. This tool therefore represents a very elegant manner to characterize the precise molecular function of PI4P in autophagy and by this having a better comprehension of the first steps of the structural assembly of the phagophore.

Role of PI4P in autophagic membranes: molecular function?

The presence of PI4P in autophagic structures has been reported in both mammals and in yeast. PI4P was shown to be present in both the inner membrane as well as the outer membrane of APs in both organisms, although some differences could be retrieved. In mammals, PI4P was found to be present in the cytosolic leaflets (facing the cytosol for the outer membrane, and facing the interior of APs for the inner membrane) of APs [START_REF] Kurokawa | Phosphatidylinositol 4-phosphate on Rab7-positive autophagosomes revealed by the freeze-fracture replica labeling[END_REF]. In yeast, however, PI4P was found to be localized in the luminal leaflet of both membranes of APs (Kurokawa et al., 2019). These findings highlight the differences between APs of different organisms. Furthermore, yeast APs were found to be invariably spherical, while in mammals, they were found to take heterogeneous shapes (Kurokawa et al., 2019[START_REF] Kurokawa | Phosphatidylinositol 4-phosphate on Rab7-positive autophagosomes revealed by the freeze-fracture replica labeling[END_REF]. The lipid distribution in membranes can have an impact in their curvature (Bigay & Antonny, 2012). Thus, although this remains to be proved, this particular distribution of PI4P

might have an impact in the distinct shapes of APs in both organisms. Our results suggest that PI4P is also present in autophagy structures in plants, yet how this lipid partitions between the two membrane of the AP and how it impacts the phagophore ultrastructure remains unknown.

We have started correlative light electron microscopy analyses (CLEM) to observe autophagic structures at a high resolution and test how their morphology is affected in the absence of PI4P. Our results indicate that in PI4K inhibition conditions, early ATG marker proteins are still recruited to a membrane upon autophagy induction; yet, confocal microscopy analyses showed that these early ATG proteins do not form puncta in the cytosol (Figure 18,G). This suggests that, in the absence of PI4P, an initial PAS is nucleated and able to recruit early ATG markers, although it is not capable to expand as to be recognized as a dot-like structure by confocal microscopy. In this manner, the high-resolution CLEM microscopy analysis using early ATG markers, as probes will provide valuable answers to this question as it will allow to visualize the morphology of these early structures.

Protein/anionic lipid interactions, fundamental inputs in mechanism of autophagy

The AP biogenesis process relies on the finely tuned spatiotemporal recruitment of ATG proteins to the site of AP formation. As mentioned before, most ATG proteins reside in the cytosol at a steady state, and are exclusively recruited to the site of AP formation upon autophagy induction. The recruitment of many of these ATG proteins occurs through protein/lipid interactions as many ATG proteins bear lipid-binding domains or are able to interact with lipids, notably anionic lipids, through electrostatic interactions (see review). These protein/lipid interactions greatly contribute to the activity and dynamics of autophagic membranes, as they allow for the rapid yet transient recruitment of peripheral membrane associated proteins that are required for the growth, shaping and more generally all the membrane dynamic events that drive autophagosome formation. Our results indicate that in the absence of PI4P, the ATG8 lipidation is harshly affected (Figure 17) which leads to a block in the formation of APs (Figure 16, Figure S16). PI4P is an anionic lipid and as such, it has been implicated in the recruitment of proteins to specific membranes through electrostatic interactions thanks to its negatively charged polar head (Figure 32) [START_REF] Noack | Functions of Anionic Lipids in Plants[END_REF].

Hence, we hypothesized that PI4P might function by recruiting a PI4P-binding protein implicated in at least acting upstream of the lipidation of ATG8.

ATG4, a phosphoinositide-binding protein?

To identify this putative PI4P-binding protein, we first looked at proteins from the ATG machinery; searching through the amino acid sequences of ATG proteins that act upstream of ATG8 lipidation, we identified that ATG4 proteins have three motifs enriched in positively charged amino acids (Figure 33,A) that are reminiscent of domains involved in electrostatic interactions with anionic lipids.

ATG4 is a cysteine protease that removes C-terminal residues of ATG8, exposing a Glycine residue to which PE is conjugated (Feng et al., 2014). ATG4 is thus part of the ATG8-PE ubiquitination system [START_REF] Klionsky | Dynamic regulation of macroautophagy by distinctive, ubiquitin-like proteins[END_REF], and as such, is considered to play a role in autophagosome expansion by contributing to the incorporation of ATG8 into the phagophore membrane (Xie et al., 2008). In Arabidopsis, two genes coding for ATG4 exist (ATG4A and ATG4B), and were shown to contribute to plant development under nutrient limiting conditions (Yoshimoto et al., 2004). Furthermore, in the atg4a4b-1 double mutant, no APs were formed, demonstrating their implication in plant autophagy (Yoshimoto et al., 2004). As mentioned above, amino acid sequences of both ATG4a and ATG4b isoforms of Arabidopsis showed regions relatively rich in positively charged amino acids such as Arginine (R) or Lysine (K) (Figure 33,A). We thus decided to test whether (1) ATG4 was membrane-bound, (2) recruited to autophagic structures, (3) this membrane recruitment was dependent on PI4P and (4) the polybasic motifs had an implication in this membrane recruitment, through a putative interaction with anionic lipids. Our results from cell fractionation assays showed that indeed both ATG4A and ATG4B are membrane-associated (Figure 18, C; data not shown for ATG4B). Confocal microscopy analysis showed that both ATG4A and ATG4B localize mostly in the cytosol but are also recruited to autophagic structures upon autophagy inducing conditions where they co-localize with ATG8 (Figure S18, A-B). Nevertheless, in the absence of PI4P, both ATG4A and ATG4B were still recruited to membranes (Figure 18, C; data not shown for ATG4B) indicating that this is not dependent, or not exclusively dependent, on PI4P.

Importantly, while it was suggested that ATG4 recruitment to autophagic membranes is dependent on its interaction with ATG8 in other organisms [START_REF] Abreu | Conserved Atg8 recognition sites mediate Atg4 association with autophagosomal membranes and Atg8 deconjugation[END_REF], we found that in the absence of PI4P, where ATG8 recruitment to the phagophore was diminished (Figure 17

Identifying the PI4P-effectors in plant autophagy

As mentioned before, we speculate that PI4P could participate in autophagy by mediating the recruitment of specific protein(s) at the PAS/phagophore. In that context, identifying the molecular partners of PI4P would provide great insights into its role in autophagy. PI4P has also been reported to play important roles in the AP lysosome fusion step in mammals (H. Wang et al., 2015). One of ATG8 homologs in mammals (GABARAP), was found to recruit the mammalian PI4KIIα to APs to produce PI4P in situ and this was required for proper autophagosome-lysosome fusion (Wang et al., 2015). In this study, the authors did not identify the molecular partners of PI4P but they suggested that PI4P might be implicated in the recruitment of SNARE complexes known to mediate the AP -lysosome fusion step. Our results indicate that PI4P is present on or close to autophagic structures (Figure 19, A-D). Moreover, in the absence of PI4K activity, ATG8 lipidation is hampered (Figure 17), which results in the absence of AP formation (Figure 16, Figure S16). We thus speculate that PI4P might be mediating the recruitment of a protein required for, or at least acting upstream of, the lipidation of ATG8. In order to identify this protein, we are planning to perform proteomic analysis on purified phagophore structures in control conditions compared to conditions with an altered level of PI4P (PAO, amiRNA:PI4Kα1 or ATG5-SAC1). We would next analyze the proteins that are not recruited in the absence of PI4P, and try to identify potential PI4P-binding candidates among them. Next, lipid-protein binding assays would be performed as to test their ability to interact with PI4P primarily, but also to assay if they are able to bind other lipids. The implication of these potential candidates in plant autophagy will later be assessed by testing features like ATG8 lipidation, autophagic flux, as well as the number and morphology of autophagic structures in their corresponding mutant backgrounds.

More generally, in other organisms it has been reported that many ATG proteins are recruited to autophagic structures thanks to their interaction with different lipids (see review).

In plants however, only some PI3P-binding proteins implicated in autophagy have been identified (Gao et al., 2015;Zhuang et al., 2013). Concerning other proteins that bind to other lipids, nothing is known. In this manner, future studies to identify other lipid-binding proteins will provide valuable insights on the function of such lipids in plant autophagy.

General Conclusions of the thesis

My PhD work has allowed to confirm our initial hypothesis: lipids are more than mere building blocks that constitute autophagic membranes. Rather, different lipids seem to have different impacts on autophagy indicating that they play distinct and specific roles in the pathway.

Moreover, my work has led to the identification of interesting lipid candidates and lipid related protein that seem to play important roles in plant autophagy. The most prominent discovery was the identification of a lipid critical for the formation of APs: the phosphoinositide PI4P.

Further, I found that PI4P is required for the early stages of AP formation in plants. Additionally, we have obtained valuable insights for a better understanding of the AP formation process in plants; notably, my results suggest that the PM plays important roles in the formation of these structures. Taken together, the results that I obtained during my thesis highlight to which point lipids and not just proteins, are key actors in the autophagic process, and in this manner, they pave the way for exciting discoveries of the roles of lipids in plant autophagy.

BFP-Sac1 dead construct. These constructs were clones into the pDONRP2rP3 using the gateway technology. Similarly, we PCR-amplified and cloned the ATG5 coding sequence in the pDONR221. All plasmids were sequenced prior to be used for multisite gateway LR cloning together with the promoter UBQ10 or the promoter p35S cloned in the pDONRP4-P1R resulting in the constructs: UBQ10p-ATG5-BFP-Sac1 WT ; UBQ10p-ATG5-BFP-Sac1 dead ; 35Sp-ATG5-BFP-Sac1 WT and 35Sp-ATG5-BFP-Sac1 dead in the expression plasmid PH7m34GW.

The constructs were verified by sequencing and then transformed into A. thaliana atg5-1 ecotype (SAIL_129_B07 ; Thompson et al., 2005;Yoshimoto et al., 2009) by floral dip ;

homozygous lines containing single construct insertions lines were selected based on hygromycin resistance.

Growth conditions, autophagy induction and chemical treatments

Seeds were vernalized in water and darkness, at 4°C, for 24h-48 hours. They were next surface sterilized in 10% bleach for 20 min, and sown on Murashige and Skoog (MS) agar medium plates (0.8% plant agar, 1% sucrose and 2.5 mM morpholinoethanesulfonic acid (Sigma), pH 5.7. Seeds were sown on a piece of mesh to facilitate root dissection from leaves.

Seedlings were grown vertically for 7 days, at 21°C, under long-day conditions (16h-light/ 8hdark photoperiod, 300µEm²s-1).

For nutrient starvation (i.e. autophagy induction) seedlings were transferred into liquid MS -NC medium (Murashige and Skoog liquid medium depleted for nitrogen and carbon: Murashige and Skoog micronutrient salts (Sigma, M0529), 3mM CaCl2, 1.5 MgSO4, 5mM KCl, 1.25mM KH2PO4, 0.5% (w/v) mannitol, 3mM 2-(N-morpholino)ethanesulphonic acid, pH 5.7), and incubated in darkness (wrapped in aluminium foil) for 3 hours, as indicated in the figures.

For rich conditions, seedlings were sampled directly from the MS plates or incubated in liquid Murashige and Skoog (MS) medium (1% sucrose and 2.5 mM morpholinoethanesulfonic acid (Sigma), pH 5.7 under normal light conditions, for 3 hours.

Chemical treatments to inhibit lipid synthesis (see Figure 8) were applied concomitantly with autophagy induction, as indicated in figures. For inhibitors affecting sterol synthesis (fenpropimorph and lovastatin treatments) seedlings were grown on normal medium for 5d before being transferred for 48h to medium containing fenpropimorph or lovastatin.

Protein extraction and Immunoblotting

For the GFP-ATG8 assay, total proteins were extracted from dissected roots exclusively.

Roots were frozen in liquid nitrogen, disrupted using a TissueLyser (Qiagen) by and homogenized in 2XLaemmli buffer (20µl/mg of fresh weight) and clarified by two consecutive centrifugations at 3000 rpm for 1 min and 10 000 rpm for 10 min, respectively. Proteins were denaturated at 55°C for 15 min. After migration on 12% SDS-PAGE (TGX Stain-Free FastCast Acrylamide kit, BioRad), equal protein quantity per lane was verified by stain-free activation of the loaded gel. After transfer to nitrocellulose (BioRad, #1704270), membranes were incubated with an anti-GFP mouse antibody (Roche, #11814460001) at a dilution of 1/5000. Peroxidase activity coupled to the Goat anti-Mouse antibody at a dilution of 1/5000 (Bio-Rad, #1706516) was revealed using Wester Lighting Plus-ECL (PerkinElmer) and detected with the Chemidoc MP Imaging system (Bio-Rad). Blots were imaged at different exposition times, long enough to observe clear distinct bands but prior to pixel saturation. Blot images were analyzed using the ImageJ 1.52i software, band intensities were calculated using the plot surface areas functionality of the software. The intensities of the free-GFP band and the total GFP (GFP-ATG8 + free-GFP) were then used to calculate ratios representatives of the autophagic flux.

Statistical analyses

Statistical analyses were performed on samples from at least three independent experiments.

All data are presented as the mean ± SD. Differences in the autophagic flux between treated and untreated plants were statistically analyzed using unpaired one sample t test. Statistical analyses were carried out using GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA).

P-values were as follows: P-value>0.05 (non significant, n.s.), * P<0.05, ** P< 0.01 and *** P<0.001

Microscopy analysis

All observation were performed in on root epidermal cells of the elongation to early differentiation zone, employing 7-day-old seedlings mounted in culture medium with chemical treatments as indicated in the figures or in the following. Roots were kept under the microscope for no longer than 10 minutes in order to avoid secondary effects due to prolonged treatment times. Confocal images were acquired with a ZEISS LSM880 confocal system. For tagBFP, a 405-nm wavelenght was used for excitation, using an MBS 405 mirror and tagBFP signal was detected using a band-pass filter of 446-499nm. For GFP and tagRFP, the Argon 488 nm and Diode 561nm wavelength laser were used for excitation, a dual-band dichroic mirror (488/561) was used for detection and emissions were detected at 499-553 nm for GFP and 597-668 nm for tagRFP. Fluorescence In multi-labeling acquisitions, detection was in sequential linescanning mode with a line average of 4. Confocal microscope images were processed using the Zen Black Software (Zeiss) for intensity optimization.

RT-qPCR analysis of PLA2 gene expression

Total RNA was extracted using the RNeasy mini kit (Qiagen). To eliminate genomic DNA contamination, an additional DNase treatment was performed according to the RNeasy kit instruction with the RNase-free DNase set (Qiagen). One microgram of total RNA was reversetranscribed into cDNA in a 20 μL reaction mixture using the Superscript II reverse transcriptase enzyme (Invitrogen). cDNA levels were then analyzed using the iQ™ Sybr Green supermix (BioRad) on the iQ iCycler thermocycler (BioRad) with the gene-specific primers listed in Table 4. The thermocycling program consisted of one hold at 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. After completion of these cycles, melting-curve data were then collected to verify PCR specificity, contamination and the absence of primer dimers. The transcript abundance in samples was determined using a comparative threshold cycle method.

The relative abundance of the reference mRNAs of CLATHRIN, UBQ10 and ATG43380 or ACTIN1 and APT1 for the different experiments presented were determined and used to normalize for differences of total RNA amount according to the method described by [START_REF] Snare Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF].

VI-REFERENCES

Fig. 1 .

 1 Fig. 1. Schematic diagram of the progression of macroautophagy in plant cells. Upon induction, autophagy starts with the nucleation of the phagophore at the phagophore assembly site. (A) Early autophagy-related (ATG) proteins, represented as red circles, are recruited to establish the initial structure following two distinct hypothetical models: (a) they are recruited on ER subdomains from which emerges an omegasome-like structure that detaches to form the phagophore; (b) they are recruited at ER-derived tubulovesicular compartments that coalesce with other membrane sources and possibly ATG9 vesicles to nucleate the phagophore. (B) The membrane of the phagophore expands and matures by addition of lipids and proteins in a cradle-like structure surrounded by ER membranes. (C) The phagophore closes yielding a double-membrane autophagosome (AP) enwrapping cargo. En route to the vacuole, the AP may maturate through interaction with endosomes. (D) The outer membrane of the AP fuses with the membrane of the vacuole.Components of the AP may be recycled from the vacuolar membrane. The inner membrane surrounding the cargo is released inside the vacuolar lumen where it forms the autophagic body (AB). The AB is rapidly degraded by vacuolar hydrolases and lipases. Molecules resulting from the degradation are exported back to the cytoplasm.

Fig. 2 .

 2 Fig. 2. Lipids in biological membranes. (A) Chemical structure of the three major classes of plant membrane lipids. Polar heads are represented in light red, acyl chains in black. For glycerolipids, the glycerol moiety is in orange, the phosphate group is in green. For sphingolipids, the long-chain base is in blue. (B) Shape of membrane lipids. Polar heads are represented in grey, hydrophobic tails in black. Cylinder-shaped lipids create bilayer while the insertion of inverted cones or conical lipids as well as their asymmetric distribution within the membrane leaflets promotes the formation of membrane curvature. (C) Hypothetical representation of lipid-and protein-driven curvature within the phagophore. A specific lipid composition and distribution could shape the phagophore and especially drive the high curvature of the membranes at the rim. BAR-domain-containing proteins (represented in red) and amphipatic proteins (represented in brown) could create, force or stabilize membrane deformation. DGDG, digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol.

  Lipids in the formation of autophagic membranes in plants | Page 7 of 13 Downloaded from https://academic.oup.com/jxb/advance-article-abstract/doi/10.1093/jxb/erx392/4626680 by Universite de Bordeaux user on 20 November 2017

Figure 1 .

 1 Figure 1. Overview of the autophagy pathwayIn nutrient rich conditions upstream regulators of autophagy (such as TOR/RAPTOR) inhibit the pathway. Upon autophagy induction, the inhibition is stopped, and the autophagosome biogenesis process initiates. The ATG1 initation complex, ATG9 vesicles and other early ATG components such as the PI3K complex relocalize to a site in close proximity to the endoplasmic reticulum (ER) to establish the pre autophagomal structure (PAS). The PAS is considered a lipo-proteic compartment from which an initial membrane emerges and constitutes the double membrane phagophore structure. Then, the ATG8 lipidation machinery (ATG5-ATG12 complex, ATG4, ATG3, ATG7) mediate the covalent conjugation of ATG8 protein to a phosphatidylethanolamine (PE) moiety. The incorporation of ATG8-PE along with other mechanisms lead to the expansion of the phagophore and an engulfment of autophagic cargo. Once the structure has reached its final size, the membranes at the rim of the phagophore eventually close to form a sealed autophagosome in a process implicating ESCRT complexes. Upon completion, ATG proteins dissociate from the autophagosome and an active removal of ATG8-PE moieties present in the outer membrane mediated by the cysteine protease ATG4 is required. Then, the outer membrane of the mature autophagosome fuses with the vacuolar membrane resulting in the delivery and subsequent degradation by vacuolar hydrolases of the autophagosme inner membrane (called « autophagic body» when present inside the vacuole) along with the autophagic cargo. Finally, the resulting macromolecules are recycled back into the cytosol.

Figure 2 .

 2 Figure 2. Blocking the degradation of autophagosomes reveal the tremendous amount of autophagy structures produced after 3 hours of autophagy induction in Arabidopsis roots Confocal microscopy images of GFP-ATG8a expressing plants. 7 day old plantlets were transferred to liquid nutrient deprived medium (-NC) in control conditions (DMSO), or supplemented with Concanamycin A (1 µM), for 3 hours. Concanamycin A 1 µM is an inhibitor of vacuolar hydrolases, and thus, treatment results in the accumulation of autophagic structures inside the vacuole. Scale bar, 20µm.

Figure 3 .

 3 Figure 3. Multiple membrane sources contributes to the phagophore nucleation and expansionMultiple membrane sources contribute first for the establishment of the initial autophagic membrane as well as for its expansion. Different types of vesicles (notably ATG9 vesicles but also endosomes or COPII vesicles) have been proposed as membrane seeds for the nucleation of the initial structure. Lipid transfer proteins implicated in autophagy (such as ATG2) mediate the direct transfer of lipids from other organelles in contact sites such as ERphagophore contact sites. Finally, on-site lipid synthesis is also an important mechanism for phagophore expansion, for example localized PI3P synthesis allows for the recruitment of downstream effectors of the pathway leading to growth of the structure.

Figure 4 .

 4 Figure 4. Schematic representation of autophagy regulation under various stressesA plethora of environmental stresses have an impact on plant autophagy. Different types of stress can either (1) induce autophagy (2), inhibit autophagy or (3) hijack autophagy. Hence, plants use autophagy to mediate different types of cellular adaptation mechanisms to cope with these stress. Autophagy is able to redirect plant metabolism in order to counterbalance environmental cues and by this maintain a healthy organism. On the other hand, when the autophagic process is inhibited, or hijacked notably by viral or bacterial pathogens, plants are unable to adapt to these stress, thus resulting in plants unable to support growth/development.

  ) was shown to negatively regulates autophagy in the context of viral infection. Arabidopsis thaliana have four distinct isoforms of GAPDH: i) chloroplastic

Figure 5 .

 5 Figure 5. Schematic representation of the autophagy pathway

Figure 6 .

 6 Figure 6. Chemical structures of the three major classes of lipids present on the membranes of plant cells: Sterols, Sphingolipids and Glycerolipids. Their respective polar heads are represented in yellow.

Figure 7 .

 7 Figure 7. Crucial membrane features are determined by the physicochemical properties of lipids A.The size of the polar head or the unsaturation degree of the acyl chains determines the « geometry » of a lipid (i.e. its shape). When inserted into membranes, lipids can create curvatures within these membranes depending on their lipid geometry B. Lipids with charged polar heads (such as anionic phospholipids) can create electrostatic fields in membranes and by this permit interactions with proteins carrying positively charged amino acids C. Lipids with saturated acyl chains pack tightly in membranes, while lipids with a high degree of unsaturation of their acyl chains do the opposite. Lipids with unsaturated acyl chains can thus create regions with lipid packing defects. Regions with lipid packing defects are used by membrane anchored proteins for their insertion into membranes.

Figure 8 .

 8 Figure 8.Table of the inhibitors of lipidsynthesizing enzymes used in the screen.

  Figure 9. GFP-ATG8a degradation and GFP accumulation reflects rates of autophagy activity

  Figure 10. Sphingolipid inhibition treatments using Fumonisin B1 (Fb1, 1 µM), D,L-threo-1-pheny-2decanoylamin-3-morpholino-propanol (PDMP, 50 µM) , and Metazachlor (Mz, 100nM), differently affect the autophagic flux A. Simplified Very Long Chain Fatty Acid (VLCFA) synthesis pathway occuring in the ER in Arabidopsis thaliana. B. Simplified Ceramide and Glucosylceramide biosynthetic pathway occuring in the ER in Arabidopsis thaliana. C,D,E. Immunoblot analyses of GFP-ATG8a degradation assay upon treatment with Fb1 1 µM treatment (C), PDMP 50 µM (D) or Metazachlor 100nm (E) compared to control conditions (DMSO). (C-D) 7 day old seedligs were transferred for 3 hours to either liquid nutrient rich (+) or nutrient deprived (-NC, -) medium, both supplemented with the aformentionned treatments. For (E), 5 day old seedlings were first transferred to DMSO-containing (control) or Mz 100nmcontaining plates for 48 hours. Then, seedlings were transferred for 3 hours to liquid nutrient rich (+) or nutrient deprived (-) medium to induce autophagy, supplemented with Metazachlor (100nm) or control conditions (DMSO) Immunoblot analyses were performed using the anti-GFP antibody (Roche) and stain free images were used as loading control. Data represents the average of at least 3 independent experiments with standard deviation. Statistics, see Experimental procedures, *, p<0,05.

  Figure 11. Sterol inhibition treatments using Lovastatin (1µM) or Fenpropimorph (165µM) as inhibitors has different impacts on the autophagic flux.

)Figure 12 .

 12 Figure 12. Inhibition of phosphatidic acid (PA)-related enzymes differently affects the autophagic flux. A. Schematic representation of metabolic pathways leading to PA. B. Effect of CI976 on the neo-synthesis of PA in Arabidopsis roots. Seedlings were grown on MS agar medium plates or MS agar medium plates complemented with 10µM CI976. Primary roots from 20 seedlings were collected 5 days after germination and incubated 4 hours with [ 14 C] acetate. Lipids were extracted, separated by HPTLC and amounts of neosynthesized [ 14 C] PA were quantified. For each replicate, the amounts of [ 14 C] PA produced in the presence of the drug were calculated by taking as 100 the amounts of [ 14 C] PA produced without treatment. Data are mean values ± SD from three biological replicates. Statistics were done by non-parametric Kruskal-Wallis rank sum test. P-values < 0.0001. Experiments were performed by W. Boyer and F. Dittrich-Domergue). C. Immunoblot analysis of GFP-ATG8a degradation assay upon treatment with Ci-976 (5 µM) or (10 µM) treatment (C), R59022 (12,5 µM) or (50 µM) (D), Propanolol (50 µM) (E ) or U73122 (5 µM) and U73343 (5 µM) (F) compared to control conditions (DMSO). Experiments were performed and results are presented as in Fig.10 A.

  Figure 13. Inhibition of phosphoinositides (PI)-related enzymes has different impacts on the autophagic flux A. Schematic representation of phosphoinositides biosynthetic pathway B. Immunoblot analysis of GFP-ATG8a degradation assay upon Wormannin (10 µM) treatment. 7 day old seedligs were transferred for 3 hours to liquid nutrient rich (+) or nutrient deprived (-) medium to induce autophagy, supplemented with Wormannin (10 µM) or control conditions (DMSO) C. Immunoblot analysis of GFP-ATG8a degradation assay upon YM-201636 (1 µM) treatment. 7 day old seedligs were transferred for 3 hours to liquid nutrient rich (+) or nutrient deprived (-) medium to induce autophagy, supplemented with YM-201636 (1 µM) or control conditions (DMSO) Immunoblot analyses were performed and results presented as in Fig.10.

Figure 14 .

 14 Figure 14. The PI4-kinase inhibitor PAO blocks autophagic flux in root cells A, C, E. Detection of GFP-ATG8a degradation and release of free GFP in the vacuole. 7 day old GFP-ATG8a seedlings were transferred from nutrient rich MS plates to either nutrient rich (+N), nutrient starved (-nitrogen and carbon; -NC) liquid medium or nutrient rich medium supplemented with 1 M AZD in PI4K inhibiting conditions (+PAO) or control conditions (DMSO). Total protein was subjected to immunoblot analysis with anti-GFP antibodies. Stain free images were used as loading control. (A) Seedlings were transferred to nutrient deprived conditions and treated with 30 M PAO or DMSO as control for 1.5h or 3h. In (C), seedlings were treated for 3h with various concentrations of PAO as indicated on the figure. In (E), seedlings were transferred to liquid medium containing DMSO or 1 M AZD supplemented with PAO at either 7.5 M or 30 M, or DMSO as control. B, D, F. Quantification of the ratio of GFP/GFP-ATG8a reflective of the rate of autophagy flux in each conditions in experiment presented in A,C and E respectively. Results are presented as the mean ± SD (n = 3 independent experiments) with one sample t and Wilcoxon test values. Statistical analysis were carried out using GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA). P-values were as follows: P-value>0.05 (non significant, n.s.), * P<0.05, ** P< 0.01 and *** P<0.001

  Figure S14. PAO treatment disrupts the plasma membrane localization of the PI4P biosensor.Seedlings were grown for 7 days in nutrient rich MS plates supplemented and then transferred to nutrient deprived (-NC) medium to work on autophagy inducing conditions. A. Confocal images of a PI4P biosensor (P7Y) treated for 3 hours with PAO 7,5 or 15 µM compared to DMSO as control. B. Confocal images of a PI4P biosensor (P7Y) treated for 30 minutes with PAO 60 µM compared to DMSO as control. Graphics represent the signal intensity of the probe within the drawn area (red line), created using the "plot profile" feature (Fiji software). Scale bar = 20 µm

Figure S15 .

 S15 Figure S15. Characterization of pi4kβ1 pi4kβ2 and amiRNA:PI4Kα1 lines. A. Genotyping of the previously characterized pi4kβ1 pi4kβ2 double KO line. PCR amplification on genomic DNA was performed using the pair of primers LP1/RP1 or LP1/RB. Positions of primers and tDNA are indicated on the schematic of the PI4Kβ1 and PI4Kβ2 genes. Boxes indicate intron, lines indicate exons. B. amiRNA:PI4Kα1 lines show a reduced PI4Kα1 expression. RT-QPCR analyses of PI4Kα1 expression upon 24 hours of β-estradiol (10 µM) treatment in amiRNA:PI4Kα1 lines compared to WT plants. Results are presented as average of mRNA level with standard deviation of N=4 in 2 independent experiments. C. Schematics of the experimental procedures followed in (B) and Fig. 12 A-C

Figure

  Figure 15. Downregulating but not compromises NBR1 degradation, a marker of autophagy activity * preliminary results A. Immunoblot analysis of NBR1 protein levels extracted from roots. Col-0 (WT), , , were ferred in liquid nutrient rich (+), nutrient starvation (-N), or nutrient starvation medium suplemented with concanaplants were being directly transferred from full MS plates to liquid rich, nutrient starvation or nutrient starvation medium supplemented with concanamycin A 1 µM (+Ca) for 16 hours. B. Quantification of the NBR1 protein levels the different conditions presented in A. Stain free gels were used as loading controls. Histone H3 band was used for normalization. Results represent the average ± SD of for , WT and non significant. C. NBR1 mRNA levels in the same conditions as in A. Results represent the average ± SD of ent experiments. D.seedlings display phenotypes reminiscent of an autophagy deficiency in nutrient starvation conditions. Col-0 (WT), and seedlings were grown for 6 days in full MS medium agar plates with

Figure 16 .

 16 Figure 16. Inhibition of PI4K activity blocks autophagosome formation A. Confocal images of roots from Arabidopsis plants expressing different isoforms of ATG8: GFP-ATG8a, GFP-ATG8e or GFP-ATG8f. 7 day old plants were transferred for 30 min to liquid nutrient rich (+N) or nutrient deprived (-NC) medium; in PI4K inhibiting conditions (PAO, 60 ) or control conditions (DMSO). Scale Bar: 20 µm. B. Quantification of autophagic structures in conditions presented in A (for quantification methods, see Materials and Methods). Results as presented as number of puncta per root area (in nm²), N>35 in at least 3 indepedent experiments. Statistical differences between conditions were assessed using Dunett's multiple comparison test on Prism. ***, P<0,001

ATG8f.

  Figure 17. PI4K inhibition blocks ATG8 lipidationA. Immunoblot analysis using anti-ATG8 antibody of WT or atg5-1 seedlings in rich conditions (R) or upon transfer for 30 minutes in nutrient depleted conditions (-NC) with PAO 60µM (P) compared to control conditions (D, DMSO). Lipidated ATG8a is present in the pellet membrane containing fraction (P100), while being excluded from the supernatant (S100) fraction as shown by the comparison of the protein profiles bewteen WT and atg5-1 seedlings where ATG8 conjugation is prevented. PIP2,7 was used as loading control and normalization factor for the membrane fraction; UGPase was used as loading control and normalization factor for the supernatant fraction. B. Quantification of ATG8 levels in conditions presented in A. Results present the average of the level of ATG8 and ATG8-PE band intensities ± SD (n = 3 independent experiments) with Kruskal-Wallis test for comparison among 3 samples of a same group of data and Mann-Whitney test to compare between DMSO and PAO conditions. Statistical analysis were carried out using GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA). P-values were as follows: P-value>0.05 (non significant, n.s.), * P<0.05, ** P< 0.01 and *** P<0.001

Figure

  Figure S17. ATG8 lipidation is affected upon gene extinction but remains unaffected in the pi4k KO background.A. ATG8 and ATG8-PE levels in the microsomal fraction of WT, or seedlings. Immunoblot analyses were performed using an anti-ATG8 antibody. 8 day old plantlets were transferred from nutrient rich (+N) conditions to nutrient deprived (-N) or nutrient deprived conditions supplemented with concanamycin A 1µM (3+Ca) MS medium, for 0.5 or 3 hours. PIP2,7 was used as a loading control and normalization factor for (B). B. Quantification of the ATG8-PE levels in conditions presented in (A). Results represent the average of 3 independent experiments ± SD. C. ATG8 and ATG8-PE levels in the microsomal fraction of WT and seedlings.Immunoblot analyses were performed using an anti-ATG8 antibody. 7 day old plantlets were transferred to liquid nutrient

Figure 18 .

 18 Figure 18. PI4K inhibition prevents the formation of punctae-like structures of early ATG markers, but not their recruitment to membranes A, C, E. ATG5-mCherry, ATG12-ATG5-mCherry (A), RFP-ATG4a (C) and YFP-ATG18a (E) accumulate in the membrane fraction in PI4P inhibition conditions. Immunobot analyses using anti-mcherry antibody (A), anti-RFP antibody (C) and anti-YFP antibody (E). 7 day old seedlings were transferred from rich conditions (R) to nutrient deprived control conditions (D) or nutrient deprived medium supplemented with PAO 60 µM (P), for 30 minutes. PIP2,7 was used as loading control and normalization factor for the membrane fraction; UGPase was used as loading control and normalization factor for the supernatant fraction. S100, supernatant fraction; P100, pellet membrane containing fraction. B, D, F. Quantification of the levels of ATG5 and ATG5-ATG12 (B), RFP-ATG4a (D) and YFP-ATG18a (F) in conditions presented in A, C, E. Results present the average ± SD of at least 3 independent experiments. Statistical differences were assessed by the one sample t test (B,C) and upaired t test (E) using GraphPad Prism 6. * P<0.05, ** P< 0.01 and *** P<0.001 G. Confocal microscopy images of ATG5-mcherry, RFP-ATG4a and YFP-ATG18a-expressing plants. 7 day old plantlets were transferred to liquid nutrient deprived control conditions (DMSO) or nutrient deprived supplemented with PAO 60 µM (PAO), for 30 minutes. H. Quantification of ATG puntae in conditions presented in G. Results as presented as number of puncta per root area (in nm²), N>35 in at least 3 indepedent experiments. Statistical differences between conditions were assessed using Dunett's multiple comparison test on GraphPad Prism 6. ***, P<0,001

  Figure S18.ATG4a and ATG4b isoforms are recruited to ATG8a-positive autophagic structures. A. Confocal images if Arabidopsis roots co-expressing GFP-ATG8a and RFP-ATG4a or RFP-ATG4b. 7 day old plantlets were transferred to liquid nutrient depleted medium (-NC) for 30 minutes. B. Graphics represent the signal intensity of the probes within the drawn area (dotted white line), created using the "plot profile" feature (Fiji software). C. RFP-ATG4a and RFP-ATG4b colocalization in percentage of puncta with both RFP-ATG4 and GFP-ATG8 signal over total ATG4 puncta. Results represent the average of at least 3 independent experiments. D. RFP-ATG4b also localizes to dot like structures in control conditions but not in PI4K inhibition conditions. Confocal images if Arabidopsis roots expressing RFP-ATG4b. 7 day old plantlets were transferred to liquid nutrient depleted medium (-NC) in control conditions (DMSO) or in PI4K inhibition conditions (PAO §0 µM) for 30 minutes. E. Quantification of RFP-ATG4b dot-like structures in conditions presented in (D). Results as presented as number of puncta per root area (in nm²), N>35 in at least 3 indepedent experiments. Statistical differences between conditions were assessed using Dunett's multiple comparison test on GraphPad Prism 6. ***, P<0,001

Figure 19 .

 19 Figure 19. Partial co-localization of autophagic structures and PI4P biosensor A. Confocal images of 7 day old plantlets co-expressing mCherry-ATG8f and the PI4P-binding probe Citrine-1xPH FAPP1-E50A-H54A . Plants were placed in liquid MS medium deprived of nutrients (-NC) for 2 and 5 hours, in control conditions or supplemented with concanamycin A (1µM). Scale bar: 20µM. B. Quantification of the co-localization events in A. Data represents the average of at least 4 independent experiments with standard deviation. C. High resolution high confocal images using Zeiss Airyscan technology of plants co-expressing GFP-ATG8a and mCherry-1xPH FAPP1 (PI4P biosensor). Plants were placed in autophagy induction conditions for 90 minutes. Open arrows indicate puncta in the cytosol (Cy), solid arrows indicate puncta at proximity from the plasma membrane (Pr) and arrowhead indicate puncta at the plasma membrane (PM). Scale Bar: 5µM D. Repartition of the three distinct types of ATG8 puncta populations described in (C). Results represent 35 data points collected in at least 4 independent experiments.

  Phospholipases (PLs) are a family of proteins catalyzing the hydrolysis of different chemical bonds of phospholipids. Plant PLs are divided into three major classes (A, C and D) depending on the site at which they cleave phospholipids. Phospholipases C (PLCs) cleave the glycerophosphate bond resulting in a diacylglycerol molecule and the entire phosphoryl head group. Phospholipases D (PLDs) cleave the phosphodiester bond thus yielding phosphatidic acid (PA). Phospholipases A (PLAs) hydrolyze acyl groups at either the sn-1 or sn-2 positions of phospholipids, and are thus called PLA1 and PLA2 respectively (Figure 22, C).

  -3.3 Exploring the potential implication of PLA2 and PLA1 in autophagy 3-1 PLA2 and PLA1 Subcellular localization Note: I Initiated this work, generated all transgenic lines and performed initial experiments. Follow-up experiments including results from Figure 24 and quantifications presented in Figure 25 were performed by Julie Castets during her Master2 internship.

Figure 25 .

 25 Figure25. The expression pattern of PLA2 gene ressembles that of ATG genes A. Relative gene abundance of PLA2 compared to that of different ATG genes: ATG8 (a,c, e and i), ATG9, ATG18 (a and h) in a wild type plant (Col-0 ecotype). Plants were placed in liquid medium in nutrient rich (+) or nutrient starvation conditions (-) to induce autophagy, for 3, 6 and 16 hours. Results were normalized using three genes: CLATHRIN, UBQ10 et ATG43380. Results from three technical replicates. B. Relative gene abundance of PLA2, ATG8a and ATG9 at different developmental stages (d = days after seed sowing). Results were normalized using two genes: ACTIN1 and APT1. Results from three technical replicates.

1 :Figure 27 .

 127 Figure 27. Overview of inhibitors screen. Inhibition of different lipid synthesizing enzymes resulted in different impacts on the autophagic flux

Figure 28 .

 28 Figure 28. Phospholipid remodeling capacities of PLA2 and lysphophopholipid acyltransferases (LP-AT) contribute to the formation of membrane curvature.Adapted fromHa KD et al., 2012. Biochim Biophys Acta. 2012 1821:1078-1088

  Figure 31. ATG5-BFPSAC1 constructThe ATG5-BFPSAC1 construct should be guided by the ATG5 moiety towards the edges of the phagophore structure where it should de-phosphorylate PI4P and this should result in a blockage in the growth of these structures.

Figure 33 .

 33 Figure 33. Polybasic regions in the ATG4a and ATG4b proteins A. Amino acid sequence of ATG4a and ATG4b proteins. Four polybasic regions are represented in blue. Lysines (K) and Arginines (R) are highlighted in yellow and green respectively B. Individual mutated forms of the PB regions C. Versions of ATG4a and ATG4b that we created to test the implication of these PB motifs in protein/lipid interaction.

  , A), that of ATG4A was increased (Figure18, C) suggesting that ATG4-ATG8 interaction is not the only component of ATG4 membrane association in plants. Hence, we speculated that the transient association of ATG4A and ATG4B with autophagic structures maybe controlled by anionic lipids other than PI4P (such as PA, PS or PI3P..). To address this hypothesis, we decided to test the possible implication of AtATG4s polybasic motifs in membrane and lipid interactions. We thus mutated the polybasic regions of both ATG4A and ATG4B (named PB1-PB4) and replaced the positively charged amino acids Arginine (R) and Lysine (K), by neutral Glutamines (Q) (Figure33, B). With these mutated versions of ATG4A and ATG4B, we created fluorescent-tagged constructs (Figure33, C) which will be used to assay the implication of such polybasic motifs in(1) the binding of ATG4 to phospholipids by performing PIP strip and liposome floatation assays, (2) the localization of ATG4 at autophagy structures, (3) autophagy activity and AP formation.

Table of contents

 of Plant autophagic responses to environmental stress and crucial role of lipids in the autophagy process………………………………………………………………………………….....

	I-1 Lipids in membrane dynamics during autophagy in plants……………………………………
	I-2

I -INTRODUCTION…………………………………………………………………………………... II -SCIENTIFIC CONTEXT AND OBJECTIVES…………………………………………………..

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  Table of the inhibitors of lipidsynthesizing enzymes used in the screen.

Table 1 Affected Lipid/ClassTarget Enzymes Concentration used Name Effects we observed

 1

	Organism
	Publication

Table S1 : primers used in section III-2

 S1

	Gene name	Gene Locus	Usage	Primers (F/R)	Sequence
				F	GGGGACAGCTTTCTTGTACAAAGTGGCTATGAAGGCTTTATGTGATAGATTTGTTC
				R	GGGGACAACTTTGTATAATAAAGTTGTGATTGTTAAAATGTTTGACGATGATCATGA
				I miR-s	gaTGATATTCGAACACACACCTGtctctcttttgtattcc
				II miR-a	gaCAGGTGTGTGTTCGAATATCAtcaaagagaatcaatga
				III miR*s	gaCAAGTGTGTGTTCCAATATCTtcacaggtcgtgatatg
				IV miR*a	gaAGATATTGGAACACACACTTGtctacatatatattcct
				I miR-s	gaTTTCACGAATACTTGAGGCTAtctctcttttgtattcc
				II miR-a	gaTAGCCTCAAGTATTCGTGAAAtcaaagagaatcaatga
				III miR*s	gaTAACCTCAAGTATACGTGAATtcacaggtcgtgatatg
				IV miR*a	gaATTCACGTATACTTGAGGTTAtctacatatatattcct

Table S2 . Artificial miRNAs against PI4K⍺1 AT1G49340 In

 S2 red: AttB1 and AttB2; in bold: artificial miRNA; in blue: hair pin sequence.

	ATG4a	AT2G44140	Cloning Complete cds
	PI4Ka	AT1G49340	Cloning Inducible miRNA 1
	PI4Ka	AT1G49340	Cloning Inducible miRNA 2
	ATG8a	AT4G21980	Cloning Complete cds
	PI4Kα1	AT1G49340	qPCR
	NBR1	AT4G24690	qPCR
		ACT2/8
		(AT1G49240)
	reference genes		qPCR normalization
		At4g33380
			Cloning cds
	PI4Kβ1	AT5G64070
			Genotyping double
	PI4Kβ2	AT5G09350	mutant

Table S1 . Primers used in this study III-3 Putative implication of a phospholipase A 2 in autophagy III-3.1 Identification of AT4G19860 and AT3G03310 as potential candidates implicated in plant autophagy

 S1

Figure 30. Distinct PI4P pools in Arabidopsis cells and PI4 Kinases and proposed site of autophagosome formation

  Schematic representation of the different PI4P pools in plant cells. (PI4P accumulation is represented in light pink). Our results suggest a contribution of the PM-localized PI4P pool in the formation of autophagy structures. The ER is known to contribute to this process as well. Hence, we propose ER-PM contact sites (EPCS) as the site of autophagosome formation in plant cells.

	Site of
	autophagosome
	formation ?	Vacuole
	Endosomes
	PI4Kα1	AP
	EPCS
		ER
		TGN
	PM
	PI4Kβ1/β2

Thus,

we could propose a model in which autophagosome biogenesis in plants takes place in ERplasma membrane contact sites (EPCS), as the close proximity to the plasma membrane that

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Remerciements

Acknowledgements

The authors kindly thank Romain Le Bars (CNRS, I2BC) for helpful discussions. This work was funded by the Centre National de la Recherche for AB, JJ, REG, NV and BSJ. Access to core facilities (Gif-sur-Yvette and Bordeaux) was supported by France BioImaging infrastructure French National Research Agency grants (ANR 106INBS-04-01) and Saclay Plant Science (Gif-sur-Yvette), ANR-11-IDEX-0003_02). This work was further funded by the Wallonia-Brussels Federation Joint Research Action (ARC grant no. 11/16-036 to HB), the Belgian Funds for Scienti c Research (FRS-FNRS) (CDR grant no. 19516174 and FRFC grant no. 6794930 to HB). HB was a Senior Research Associate of the FRS-FNRS. This work was primed by the TRANSAUTOPHAGY COST ACTION CA15138 European Network of Multidisciplinary Research and Translation of Autophagy knowledge.

este logro se los dedico a ustedes.

MATERIALS AND METHODS

Arabidopsis lines

All experiments were performed in Arabidopsis of the ecotype Col-0, as wild-type, or derived of the ecotype Col-0. The following transgenic lines were used as previously described 35S::GFP-ATG8a (Thompson et al., 2005), pUbi::GFP-ATG8e (Stephani et al., 2020), pUbi::GFP-ATG8f (Stephani et al., 2020), ATG5-mcherry (Le Bars et al., 2014), YFP-ATG18a (Zhuang et al., 2017), P7Y (1xPH OSBP ) [START_REF] Michaeli | ATG8-INTERACTING PROTEIN1 Is Involved in Autophagy-Dependent Vesicular Trafficking of Plastid Proteins to the Vacuole[END_REF], pi4kβ1 pi4kβ2 (SALK_040479 9 SALK_098069; Jandaa et al., 2014), atg5-1 (SAIL_129_B07; Thompson et al., 2005;Yoshimoto et al., 2009).

To generate Arabidopsis lines stably co-expressing mCherry-ATG8f and PI4P-specific fluorescent probes, mCherry-ATG8f lines (Zhuang et al., 2013) were crossed with either P7Y (1xPH OSBP ), P7Y (2xPH FAPP1 ), or with the mutated version of the FAPP1 probe (2xPH FAPP1-E50A- H54A ) [START_REF] Simon | A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants[END_REF]. The double homozygous F3 families were selected by hygromycin/Basta resistance. Similarly, plants co-expressing ATG5-mCherry and PI4P

probes; ATG5-mCherry (Le Bars et al., 2014) was crossed with the mutated version of the FAPP1 line (2xPH FAPP1-E50A-H54A ). TagRFP-ATG4a plants were generated as following. The ATG4a coding sequence was PCR-amplified using primers listed in Table S1 and Designer [START_REF] Schwab | Highly Specific Gene Silencing by Artificial MicroRNAs in Arabidopsis[END_REF]; see Table S2). The amiRNA:pi4kIIIa1 precursor fragment sequences were PCR amplified from the pRS300 plasmid as previsouly described [START_REF] Schwab | Highly Specific Gene Silencing by Artificial MicroRNAs in Arabidopsis[END_REF] using specific primers listed in Table S1. PCR products were cloned into the pENTR/D-TOPO vector (pENTR Directional TOPO Cloning kit, Invitrogen). Clones were verified by sequencing and then transferred by gateway LR cloning into the destination vector PMDC7 which allows expression of the transgene under the control of β-estradiol [START_REF] Curtis | A Gateway Cloning Vector Set for High-Throughput Functional Analysis of Genes in Planta[END_REF]. The constructs were transformed into A. thaliana Col-0 ecotype by floral dip; homozygous lines containing single construct insertions lines were selected based on hygromycin resistance; 15 lines were obtained for each amiRNA. In order to test amiRNA After transfer to nitrocellulose (BioRad, #1704270), membranes were incubated with an anti-GFP mouse antibody (Roche, #11814460001) at a dilution of 1/5000. Peroxidase activity coupled to the Goat anti-Mouse antibody at a dilution of 1/5000 (Bio-Rad, #1706516) was revealed using Wester Lighting Plus-ECL (PerkinElmer) and detected with the Chemidoc MP Imaging system (Bio-Rad). Blots were imaged at different exposition times, long enough to observe clear distinct bands but prior to pixel saturation. Blot images were analyzed using the ImageJ 1.52i software; band intensities were calculated using the plot surface areas functionality of the software. The intensities of the free-GFP band and the total GFP (GFP-ATG8 + free-GFP) were then used to calculate ratios representatives of the autophagic flux.

Subcellular Fractionation and protein analyses

Upon treatment indicated in the figures, seedlings were frozen in liquid nitrogen, disrupted using a TissueLyser (Qiagen) and homogenized in the following buffer: HEPES 50 mM pH 7.5, Sucrose 0.225 M, MgCl2 2.5mM, DTT 0.5 mM, PVP (Sigma P2307) 0.25% [w/v], PMSF 1 mM, antiprotease mix (cOmplete™ Protease Inhibitor Cocktail). Homogenates were filtered through a Miracloth layer and centrifuged twice at 1600 xg, for 15 min at 4 °C, for debris elimination.

Clarified lysates (total lysate, TL) were centrifugated for 20 min at 55,000 rpm (eq. 100,000 xg) in the "Sorvall Discovery M150 SE Ultra-Centrifuge (Hitachi), using the S100 AT6-0257 rotor to separate the membrane fraction (pellet, P100) containing membrane-anchored proteins from the soluble fraction (supernatant, S100). Pellets were resuspended in homogenization buffer (P100). To analyse samples from subcellular fractionation, the protein concentration of each sample (TL, P100, S100) was determined using Bio-Rad Protein Assay Dye Reagent Concentrate, (BioRad,ref #5000006) and measuring sample absorption at 595 nm. Equal amounts of proteins were prepared and denatured using Laemli buffer, loaded onto 10-12% SDS-PAGE and analysed by western blot as described above.

ATG5-mCherry was detected using a HRP anti-RFP antibody (Abcam, ab34767; 1/1000).

YFP-ATG18 was detected using anti-GFP (Roche ;1/2000). Peroxidase activity coupled to the Goat anti-Rabbit antibody at a dilution of 1/5000 (Bio-Rad #1721019) was used for revelation of YFP-ATG18. tagRFP-ATG4 was detected using a mouse anti-RFP (Agrisera, AS153028, 1/1000). Peroxidase activity coupled to the Goat anti-mouse antibody at a dilution of 1/5000 (Bio-Rad #1706516) was used for revelation of tagRFP-ATG4. Anti-PIP2.7 (Agrisera, AS09 469, 1/5000) and anti-UGP (Agrisera, AS05086, 1/10000) antibodies were used as loading controls and markers of the membrane fraction (P100) and soluble fraction (S100), respectively. Peroxidase activity coupled to the Goat anti-Rabbit antibody at a dilution of 1/5000 (Bio-Rad #1721019) was used for revelation of both PIP2.7 and UGP.

The transcript abundance in samples was determined using a comparative threshold cycle method. The relative abundance of the reference mRNAs of ACT2/8 and AT4G33380 in each sample was determined and used to normalize for differences of total RNA amount according to the method described by [START_REF] Snare Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF].

How lipids instruct plant autophagy?

This was the initial question of my thesis. The autophagic process relies on the formation of autophagic membranes that mediate the engulfment and delivery of the cargo to be degraded to the vacuole. In order to form these membranes, members of the core autophagy machinery (ATG genes) are sequentially recruited to these membranes and mediate different steps in the formation of autophagic membranes. However, recent findings have highlighted the importance of the other structural components of these membranes: lipids. The lipid composition of autophagic membranes dictates critical features that ensure their biological functions. For example, most ATG proteins are only transiently recruited to autophagic structures and their recruitment is in many cases the result of protein-lipid interaction.

Furthermore, AP biogenesis is a highly dynamic process, characterized by a wide range of membrane dynamic events that mediate its progression but in the same time maintain the particular shapes adopted by these structures during their formation. In this manner, the objectives of my PhD were to identify the lipids that participate in the formation of these autophagic membranes and characterize their function to get a better understanding of AP formation in plants.

IV-1 Regarding the identification and functional characterization of lipids required for autophagy in plants cells

To functionally characterize the impact of lipids in autophagy, we performed two types of initial analyses in parallel: (1) an inhibitor screen to identify critical lipids for autophagy activity and

(2) an in-depth analysis of gene co-expression to identify lipid-related genes potentially involved in autophagy as candidates for regulating lipid metabolism, trafficking, or membrane remodeling events upon autophagy induction. Manipulating these proteins by reverse genetics (KO, OE) would allow to modulate lipid-related events during autophagy and observe their impact on the pathway thus characterizing their function in plant autophagy.

To identify lipids required for autophagy in plant cells, we performed a screen in which we tested the effect of blocking the synthesis of different lipids on autophagic activity. Results from this screen showed that blocking the synthesis of distinct lipids (from distinct lipid classes) differently affected autophagy. Some treatments increased considerably the autophagic flux, others decreased it, and some had simply no effect (Figure 27). These GFP-ATG8a x pi4kβ1 pi4kβ2 GFP-ATG8a Proteins that bear lipid binding domains (LBD) can bind to membranes that contain these lipids. On the other hand, lipids that bear charged polar heads (like anionic phospholipids) can interact with positively charged proteins by electrostatic interactions.

V-Experimental procedures

Arabidopsis lines

All experiments performed in the manuscript were performed in Arabidopsis of the ecotype Col-0, as wild-type, or derived of the ecotype Col-0. The following transgenic lines were used as previously described 35S::GFP-ATG8a (Thompson et al., 2005).

Arabidopsis lines stably expressing PLA1-tagRFP, PLA2-tagRFP or PLA2-tagBFP were generated as following. PLA1 and PLA2 coding sequences, as well as each promoter sequences, were PCR-amplified using primers listed in Table S2. The promoter sequences were cloned into the pDONRP4-P1R plasmid and the coding sequences were cloned into the homozygous lines containing single construct insertions lines were selected based on hygromycin resistance. To generate amiRNA PLA2 lines, an artificial microRNA sequence specific to the the PLA2 gene (AT4G19860) was designed using the software WMD3 Web MicroRNA Designer [START_REF] Schwab | Highly Specific Gene Silencing by Artificial MicroRNAs in Arabidopsis[END_REF] (See Table S2). The amiRNA:PLA2 precursor fragment sequences were PCR amplified from the pRS300 plasmid as previsouly described [START_REF] Schwab | Highly Specific Gene Silencing by Artificial MicroRNAs in Arabidopsis[END_REF] using specific primers listed in Table S2. PCR products were cloned into the pENTR/D-TOPO vector (pENTR Directional TOPO Cloning kit, Invitrogen). Clones were verified by sequencing and then transferred by gateway LR cloning into the destination vector PMDC7 hich all e e i n f he an gene nde he c n l f -estradiol [START_REF] Curtis | A Gateway Cloning Vector Set for High-Throughput Functional Analysis of Genes in Planta[END_REF]. The constructs were transformed into A. thaliana GFP-ATG8a plants by floral dip; homozygous lines containing single construct insertions lines were selected based on hygromycin resistance. In order to test amiRNA efficiency, we compared the expression of PLA2 in each line compared to wild type upon treatment with 10 M -oestradiol in various conditions using RT-qPCR.

ATG5-BFP construct engineering

The sequence corresponding to the catalytic site of Sac1(Sac1 WT ) or SAC1-dead (Sac1 dead mutation: C392S, von Filseck et al., 2015) was PCR amplified from plasmids kindly provided by S. Mongrand [START_REF] Gronnier | GIPC : Glycosyl Inositol Phospho Ceramides , the major sphingolipids on earth[END_REF] using primers listed in Table S2. Using overlapping PCR, each sequence was fused to the mTagBFP2 sequence to generated a BFP-Sac1 WT or