Marie-Hélène Montané
email: marie-helene.montane@univ-amu.fr

Benoît Menand
email: benoit.menand@univ-amu.fr

TOR inhibitors: from mammalian outcomes to pharmacogenetics in plants and algae Review paper

Keywords: TOR, mammals, plants, algae, rapamycin, ATP-competitive TOR inhibitor

Introduction

Rapamycin together with the structurally related drug FK506 are immunosuppressive agents that are reciprocal antagonists of lymphocyte cell activation [START_REF] Sigal | Cyclosporin A, FK-506, and rapamycin: pharmacologic probes of lymphocyte signal transduction[END_REF]. Rapamycin stood out for its role in second phase of lymphocyte activation by inhibiting cell cycle and subsequently proliferation [START_REF] Aagaard-Tillery | Inhibition of Human B-Lymphocyte Cell-Cycle Progression and Differentiation by Rapamycin[END_REF]. A recent overview of rapamycin [START_REF] Yoo | An overview of rapamycin: from discovery to future perspectives[END_REF] describes the respective mechanisms of immunosuppressive action of FK506 that interferes with the phosphatase calcineurin and of rapamycin that interferes with the serine/threonine kinase "Target Of Rapamycin" (TOR). Both compounds bind to a single domain of the cytosolic immunophilin FKBP12 (12 kDa FK506 Binding Protein). Briefly, the FKBP12-rapamycin duo binds to the so-called FRB (FKBP12-Rapamycin Binding) domain of TOR therefore creating a ternary complex that inhibits TOR kinase activity through allosteric interaction. Throughout eukaryotes, TOR progressively emerged as a hub for orchestrating cellular anabolic and catabolic processes that basically characterize growth homeostasis, i.e., cell/organ size and cell proliferation as well as cell components turnover. "In simple terms, cell growth is the accumulation of mass. But this description short changes a process that is vastly more complex and interesting" [START_REF] Thoreen | The molecular basis of mTORC1-regulated translation[END_REF]. TOR interconnects numerous inputs and outputs of anabolism functions while repressing autophagy, ensuring growth homeostasis, i.e, the building up, the "stability" or survival of cells up to their aging and senescence or in response to any imbalance caused by stress, disease or energy changes [START_REF] Saxton | mTOR Signaling in Growth, Metabolism, and Disease[END_REF][START_REF] Thoreen | The molecular basis of mTORC1-regulated translation[END_REF].

Rapamycin was decisive for the discovery of TOR protein, basic TOR complexes (TORCs) components and targets [START_REF] Alessi | New Insights into mTOR Signaling: mTORC2 and Beyond[END_REF][START_REF] Huang | Rapamycins -Mechanism of action and cellular resistance[END_REF], yet the recent development of ATPcompetitive TOR inhibitors (active site TOR inhibitors, asTORis) brought new tools to study more in depth the TOR pathway. Furthermore, since the treatment of cancer by rapamycin and its derivatives rapalogs gave disappointing results, these second generation inhibitors also provided new possibilities of clinical trials aiming to cure cancer and other pathologies [START_REF] Martelli | Drug discovery targeting the mTOR pathway[END_REF]. In the context of studying TOR functions in plants, we aim to state here the use of rapamycin and of asTORis with an emphasis on their potential for pharmacogenetic studies in plant and algae.

Rapamycin and TOR complexes from yeast and mammals to plants and algae

For the historical steps on the discovery and naming of TOR, we invite the reader to rely on very informative articles of DA Sabatini and MN Hall (Hall, 2016;[START_REF] Sabatini | Twenty-five years of mTOR: Uncovering the link from nutrients to growth[END_REF]. The TOR protein kinase was first identified from a genetic screen of Saccharomyces cerevisiae (referred hereafter as yeast) lines that were resistant to rapamycin [START_REF] Heitman | Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast[END_REF]. Rapamycin-resistant lines mostly carried recessive missense mutations resulting in amino acid substitutions in the FKBP12 protein, but dominant missense mutations in two genes named TOR1 and TOR2 (Target Of Rapamycin 1 and 2) were also identified. Further studies revealed that mutations of a conserved Serine residue within the FRB domain of TOR1 or TOR2 confers dominant resistance to rapamycin [START_REF] Mclaughlin | Interaction between Fkbp12-Rapamycin and Tor Involves a Conserved Serine Residue[END_REF]. Soon after, three groups identified the "physical target of rapamycin" in mammals by biochemical approaches using rapamycin and FKBP12. TOR is a member of the atypical Ser/Thr-protein kinase of the PIKKs family that all play vital role in growth and survival and also includes essential regulators of the DNA damage response such as ATM (Ataxia-Telangiectasia Mutated), ATR (ATM-and Rad3-Related) and DNA-PK (DNA-dependent Protein Kinase) (De [START_REF] Cicco | Regulation of the Target of Rapamycin and Other Phosphatidylinositol 3-Kinase-Related Kinases by Membrane Targeting[END_REF]. Rapamycin has been an indispensable tool for studying the roles of the TOR pathway in both yeast and animals but rapamycin effects are more limited in animals on protein synthesis, autophagy and proliferation (Fig. 1A) and varied widely among cell types [START_REF] Mukhopadhyay | The Enigma of Rapamycin Dosage[END_REF][START_REF] Sarbassov | Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB[END_REF][START_REF] Thoreen | The molecular basis of mTORC1-regulated translation[END_REF][START_REF] Zhao | mTOR inhibition activates overall protein degradation by the ubiquitin proteasome system as well as by autophagy[END_REF]. Both genetic and biochemical studies identified two basic TOR complexes:

The rapamycin sensitive TORC1 containing RAPTOR/KOG1 (mammalian Regulatory Associated Protein of TOR/yeast Kontroller Of Growth 1) and LST8 (Lethal with SEC13 protein 8), and the rapamycin-insensitive TORC2 containing LST8 and RICTOR/AVO3 (mammalian Rapamycin-Insensitive Companion of mTOR/yeast Adheres-VOraciously-to-tor-2 protein 3). TORC2 components and downstream effectors have been difficult to characterize due to the absence of specific drugs that selectively inhibit this complex [START_REF] Gaubitz | TORC2 Structure and Function[END_REF][START_REF] Sparks | Targeting mTOR: prospects for mTOR complex 2 inhibitors in cancer therapy[END_REF] and because under prolonged (chronic and not acute) rapamycin treatment, TORC2 assembly was impaired [START_REF] Sarbassov | Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB[END_REF]. TORC2 is involved in cell survival and cytoskeleton regulation through different AGC family kinases including a key readout target kinase AKT, which phosphorylation requires SIN1/AVO1 (mammalian Stress-activated protein kinase-INteracting protein 1/yeast Adheres-VOraciously-to-target-of-rapamycin-2 protein 1), another essential component of TORC2 [START_REF] Gaubitz | TORC2 Structure and Function[END_REF]. Noticeably, SIN1 isoforms led to suggest occurrence of 3 different TORC2, showing plasticity of TOR complexes. A detailed composition of yeast and mammals TORC1 and TORC2 and the full range of downstream targets through which TOR drives cell growth has recently fully emerged and is extensively reviewed elsewhere [START_REF] Ben-Sahra | mTORC1 signaling and the metabolic control of cell growth[END_REF][START_REF] Eltschinger | TOR Complexes and the Maintenance of Cellular Homeostasis[END_REF][START_REF] Gaubitz | TORC2 Structure and Function[END_REF][START_REF] Gonzalez | The TOR Signaling Pathway in Spatial and Temporal Control of Cell Size and Growth[END_REF][START_REF] Jhanwar-Uniyal | Discrete signaling mechanisms of mTORC1 and mTORC2: Connected yet apart in cellular and molecular aspects[END_REF][START_REF] Saxton | mTOR Signaling in Growth, Metabolism, and Disease[END_REF].

The control of cell growth by TORC1 in response to nutrients was early demonstrated in yeast [START_REF] Barbet | TOR controls translation initiation and early G1 progression in yeast[END_REF] and later on transcriptional profiling showed that mammalian TORC1 upregulates sets of genes involved in lipid/sterol, nucleotide and protein synthesis, as well as genes involved in mitochondrial oxidative function, glycolysis and the pentose phosphate pathway and conversely down-regulated genes involved in starvation and energy production [START_REF] Duvel | Activation of a Metabolic Gene Regulatory Network Downstream of mTOR Complex 1[END_REF][START_REF] Peng | The immuno suppress ant rapamycin mimics a starvation-like signal distinct from amino acid and glucose deprivation[END_REF]. Briefly, under adequate conditions including growth factors, amino acids and AMP to ATP and/or ADP to ATP ratios, TORC1 phosphorylates two foremost targets involved in protein synthesis commitment and elongation, the eIF4E-Binding Protein1 (4E-BP1) and the ribosomal protein S6 Kinases (S6Ks) respectively. However, it is worth mentioning that the clear cut contribution of each TORC1-S6K1/S6K2 and TORC1-4E-BP1 axis in regulating cell cycle and proliferation as well as translation has been hard to delineate [START_REF] Cunningham | mTOR controls mitochondrial oxidative function through a YY1-PGC-1alpha transcriptional complex[END_REF]Dowling et al., 2010a;[START_REF] Magnuson | Regulation and function of ribosomal protein S6 kinase (S6K) within mTOR signalling networks[END_REF][START_REF] Meyuhas | Ribosomal protein S6 kinase from TOP mRNAs to cell size[END_REF][START_REF] Thoreen | The molecular basis of mTORC1-regulated translation[END_REF]. First, their kinetics of phosphorylation do not last the same and their different action in the regulation of protein synthesis machinery, which involves additional TOR targets, made it complex to decipher (Dowling et al., 2010a;[START_REF] Magnuson | Regulation and function of ribosomal protein S6 kinase (S6K) within mTOR signalling networks[END_REF][START_REF] Meyuhas | Ribosomal Protein S6 Phosphorylation: Four Decades of Research[END_REF][START_REF] Thoreen | The molecular basis of mTORC1-regulated translation[END_REF]. As such, the TORC1 target LARP1 (La-Related Protein 1) is a translation repressor that, according to a recent model, binds to the 5' end of mRNAs and thus competes with the translation initiation complex eIF4F (including eIF4E) and to some extent with S6K1 [START_REF] Vasseur | La-related protein 1 (LARP1) repression of TOP mRNA translation is mediated through its cap-binding domain and controlled by an adjacent regulatory region[END_REF]. TORC1 controls protein turnover through regulating UPS (Ubiquitin Proteasome System)-and UPS targeted-proteins abundance [START_REF] Rousseau | An evolutionarily conserved pathway controls proteasome homeostasis[END_REF][START_REF] Zhao | Control of proteasomal proteolysis by mTOR[END_REF] as well as by canonical autophagy induction through regulating activity of the kinases ULK1 and 2/ATG1 (Human Uncoordinated-51-like autophagy activating kinase 1and 2/yeast AuTophaGy related 1) [START_REF] Velazquez | So Many Roads: the Multifaceted Regulation of Autophagy Induction[END_REF][START_REF] Zhao | mTOR inhibition activates overall protein degradation by the ubiquitin proteasome system as well as by autophagy[END_REF]. Remarkably, due to reversible control of ULK1 by mTOR and AMP-activated Protein Kinase (AMPK) that senses low energy levels, mammalian growth homeostasis is orchestrated through dynamic signaling interplay of the triad of kinases, AMPK-TOR-ULK1. Under low energy, if ULK1 is activated through phosphorylation by AMPK, it can be impeded by TORC1 and in turn, ULK1-mediated phosphorylation can decrease activity of AMPK, establishing a negative feedback loop targeting the AMPK-mTOR signaling axis [START_REF] Dunlop | The kinase triad, AMPK, mTORC1 and ULK1, maintains energy and nutrient homoeostasis[END_REF][START_REF] Luo | Rapamycin inhibits mSin1 phosphorylation independently of mTORC1 and mTORC2[END_REF]. Also, a positive regulation loop occurs through phosphorylation by TOR and ULK1 of a component in autophagosome formation, which joins regulation of effectors by phosphorylation to regulation by ubiquitylation [START_REF] Nazio | mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6[END_REF].

More extensively, the crosstalk between different branches of the TOR network is nowadays upgraded by the emerging view that negative feedback loops where downstream targets become upstream regulators might be critical in the TOR pathway [START_REF] Eltschinger | TOR Complexes and the Maintenance of Cellular Homeostasis[END_REF]. As such, the negative feedback loop of the TOR-S6K-IRS1 (Insulin Receptor Substrate 1) axis in response to TORC1 activation that is mediated by S6K1 attenuates PI3K-AKT signaling by phosphorylating IRS1 and RICTOR leading to AKT kinase inhibition. These two examples of feedback loops state the importance of characterizing cell developmental or metabolic status when deciphering the role of specific TOR pathway effectors as physiology "customizes" TOR signaling backbone status. For instance, in the field of TOR-driven aging, cell entry into senescence is decelerated by rapamycin, preventing irreversible loss of proliferation capacity through inhibiting the senescence-associated secretory phenotype of cells without affecting cell cycle arrest [START_REF] Wang | Rapamycin and the inhibition of the secretory phenotype[END_REF]. This led defining new concepts and so new terms in order to delineate clear-cut functions of effectors in cell cycle arrest and/or senescence [START_REF] Blagosklonny | Cell cycle arrest is not yet senescence, which is not just cell cycle arrest: terminology for TOR-driven aging[END_REF]. Another important feature is that TOR basic targets S6Ks, 4E-BPs, ULK1 or components of TOR complexes (SIN1, RAPTOR, RICTOR) very often carry multiple phosphorylation sites, which likewise helps connecting different signaling pathways to the TOR pathway to maintain cell homeostasis but makes analysis more complex [START_REF] Batool | Reappraisal to the study of 4E-BP1 as an mTOR substrate -A normative critique[END_REF][START_REF] Meyuhas | Ribosomal Protein S6 Phosphorylation: Four Decades of Research[END_REF][START_REF] Tavares | The S6K protein family in health and disease[END_REF]. At last but not least, the recent discovery of new TOR complexes that do not contain RAPTOR or RICTOR reveals the extent of TOR function. As such, a complex TOR-RanBP2 (Ran Binding Protein 2) that ensures dynamic flux of nuclear import of ribosomal proteins [START_REF] Kazyken | The nuclear import of ribosomal proteins is regulated by mTOR[END_REF], a complex TOR-GIT1 (G-protein-coupled receptor kinaseinteracting protein 1) essential for astrocyte survival [START_REF] Smithson | Proteomic analysis reveals GIT1 as a novel mTOR complex component critical for mediating astrocyte survival[END_REF], a rapamycin insensitive TORC3 including at least LST8 and an unknown protein phosphorylating mSIN1 [START_REF] Luo | Rapamycin inhibits mSin1 phosphorylation independently of mTORC1 and mTORC2[END_REF], or a new rapamycin sensitive TORC acting on mRNA translation [START_REF] Meyuhas | Ribosomal Protein S6 Phosphorylation: Four Decades of Research[END_REF] have been identified. Another cytoplasmic TORC3 activated in cancer solely contains mTOR, 4E-BP1 and the transcription factor ETV7 (leukemia virus E26 Transformation-specific Variant 7) but not the TORC1/2 crucial components LST8, RAPTOR, RICTOR or SIN1 [START_REF] Harwood | ETV7 is an essential component of a rapamycininsensitive mTOR complex in cancer[END_REF]. TOR complexes can have various intracellular localization, close to either the nucleus or the perinuclear region, lysosomes, mitochondria-associated endoplasmic reticulum membranes or plasma membrane depending on nutrient status [START_REF] Betz | Where is mTOR and what is it doing there?[END_REF][START_REF] Jhanwar-Uniyal | Discrete signaling mechanisms of mTORC1 and mTORC2: Connected yet apart in cellular and molecular aspects[END_REF]. This also holds true for the target S6K [START_REF] Tavares | The S6K protein family in health and disease[END_REF] and altogether this makes TORCs eclectic, in coherence with the role of TOR in cell growth homeostasis. Altogether, the discovery of new TORCs, their diverse intracellular localization, the interaction of TOR-and other-signaling pathways and the multiple phosphorylation sites of TOR pathway components reflect the deployment of TOR signaling and the importance to decipher its role in clearly defined cellular contexts.

In plants, early studies benefited from the conservation of TOR among species and the libraries of Arabidopsis insertion mutants which helped find knock-out mutants of homologs of yeast and mammalian genes encoding basic members of TORCs. Thus, Arabidopsis genome contains one TOR gene (AtTOR) (Menand-2002), two RAPTOR genes [START_REF] Anderson | The Arabidopsis AtRaptor genes are essential for post-embryonic plant growth[END_REF][START_REF] Deprost | An Arabidopsis homolog of RAPTOR/KOG1 is essential for early embryo development[END_REF][START_REF] Mahfouz | Arabidopsis TARGET OF RAPAMYCIN interacts with RAPTOR, which regulates the activity of S6 kinase in response to osmotic stress signals[END_REF][START_REF] Rexin | TOR signalling in plants[END_REF][START_REF] Salem | RAPTOR Controls Developmental Growth Transitions by Altering the Hormonal and Metabolic Balance[END_REF] and two LST8 genes [START_REF] Moreau | Mutations in the Arabidopsis homolog of LST8/GbetaL, a partner of the target of Rapamycin kinase, impair plant growth, flowering, and metabolic adaptation to long days[END_REF]. Arabidopsis raptor mutants are still under study and sporadic embryonic arrest has been controversial likely due to poor quality of some insertion mutants [START_REF] Rexin | TOR signalling in plants[END_REF], making it different from mammals where RAPTOR ablation is associated with male sterility (Xiong et al., 2017b). LST8s function is still in progress since only lst8-1 mutant phenotype and not lst8-2 is documented, yet altered growth and particularly metabolomic phenotype of lts8-1 reminds amino acid accumulation observed in yeast lst8 mutants [START_REF] Moreau | Mutations in the Arabidopsis homolog of LST8/GbetaL, a partner of the target of Rapamycin kinase, impair plant growth, flowering, and metabolic adaptation to long days[END_REF]. Thus, in the absence of RICTOR homologs, only basic TORC1 is characterized in plants and algae until now (Dobrenel et al., 2016a;[START_REF] Perez-Perez | The TOR Signaling Network in the Model Unicellular Green Alga Chlamydomonas reinhardtii[END_REF][START_REF] Van Dam | Evolution of the TOR Pathway[END_REF]. The main plant TOR targets include S6K1 and S6K2, which are both related to mammalian S6K1, and the PP2A (Protein Phosphatase 2A) regulatory subunit TAP46 [START_REF] Ahn | The PP2A regulatory subunit Tap46, a component of the TOR signaling pathway, modulates growth and metabolism in plants[END_REF][START_REF] Henriques | Arabidopsis S6 kinase mutants display chromosome instability and altered RBR1-E2F pathway activity[END_REF][START_REF] Mahfouz | Arabidopsis TARGET OF RAPAMYCIN interacts with RAPTOR, which regulates the activity of S6 kinase in response to osmotic stress signals[END_REF][START_REF] Xiong | Rapamycin and glucose-target of rapamycin (TOR) protein signaling in plants[END_REF]. TOR negatively regulates autophagy also in plants and green algae [START_REF] Liu | TOR Is a Negative Regulator of Autophagy in Arabidopsis thaliana[END_REF][START_REF] Perez-Perez | Inhibition of Target of Rapamycin Signaling and Stress Activate Autophagy in Chlamydomonas reinhardtii[END_REF] and even though convergence of UPS and autophagy has been demonstrated in plants [START_REF] Marshall | Autophagic Degradation of the 26S Proteasome Is Mediated by the Dual ATG8/Ubiquitin Receptor RPN10 in Arabidopsis[END_REF] TOR dependent regulation of UPS is still unknown. Strikingly, the catalytic subunit KIN10 of SnRK1 (Snf1-Related protein Kinase 1), the plant homolog of mammalian AMPK/yeast SNF1, regulates autophagy through inhibiting TOR and SnRK1 is not regulated by the AMP/ATP ratio similarly to yeast SNF1 and contrarily to mammalian AMPK (Soto-Burgos and [START_REF] Soto-Burgos | SnRK1 activates autophagy via the TOR signaling pathway in Arabidopsis thaliana[END_REF]. The position of the ATG1/13 kinase complex in autophagy is also central in plants with four isoforms of the ATG1 kinase, two of its partner ATG13 [START_REF] Suttangkakul | The ATG1/ATG13 protein kinase complex is both a regulator and a target of autophagic recycling in Arabidopsis[END_REF] and accessory ATG proteins such as ATG11 [START_REF] Li | Arabidopsis ATG11, a scaffold that links the ATG1-ATG13 kinase complex to general autophagy and selective mitophagy[END_REF] reported in Arabidopsis, yet ATG1 phosphorylation by TOR has not been demonstrated (Wang et al., 2018a). Thus to control autophagy in plants, TOR might target ATG13 to regulate ATG1 similarly to yeast [START_REF] Kamada | Tor Directly Controls the Atg1 Kinase Complex To Regulate Autophagy[END_REF] rather than regulating both ATG13 and ULK1 as in mammals [START_REF] Kim | AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1[END_REF]. Interestingly, Arabidopsis ATG1 has a dual role through acting as a regulator and as a substrate of autophagy, likely a particular feature of plants [START_REF] Bassham | Function and regulation of macroautophagy in plants[END_REF][START_REF] Suttangkakul | The ATG1/ATG13 protein kinase complex is both a regulator and a target of autophagic recycling in Arabidopsis[END_REF]. However, as plant autophagy effectors and processes are still under study [START_REF] Masclaux-Daubresse | Regulation of nutrient recycling via autophagy[END_REF]Wang et al., 2018a), this field requires more investigation. Other plant TOR targets were also identified [START_REF] Shi | TOR signaling in plants: conservation and innovation[END_REF], including the transcription factors E2FA and E2FB which phosphorylation in vitro is lost by treatment with ATP-competitive inhibitors (Torins, see below) [START_REF] Li | Differential TOR activation and cell proliferation in Arabidopsis root and shoot apexes[END_REF][START_REF] Xiong | Glucose-TOR signalling reprograms the transcriptome and activates meristems[END_REF], or the hormone abscisic acid-receptor PYL1 (PYrabactin resistance 1-Like 1), which activity is associated with stress and senescence (Wang et al., 2018b). In the absence of plant homologs of 4E-BP1, the axis TORC1-S6Ks is nowadays the most studied link between TOR and translation in plants, mainly through read out of ribosomal protein S6 phosphorylation (Dobrenel et al., 2016b;[START_REF] Mahfouz | Arabidopsis TARGET OF RAPAMYCIN interacts with RAPTOR, which regulates the activity of S6 kinase in response to osmotic stress signals[END_REF][START_REF] Xiong | Rapamycin and glucose-target of rapamycin (TOR) protein signaling in plants[END_REF]. However, the recent discovery of a Conserved Binding of eif4E1 (CBE1) plant protein [START_REF] Patrick | Discovery and characterization of conserved binding of eIF4E 1 (CBE1), a eukaryotic translation initiation factor 4E-binding plant protein[END_REF] opens new possibilities of link between TOR and translation initiation in plants.

In Chlamydomonas reinhardtii, recent phosphoproteomic studies identified TOR-inhibition dependent phosphorylation of proteins including ATG7, S6K, the ribosomal protein S6 and LARP1 [START_REF] Roustan | Quantitative Phosphoproteomic and System-Level Analysis of TOR Inhibition Unravel Distinct Organellar Acclimation in Chlamydomonas reinhardtii[END_REF][START_REF] Werth | Investigating the effect of target of rapamycin kinase inhibition on the Chlamydomonas reinhardtii phosphoproteome: from known homologs to new targets[END_REF] showing conservation of effectors in algae and opening new avenues of TOR pathway characterization. In the red alga Cyanidioschyzon merolae, a phosphoproteomic analysis with the a transgenic strain overexpressing yeast FKBP12 identified GLG1, an authentic GLycoGenin which phosphorylation is cancelled by rapamycin [START_REF] Pancha | Target of rapamycin-signaling modulates starch accumulation via glycogenin phosphorylation status in the unicellular red alga Cyanidioschyzon merolae[END_REF]. As systems biology and omics now start connecting TOR pathway with specific aspects of plant and algae physiology, new TOR targets could be discovered soon in photosynthetic organisms [START_REF] Caldana | Systemic analysis of inducible target of rapamycin mutants reveal a general metabolic switch controlling growth in Arabidopsis thaliana[END_REF]Dobrenel et al., 2016a;[START_REF] Mubeen | Target of Rapamycin Inhibition in Chlamydomonas reinhardtii Triggers de-novo Amino Acid Synthesis by Enhancing Nitrogen Assimilation[END_REF]. New TOR complexes might exist in plants and algae, as discovered in animals, but their future identification would need more specific biochemical or genetic studies. Altogether, these data show that the TOR pathway includes conserved and specific effectors in photosynthetic organisms and thus its study benefits from outcomes from yeast and mammalian studies, as well as from plant and/or algae specific investigations.

Rapamycin-FKBP12 -TOR inhibition in plants and algae: not an easy game

In algal species, rapamycin sensitivity is species-dependent and growth inhibition level (GI %) and doses (nM) are highly variable as they range from (40%; 100 nM) for Chlamydomonas reinhardtii [START_REF] Crespo | Inhibition of target of rapamycin signaling by rapamycin in the unicellular green alga Chlamydomonas reinhardtii[END_REF], to (40%; 50,000 nM) for Euglena gracilis [START_REF] Mukaida | The effect of rapamycin on biodieselproducing protist Euglena gracilis[END_REF], to (slight effect; 10,000 nM) for the diatom Phaeodactylum tricornutum [START_REF] Prioretti | The inhibition of TOR in the model diatom Phaeodactylum tricornutum promotes a get-fat growth regime[END_REF] up to (0%; 1,000 nM) for the red algae C. merolae [START_REF] Imamura | Expression of budding yeast FKBP12 confers rapamycin susceptibility to the unicellular red alga Cyanidioschyzon merolae[END_REF]. However, chlorophyll content decreased in E. gracilis and C. merolae from 1,000 nM but not in C. reinhardtii [START_REF] Mukaida | The effect of rapamycin on biodieselproducing protist Euglena gracilis[END_REF]. A rapamycin resistant FKBP12 loss-of-function mutant in C. reinhardtii allowed to demonstrate that rapamycin inhibits proliferation via the rapamycin-FKBP12 interaction, a strong argument for the further use of rapamycin in this alga [START_REF] Crespo | Inhibition of target of rapamycin signaling by rapamycin in the unicellular green alga Chlamydomonas reinhardtii[END_REF]. These few data show that rapamycin is not a general potent TOR inhibitor in algae species, reminding the variety of background responses of mammalian cell lines. In vascular plants, rapamycin hardly inhibits growth of various genera including Arabidopsis, Nicotiana, cotton or potato plantlets with some peculiar cases like tomato where partial growth inhibition has been observed [START_REF] Deng | The TOR Pathway Is Involved in Adventitious Root Formation in Arabidopsis and Potato[END_REF][START_REF] Deng | Target of Rapamycin Is a Key Player for Auxin Signaling Transduction in Arabidopsis[END_REF][START_REF] Mahfouz | Arabidopsis TARGET OF RAPAMYCIN interacts with RAPTOR, which regulates the activity of S6 kinase in response to osmotic stress signals[END_REF][START_REF] Menand | Expression and disruption of the Arabidopsis TOR (target of rapamycin) gene[END_REF][START_REF] Montane | ATP-competitive mTOR kinase inhibitors delay plant growth by triggering early differentiation of meristematic cells but no developmental patterning change[END_REF][START_REF] Ren | Target of rapamycin signaling regulates metabolism, growth, and life span in Arabidopsis[END_REF][START_REF] Song | The crosstalk between Target of Rapamycin (TOR) and Jasmonic Acid (JA) signaling existing in Arabidopsis and cotton[END_REF][START_REF] Sormani | Saccharomyces cerevisiae FKBP12 binds Arabidopsis thaliana TOR and its expression in plants leads to rapamycin susceptibility[END_REF][START_REF] Xiong | Tomato FK506 Binding Protein 12KD (FKBP12) Mediates the Interaction between Rapamycin and Target of Rapamycin (TOR)[END_REF]. Insensitivity or weak sensitivity to rapamycin has been attributed to low ability of plant FKBP12 proteins to form the inhibitory ternary complex with rapamycin due to lack of conservation of aminoacid residues critical for interaction with rapamycin (Supplementary Fig. S1) [START_REF] Choi | Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP[END_REF][START_REF] Sormani | Saccharomyces cerevisiae FKBP12 binds Arabidopsis thaliana TOR and its expression in plants leads to rapamycin susceptibility[END_REF][START_REF] Xu | Molecular characterization of a plant FKBP12 that does not mediate action of FK506 and rapamycin[END_REF]. A similar situation was described in red algae [START_REF] Imamura | Expression of budding yeast FKBP12 confers rapamycin susceptibility to the unicellular red alga Cyanidioschyzon merolae[END_REF]. There is no straightforward evolutionary explanation for this particular feature of FKBP12s of plants and some algae, but we could speculate the selection of new FKBP12 endogenous peptidyl-prolyl isomerase functions or a selective advantage to resist to the soil Streptomycete that produces rapamycin [START_REF] Vezina | Rapamycin (Ay-22,989), a New Antifungal Antibiotic .1. Taxonomy of Producing Streptomycete and Isolation of Active Principle[END_REF]. Neither the yeast two-hybrid analysis nor an in vitro interaction assay could demonstrate a rapamycin-dependent interaction between Arabidopsis FKBP12 (AtFKBP12) and the AtTOR-FRB domain but this domain was able to form a complex with human (Hs) or yeast FKBP12 [START_REF] Mahfouz | Arabidopsis TARGET OF RAPAMYCIN interacts with RAPTOR, which regulates the activity of S6 kinase in response to osmotic stress signals[END_REF][START_REF] Menand | Expression and disruption of the Arabidopsis TOR (target of rapamycin) gene[END_REF][START_REF] Sormani | Saccharomyces cerevisiae FKBP12 binds Arabidopsis thaliana TOR and its expression in plants leads to rapamycin susceptibility[END_REF]. As a consequence, Arabidopsis plants could be made sensitive to rapamycin by overexpression of yeast or human FKBP12 [START_REF] Deng | Target of Rapamycin Is a Key Player for Auxin Signaling Transduction in Arabidopsis[END_REF][START_REF] Leiber | The TOR pathway modulates the structure of cell walls in Arabidopsis[END_REF][START_REF] Ren | Target of rapamycin signaling regulates metabolism, growth, and life span in Arabidopsis[END_REF][START_REF] Sormani | Saccharomyces cerevisiae FKBP12 binds Arabidopsis thaliana TOR and its expression in plants leads to rapamycin susceptibility[END_REF][START_REF] Xiong | Rapamycin and glucose-target of rapamycin (TOR) protein signaling in plants[END_REF]. A yeast-FKBP12 overexpressing line in the red alga C. merolae similarly confers sensitivity to 10-500 nM rapamycin [START_REF] Imamura | Expression of budding yeast FKBP12 confers rapamycin susceptibility to the unicellular red alga Cyanidioschyzon merolae[END_REF]. However, to our opinion, the dogma of plant TOR kinase inhibition by rapamycin through transgenic FKBP12 overexpression deserves little bit more attention.

Several groups reported that Arabidopsis seedlings grown on solid media are insensitive to rapamycin up to ca. 10 µM [START_REF] Deng | Target of Rapamycin Is a Key Player for Auxin Signaling Transduction in Arabidopsis[END_REF][START_REF] Mahfouz | Arabidopsis TARGET OF RAPAMYCIN interacts with RAPTOR, which regulates the activity of S6 kinase in response to osmotic stress signals[END_REF][START_REF] Ren | Target of rapamycin signaling regulates metabolism, growth, and life span in Arabidopsis[END_REF][START_REF] Sormani | Saccharomyces cerevisiae FKBP12 binds Arabidopsis thaliana TOR and its expression in plants leads to rapamycin susceptibility[END_REF]. Such concentration range is 100-1000 times the concentration that inhibits proliferation of yeast (100 nM block cells in G1 with large unbudded cells as the terminal phenotype [START_REF] Heitman | Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast[END_REF] or that reduces cell size and proliferation of lymphocytes B cells (EC50 0.005-0.5 nM and maximal inhibition of ca. 50-70% up to 100 nM) or of mouse embryonic fibroblasts (50-250 nM) [START_REF] Sarbassov | Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB[END_REF][START_REF] Thoreen | Rapamycin inhibits mTORC1, but not completely[END_REF][START_REF] Wicker | Suppression of B cell activation by cyclosporin A, FK506 and rapamycin[END_REF]. Later, AtTOR-dependent phosphorylation at P-T449 (equivalent to T389 in animal) of AtS6K1 overproduced in transfected protoplasts was found inhibited by far much lower concentrations of rapamycin when FKBP12 was co-expressed compared to the AtS6K alone. Indeed, 100 to 1000 times lower concentration of rapamycin was needed to erase S6K1 P-T449 when S6K1 was overexpressed in combination with either AtFKBP12 or HsFKBP12 respectively [START_REF] Xiong | Rapamycin and glucose-target of rapamycin (TOR) protein signaling in plants[END_REF]. This showed that a high amount of HsFKBP12 "optimize" the titration of rapamycin to inhibit plant TOR. Thus, the affinity to rapamycin and the stoichiometry of each component of the ternary complex might influence the stability of TOR complex conformation shift and therefore the outcome of TOR inhibition. In other words, the poorest the interaction FKBP12-rapamycin is, the highest the rapamycin concentration is required to erase AtS6K1 P-T449. Thus, if we consider that this is the rule despite the peculiar physiological context of protoplasts incubated in mannitol and KCl in which it has been studied (no nutrients), a low amount of AtFKBP12 together with a poor binding of endogenous AtFKBP12 to AtTOR can explain why plants are poorly sensitive to rapamycin. Deng et al. similarly developed transgenic plants overexpressing FKBP12 coming from Arabidopsis, yeast and human but showed that AtFKBP12 overexpression could not make plants sensitive to rapamycin (Fig. 1C) [START_REF] Deng | Target of Rapamycin Is a Key Player for Auxin Signaling Transduction in Arabidopsis[END_REF]. This discrepancy with the data of Xiong and Sheen [START_REF] Xiong | Rapamycin and glucose-target of rapamycin (TOR) protein signaling in plants[END_REF], shows the possible drawback of building transgenic lines to overexpress FKBP12 (FKBP12 OX ). Yet, the overexpressed yeast FKBP12 was more efficient than HsFKBP12 to increase plant sensitivity to rapamycin (Fig. 1C) [START_REF] Deng | Target of Rapamycin Is a Key Player for Auxin Signaling Transduction in Arabidopsis[END_REF].

Growth conditions also influence rapamycin sensitivity as Arabidopsis seedlings grown in liquid culture were reported sensitive to rapamycin [START_REF] Deng | Target of Rapamycin Is a Key Player for Auxin Signaling Transduction in Arabidopsis[END_REF][START_REF] Xiong | Glucose-TOR signalling reprograms the transcriptome and activates meristems[END_REF][START_REF] Xiong | Rapamycin and glucose-target of rapamycin (TOR) protein signaling in plants[END_REF]). An interesting explanation for the discrepancy between plants grown on liquid and solid media was proposed by M Ren and colleagues who suggested that a hypoxia stress could facilitate rapamycin action in Arabidopsis [START_REF] Deng | Target of Rapamycin Is a Key Player for Auxin Signaling Transduction in Arabidopsis[END_REF]. Indeed, growth is dramatically slowed down in liquid media as after 9 days, seedlings roots were around 2-3 cm long [START_REF] Xiong | Rapamycin and glucose-target of rapamycin (TOR) protein signaling in plants[END_REF], which is around 2-3 times less than vertically grown seedlings on solidified medium [START_REF] Montane | ATP-competitive mTOR kinase inhibitors delay plant growth by triggering early differentiation of meristematic cells but no developmental patterning change[END_REF][START_REF] Ren | Target of rapamycin signaling regulates metabolism, growth, and life span in Arabidopsis[END_REF]. It is therefore likely that hypoxic stress might upregulate AtFKBP12 expression as FKBPs are reported to have a role in stress response [START_REF] Dong | Genome-wide analyses of genes encoding FK506-binding proteins reveal their involvement in abiotic stress responses in apple[END_REF][START_REF] Geisler | Tete-a-tete: the function of FKBPs in plant development[END_REF] and/or that AtTOR is largely inhibited in this condition which might make AtTOR activity easier to be inhibited by rapamycin. Interestingly, the sensitivity of Arabidopsis seedlings to the rapamycin structurally related compound FK506 (that binds FKBP12 but not TOR), was tested in WT and yeast-FKBP12 OX lines [START_REF] Zhang | ScFKBP12 bridges rapamycin and AtTOR in Arabidopsis[END_REF]. The authors concluded that FK506 has no effect on seedlings growth but the seedling phenotype shown after 25 days on half strength Murashige and Skoog (MS) medium containing 20 µM FK506 appears to us different from the control. Indeed, roots hardly grew in the solid medium but grew in the air outside the solid matrix, a feature already observed after 9 days on non-optimal full strength 1xMS medium [START_REF] Ren | Target of rapamycin signaling regulates metabolism, growth, and life span in Arabidopsis[END_REF]. This altered growth is characteristic of a stress due to non-optimal or to toxic drug containing medium and might reveal potential TOR-rapamycin-independent phenotypes. Thus, we think that more detailed analysis is required before ruling out that FK506 affects or not the physiology of yeast-FKBP12 OX lines.

Additionally, Arabidopsis FKBP12 was shown to interact with a nuclear protein that controls endoreduplication and therefore might control this process as in mammals [START_REF] Vespa | The immunophilin-interacting protein AtFIP37 from Arabidopsis is essential for plant development and is involved in trichome endoreduplication[END_REF]. As in the case of calcineurin, HsFKBP12 is also a subunit of the transforming growth factor B (TGFβ) type Ι receptor, a transmembrane Ser/Thr kinase that regulates cell growth and differentiation [START_REF] Gold | Type I (RI) and type II (RII) receptors for transforming growth factor-beta isoforms are expressed subsequent to transforming growth factor-beta ligands during excisional wound repair[END_REF]. HsFKBP12 -/-cells show cell cycle arrest due to impaired regulation of TGF-β receptor signaling [START_REF] Aghdasi | FKBP12, the 12-kDa FK506-binding protein, is a physiologic regulator of the cell cycle[END_REF]. Thus, it appears relevant to wonder whether in the absence of rapamycin AtFKBP12 is involved in cell cycle regulation or other signaling regulations that might interfere with TOR signaling studies with FKBP12 OX lines. Indeed, expressing an heterologous FKBP12 gene might also change plant physiology as the PaFKBP12 gene from the Antarctic moss Polytrichastrum alpinum ectopically expressed in Arabidopsis increases plant stress tolerance [START_REF] Alavilli | Enhanced multiple stress tolerance in Arabidopsis by overexpression of the polar moss peptidyl prolyl isomerase FKBP12 gene[END_REF]. Thus, even though FKBP12 OX lines do not have a macroscopic phenotype [START_REF] Deng | Target of Rapamycin Is a Key Player for Auxin Signaling Transduction in Arabidopsis[END_REF][START_REF] Sormani | Saccharomyces cerevisiae FKBP12 binds Arabidopsis thaliana TOR and its expression in plants leads to rapamycin susceptibility[END_REF], it does not preclude conditional cell/tissue responses compared to WT.

Not least, when regarding the other component of the duo FKBP-Rapamycin, a concentration range of 0.5-5 µM rapamycin was shown to interfere with interactions of the core particle 20S with its cellular activators and consequently to inhibit proteasome by attenuating major peptidase activities [START_REF] Osmulski | Rapamycin allosterically inhibits the proteasome[END_REF]. The authors hypothesized that interactions of the proteasome with rapamycin induce a maximal conformation shift of the proteasome, which results in compromised gating of substrates. Such an off target of rapamycin might interfere with other functions independently of the effects of TOR inhibition. Whether proteasome is an off target of rapamycin also in plants is unknown but should be carefully considered as proteolysis is enhanced following TOR inhibition [START_REF] Zhao | mTOR inhibition activates overall protein degradation by the ubiquitin proteasome system as well as by autophagy[END_REF] and UPS and autophagy converge in Arabidopsis [START_REF] Marshall | Autophagic Degradation of the 26S Proteasome Is Mediated by the Dual ATG8/Ubiquitin Receptor RPN10 in Arabidopsis[END_REF]. At last, another hint is the putative role of metabolites interacting with TOR such as phosphatidic acid that might impede interaction rapamycin-TOR and explain the strong variation of rapamycin dose to inhibit TOR in different mammalian cell lines [START_REF] Mukhopadhyay | The Enigma of Rapamycin Dosage[END_REF]. Anyhow, altogether this underscores that FKBP12 and/or rapamycin dosage as well as the cell physiology might be carefully controlled as they influence TOR inhibition-dependent results. This also opens the question of the selectivity of rapamycin. Therefore, compared to yeast and mammals, the conditional FKBP12 overexpressiondependent allosteric inhibition of TOR by rapamycin in plants might easily turn to be a conundrum.

Another aspect to underline is that for each combination of FKBP12 OX lines, the dose response to rapamycin shows that growth cannot be completely inhibited. Seedlings growth of yeast-FKBP12 OX lines was partially inhibited by 1-20 µM rapamycin [START_REF] Deng | Target of Rapamycin Is a Key Player for Auxin Signaling Transduction in Arabidopsis[END_REF][START_REF] Ren | Target of rapamycin signaling regulates metabolism, growth, and life span in Arabidopsis[END_REF] or by even lower range of 10-100 nM [START_REF] Zhang | ScFKBP12 bridges rapamycin and AtTOR in Arabidopsis[END_REF]. Anyhow, routine rapamycin concentration ranges are 4-10 µM to inhibit such lines [START_REF] Leiber | The TOR pathway modulates the structure of cell walls in Arabidopsis[END_REF][START_REF] Sormani | Saccharomyces cerevisiae FKBP12 binds Arabidopsis thaliana TOR and its expression in plants leads to rapamycin susceptibility[END_REF][START_REF] Xiong | Rapamycin and glucose-target of rapamycin (TOR) protein signaling in plants[END_REF], which makes it hard to appreciate rapamycin potency in plants. However, growth inhibition of yeast-FKBP12 OX plants by rapamycin never exceeds a plateau value of ca. 50% (Fig. 1C), which reminds the incomplete efficacy of rapamycin action observed in mammals (Fig. 1A andC). Similarly, in the green algae C. reinhardtii, which is naturally sensitive to rapamycin (Supplementary Fig. S1) in both solid and liquid media, maximal growth inhibition was also ca. 50% (Fig. 1E) [START_REF] Crespo | Inhibition of target of rapamycin signaling by rapamycin in the unicellular green alga Chlamydomonas reinhardtii[END_REF][START_REF] Juppner | The target of rapamycin kinase affects biomass accumulation and cell cycle progression by altering carbon/nitrogen balance in synchronized Chlamydomonas reinhardtii cells[END_REF][START_REF] Roustan | Quantitative Phosphoproteomic and System-Level Analysis of TOR Inhibition Unravel Distinct Organellar Acclimation in Chlamydomonas reinhardtii[END_REF]. Therefore, in both yeast-FKBP12 OX plant lines and rapamycin-sensitive WT algae challenged till now, full growth inhibition cannot be reached with rapamycin (Fig. 1C andD), making rapamycin efficacy not maximal. With that in mind, we can conclude that rapamycin can be used with more confidence in Chlamydomonas reinhardtii than in plants and other algae as, even if TOR inhibition is probably partial, transgenic over-expression of FKBP12s is avoided.

Inhibition of TOR by ATP competitive inhibitors: a new deal

Within this highly dynamic research field, studies of TOR kinase have considerably increased over the last 10 years and limitations of allosteric rapamycin-and rapalogs-based clinical strategies have pushed toward the development of orthosteric ATP-competitive mTOR inhibitors that were called asTORis (active site TOR inhibitors), TORKis or TKIs (TOR Kinase Inhibitor) [START_REF] Martelli | Drug discovery targeting the mTOR pathway[END_REF]. In contrast to rapamycin, they target the kinase domain of mTOR and are able to fully inhibit mammalian TORC1 activity in a dose dependent manner but also TORC2 and other TOR complexes (Fig. 1A andB) [START_REF] Chresta | AZD8055 is a potent, selective, and orally bioavailable ATPcompetitive mammalian target of rapamycin kinase inhibitor with in vitro and in vivo antitumor activity[END_REF]Dowling et al., 2010b;[START_REF] Harwood | ETV7 is an essential component of a rapamycininsensitive mTOR complex in cancer[END_REF][START_REF] Kang | mTORC1 phosphorylation sites encode their sensitivity to starvation and rapamycin[END_REF].

Here we would like to remind that the terms of potency, efficacy, selectivity, metabolic stability, off rate (also called associated residence time), and off targets as well as pharmacokinetic characteristics of a drug at the organism level (PK), altogether define a drug singularity. Potency and efficacy are parameters that are derived from graded dose-effect curves and that can be used to compare drugs that elicit the same pharmacological effect (Mosby's Medical Dictionary, 2009).

Potency, which is a measure of the sensitivity of a target organ or tissue to a drug, is a relative term that relates the amount of one drug required to produce a desired level of effect to the amount of a different drug required to produce the same effect. On the semi-logarithmic graded dose-effect plot, the curve of the most potent agent tends to be in the left side of the graph and the median effective concentration (EC50) is lower. A drug's potency is influenced by its affinity for its receptor and therefore independent of its maximal effect. Efficacy (or intrinsic activity) is the drug property that allows the receptor-bound drug to produce its pharmacological effect. The relative efficacy of two drugs that elicit the same effect can be measured by comparing the maximum effects of the drugs. A drug can have high potency but poor efficacy, meaning that the response is seen at very low doses and remains small even at high doses. This is the case of rapamycin which is a highly potent but poorly efficient TOR allosteric inhibitor (Fig. 1A, C andE) compared to active site TOR inhibitors which are highly efficient (Fig. 1B, D andF). If a drug has one effect, and only one effect on all biological systems it possesses the property of specificity. In experience, the vast majority of drugs are selective rather than specific [START_REF] Davis | Comprehensive analysis of kinase inhibitor selectivity[END_REF]. "A drug with the appropriate balance of avoidance of undesirable targets (narrow selectivity) and coverage of one or more targets of interest (broad selectivity) is a continual drug development challenge. In many cases this objective is attained through trial and error, but there are rational approaches that can guide the tuning of selectivity, and examples have been published that illustrate a number of generalizable strategy" [START_REF] Huggins | Rational approaches to improving selectivity in drug design[END_REF]. Thus, "a Selectivity score (S) for each drug can be calculated by dividing the number of kinases found to bind with dissociation constant <3 μM" (or sometimes 10 µM) "by the total number of distinct kinases tested. The selectivity score is an unbiased measure that enables quantitative comparisons between compounds and the detailed differentiation and analysis of interaction patterns" [START_REF] Karaman | A quantitative analysis of kinase inhibitor selectivity[END_REF].

At last, in pharmacology, an inhibiting or effective concentration (IC or EC) refers to a concentration of a drug that produces a biological response in case of enzymology in vitro assays or when unicellular organisms or mammalian cell cultures are tested. IC refers to an assay where there is decrease in activity whereas EC rather refers to a drug that activates a system. The term effective dose (ED) refers to in vivo studies when used in living organisms such as animals to usually determine the median effective dose (ED50) and/or the median lethal dose (LD50). Usually, in the context of studies involving asTORis, potency values obtained by means of in vitro enzyme-based assays (IC in vitro ) are generally different than potency values (IC cell ) obtained by cell-based assays treating cells before measuring various enzymatic products (also called cell potency values) because many targets can be modified at more than one phosphorylation site or in more than one way, e.g., ubiquitylation or acetylation [START_REF] Carlson | Development of LanthaScreen cellular assays for key components within the PI3K/AKT/mTOR pathway[END_REF]. Dissecting biochemical effects using in vitro grown cell lines might also give rise to different EC values depending on the cell physiology/line and is a far much different task than looking for clinical outcomes. Indeed the efficient doses (ED) are usually higher than those of in vitro studies likely to encompass drawbacks linked to PK properties, metabolic stability and putative off targets effect in organisms. A tool such as KInhibition portal (https://kinhibition.fredhutch.org) might help choosing a set of selective drugs among thousands depending on the objective [START_REF] Bello | KInhibition: A Kinase Inhibitor Selection Portal[END_REF]. Thus, designing a scale of inhibitor "strength" of a set of inhibitors solely from enzymatic properties (in vitro IC50) to calibrate experiments with living cells or organisms might be hazardous [START_REF] Michel | Selectivity of pharmacological tools: implications for use in cell physiology. A review in the theme: Cell signaling: proteins, pathways and mechanisms[END_REF]. Chemical structure activity relationship through docking studies using the TOR kinase domain with the dual PI3K/PIKK inhibitor NVP-BEZ235 (BEZ235), the TOR selective inhibitor PP242, and the TOR specific inhibitor KU-0063794 showed that drugs in development utilize a novel pharmacophore space to achieve specificity of TOR inhibition [START_REF] Sturgill | Activating mutations in TOR are in similar structures as oncogenic mutations in PI3KCalpha[END_REF]. So around the year 2009, several compounds were reported as asTORis (Fig. 2): PP242 [START_REF] Feldman | Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2[END_REF], Torin1 (Liu et al., 2010;Thoreen et al., 2009), KU-0063794 (Garcia-Martinez et al., 2009), WYE-354, 600 and 687 [START_REF] Yu | Biochemical, cellular, and in vivo activity of novel ATP-competitive and selective inhibitors of the mammalian target of rapamycin[END_REF] and others reviewed by Benjamin and colleagues [START_REF] Benjamin | Rapamycin passes the torch: a new generation of mTOR inhibitors[END_REF]. These compounds were generally developed from dual PI3K/PIKK inhibitors or inhibitors more largely involved in the PI3K/AKT axis and have different core structure [START_REF] Andrs | Phosphatidylinositol 3-Kinase (PI3K) and phosphatidylinositol 3-kinase-related kinase (PIKK) inhibitors: importance of the morpholine ring[END_REF][START_REF] Garcia-Echeverria | Blocking the mTOR pathway: a drug discovery perspective[END_REF]Liu et al., 2012a). They were TOR selective, having IC50 for TOR lower than for PI3Ks and also for other PIKKs [START_REF] Benjamin | Rapamycin passes the torch: a new generation of mTOR inhibitors[END_REF]. For instance, Torin1 efficiency towards TOR was compared to the effect of the dual PI3K/PIKK inhibitors PI103 and BEZ235 and its high selectivity towards a panel of kinases including PI3Ks and PIKKs was shown. Torin1 has a quinolone core structure expected to share the same binding mode as BEZ235 with mTOR, PI3K and other PIKK family members while PP242 has a pyrazolopyrimidine core structure derived from PP2 [START_REF] Feldman | Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2[END_REF]. KU-0063794 and WYE-354 also derive from other dual PI3K/PKK inhibitors such as PI-103 and LY294002 and contain a morpholino-substituted heterocycle. These asTORis were reported more efficient than rapamycin by measuring their capacity to phosphorylate TOR targets and also inhibit cell proliferation more efficiently. So new TORC1-dependent functions and previously found rapamycin resistant were deciphered due to higher efficacy of asTORis [START_REF] Feldman | Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2[END_REF][START_REF] Thoreen | Rapamycin inhibits mTORC1, but not completely[END_REF] leading to new advances in TOR pathway knowledge [START_REF] Guertin | The pharmacology of mTOR inhibition[END_REF]. For instance, the more effective TOR inhibition unveiled rapamycin resistant levels of regulation in cap-dependent initiation of translation, protein synthesis and proliferation (Dowling et al., 2010b). This also helped show that mTOR activates cap-dependent translation of cyclins and represses cap-independent translation of p27/KIP1, an inhibitor of CDK (Cyclin Dependent Kinase), therefore activating cell proliferation (Thoreen et al., 2009). These differential effects of rapamycin on substrates phosphorylation compared to that of the ATP competitive inhibitor Torin1 were studied through designing peptides from well-known TORC1 targets containing phosphorylation sites [START_REF] Kang | mTORC1 phosphorylation sites encode their sensitivity to starvation and rapamycin[END_REF]. When Torin1 blocks the phosphorylation of all TORC1 dependent phosphorylated sites in all TOR protein targets, some are not dephosphorylated by rapamycin (called strong target) and some are (called poor target). This concept of substrate quality is a property of TOR effector sites, which can explain that their differential phosphorylation vary with growth conditions. Furthermore, poor and strong targets can be found in the same protein (Fig. 3). In parallel, strong targets were also found phosphorylated in cells growing under partially depleted nutrient conditions (Fig. 3). Hence, rapamycin can be highly potent and selective for some poor mTOR targets such as S6K T389 and 4E-BP1 S65, but its intrinsic activity or efficacy cannot be maximal since TOR is still able to phosphorylate the strong mTORC1 targets such as 4E-BP1 T37/46 and ULK1 S758 in presence of rapamycin [START_REF] Kang | mTORC1 phosphorylation sites encode their sensitivity to starvation and rapamycin[END_REF]. Therefore, due to the incomplete intrinsic efficacy, rapamycindependent TOR inhibition by an acute dose might lead to error-prone interpretation of data especially when targets are not well identified and/or when a chronic dose of rapamycin is applied [START_REF] Sarbassov | Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB[END_REF].

A comparison of selectivity, potency and metabolic stability of four asTORis mentioned above carrying different structure, i.e., Torin1, PP242, WYE-354 and KU-0063794 (Fig. 2) was reported and it appears to us to be a good example of how selectivity of a drug to TOR is demonstrated through tests involving many different assays (Liu et al., 2012a). They all exhibited highly potent and similar IC50 values against the recombinant mTOR kinase domain but their relative cellular potency EC50 against the TORC1 complex was: Torin1 > KU-63794 > WYE-354 > PP242. Their relative selectivity score toward a panel of 442 kinases was: KU-0063794 > WYE-354 > Torin1 > PP242. Other kinase assays showed that Torin1 concentration above 1 µM was able to inhibit the other PIKKs: ATM, ATR and DNA-PK. The metabolic stability was also better with KU-0063794 and WYE-354 than with Torin1 and PP242. However, Torin1 had a slower off-rate as the duration of S6K1 (pS6K-Thr-389) and PI3K-dependent (p-AKT-Thr-308) phosphorylation last 16 hours vs 1 hour for the 3 other drugs after extensive washing out the drug. Altogether, authors' conclusion of the study of these four asTORis was to avoid PP242 and to cautiously interpret data when Torin1, KU-0063794 and WYE354 are used at concentrations above 1 µM. Proliferation assays on mouse embryonic fibroblasts (MEFs) showed that in the range of 10-500 nM, rapamycin induces a plateau value of ca. 50-60% inhibition without any dose dependence, whereas Torin1 induces 40% to 100 % inhibition between 10 and 250 nM when IC50 kinase values were 1-10 nM (Thoreen et al., 2009). Hence, Table 1 shows that IC50 values for in vitro TOR kinase activity do not fully predict the IC50 of proliferation. These differences likely deal with intrinsic PK, metabolic stability of the drug, or posttranslational modifications of the target as well as drug efflux or inactivation by cells as in yeast (Liu et al., 2012b).

Improvement of the pharmacokinetic properties of KU-0063794 led to the development of AZD-8055, which is very highly selective for mTOR over PI3Ks and PIKKs [START_REF] Chresta | AZD8055 is a potent, selective, and orally bioavailable ATPcompetitive mammalian target of rapamycin kinase inhibitor with in vitro and in vivo antitumor activity[END_REF][START_REF] Garcia-Echeverria | Blocking the mTOR pathway: a drug discovery perspective[END_REF][START_REF] Marshall | Benefits of mTOR kinase targeting in oncology: pre-clinical evidence with AZD8055[END_REF] and more recently to the less selective but more stable sister compound AZD2014 [START_REF] Pike | Optimization of potent and selective dual mTORC1 and mTORC2 inhibitors: the discovery of AZD8055 and AZD2014[END_REF] (Fig. 2), both used in clinical trials [START_REF] Garcia-Echeverria | Blocking the mTOR pathway: a drug discovery perspective[END_REF]. A continuous development of intermediate compounds of this series that showed very high specificity towards PI3Ks although lower potency towards TOR led to the design of AZD3147, a new highly selective inhibitor of TORC1 and TORC2 [START_REF] Pike | Discovery of AZD3147: A Potent, Selective Dual Inhibitor of mTORC1 and mTORC2[END_REF] that can circumvent the discontinuity of clinical trials [START_REF] Martelli | Drug discovery targeting the mTOR pathway[END_REF]. Similarly, other derived compounds were developed in parallel like WYE-125132 (WYE-132) or Torin2 [START_REF] Liu | Characterization of Torin2, an ATP-competitive inhibitor of mTOR, ATM, and ATR[END_REF][START_REF] Yu | Beyond rapalog therapy: preclinical pharmacology and antitumor activity of WYE-125132, an ATP-competitive and specific inhibitor of mTORC1 and mTORC2[END_REF].

WYE-132 has better pharmacokinetics properties than WYE-354 and is highly selective for mTOR over PI3K and the PIKK ATR [START_REF] Yu | Beyond rapalog therapy: preclinical pharmacology and antitumor activity of WYE-125132, an ATP-competitive and specific inhibitor of mTORC1 and mTORC2[END_REF]. Torin2 has improved pharmacological and solubility properties compared to its structural analogue Torin1 but also significant activity against mTOR, ATM, ATR, and DNA-PK, as well as both in vitro and in vivo antitumor efficacy, being therefore a potent broadly active pan-PIKK kinase inhibitor [START_REF] Liu | Characterization of Torin2, an ATP-competitive inhibitor of mTOR, ATM, and ATR[END_REF]. Indeed, if Torin2 most potently inhibits mTORC1 and mTORC2 in vivo at concentrations of less than 10 nM, it also inhibits ATR, ATM, and DNA-PK at concentrations between 20 and 100 nM and PI3K at concentrations above 200 nM. This is in contrast with Torin1 which only exhibits moderate inhibition of DNA-PK (250 nM) but is inactive against other PIKK-family kinases. Unexpectedly, Torin2 also has a lower residence time than Torin1 (4h vs 16h) leading to suggest that Torins might induce a TOR conformation change in the kinase that is energetically difficult to recover from rather than different binding affinities. This adds a specific feature to Torins that distinguish them from other asTORis, showing that Torin1 and Torin2 although structurally close cannot only be compared in terms of potency. Moreover, the intentional development of new dual Torin2 analogs that inhibit both mTOR and ATR [START_REF] Shaik | Design, Synthesis, and Docking Studies of New Torin2 Analogs as Potential ATR/mTOR Kinase Inhibitors[END_REF] for clinical purposes shows that driving drug selectivity to specificity is a difficult chemistry task.

Therefore, the crosstalk between TOR pathway and other pathways such as the DNA damage response [START_REF] Li | Relationship between ATM and ribosomal protein S6 revealed by the chemical inhibition of Ser/Thr protein phosphatase type 1[END_REF][START_REF] Silvera | mTORC1 and -2 Coordinate Transcriptional and Translational Reprogramming in Resistance to DNA Damage and Replicative Stress in Breast Cancer Cells[END_REF] involving other PIKK close to TOR might hamper the discovery of true TOR targets with Torin2 and Torin2 analogs. In addition, Torin2 was reported as an antimalarial agent 1,000-fold selective to malaria parasites over mammalian cells whereas TOR homolog is not found in Plasmodium falciparum [START_REF] Hanson | Torins are potent antimalarials that block replenishment of Plasmodium liver stage parasitophorous vacuole membrane proteins[END_REF]. Therefore, Torin2 is particularly known to target other eukaryotic proteins than TOR and should be used with caution for biological studies.

Nowadays, the use of more than one selective and potent inhibitor through targeting for instance two pathways or sub-pathways is more and more explored in medicine rather than using dual PI3K/mTOR inhibitors of a single pathway, which can have the "possible drawback of association with greater toxicity" [START_REF] Simioni | Activity of the novel mTOR inhibitor Torin-2 in B-precursor acute lymphoblastic leukemia and its therapeutic potential to prevent Akt reactivation[END_REF]. Furthermore, novel compounds are continuously searched to circumvent the discontinuity of potent and selective compounds yet cytostatic or unstable in clinical trials [START_REF] Andrs | Phosphatidylinositol 3-Kinase (PI3K) and phosphatidylinositol 3-kinase-related kinase (PIKK) inhibitors: importance of the morpholine ring[END_REF][START_REF] Chen | Inhibition of tumor cell growth, proliferation and migration by X-387, a novel active-site inhibitor of mTOR[END_REF][START_REF] Estrada | Pyrimidoaminotropanes as potent, selective, and efficacious small molecule kinase inhibitors of the mammalian target of rapamycin (mTOR)[END_REF][START_REF] Fraser | ECF309: a potent, selective and cell-permeable mTOR inhibitor[END_REF][START_REF] Mortensen | Discovery of mammalian target of rapamycin (mTOR) kinase inhibitor CC-223[END_REF][START_REF] Nowak | Discovery of potent and selective inhibitors of the mammalian target of rapamycin (mTOR) kinase[END_REF][START_REF] Park | Virtual screening and biochemical evaluation to identify new inhibitors of mammalian target of rapamycin (mTOR)[END_REF][START_REF] Pei | Discovery and Biological Profiling of Potent and Selective mTOR Inhibitor GDC-0349[END_REF][START_REF] Slotkin | MLN0128, an ATP-competitive mTOR kinase inhibitor with potent in vitro and in vivo antitumor activity, as potential therapy for bone and soft-tissue sarcoma[END_REF][START_REF] Walters | mTORC Inhibitors as Broad-Spectrum Therapeutics for Age-Related Diseases[END_REF][START_REF] Zheng | mTOR Inhibitors at a Glance[END_REF]. Thus, although selectivity is not always the major criteria in clinical trials, it is an essential criteria for the choice of an inhibitor to elucidate the role of a particular kinase in biological tissues and in vitro studies [START_REF] Arrowsmith | The promise and peril of chemical probes[END_REF]. This illustrates why potency and efficacy of selective inhibitors should be carefully examined and that the concept of inhibitor strength can be misleading according to experts [START_REF] Michel | Selectivity of pharmacological tools: implications for use in cell physiology. A review in the theme: Cell signaling: proteins, pathways and mechanisms[END_REF]. This also underlines that testing/using more than one TOR ATP competitive inhibitor should help identifying and confirming TOR-dependent regulated functions.

ATP-competitive TOR inhibitors in plant and algae

The first asTORis that have been used in plants were among those presented above, i.e., KU-0063794, AZD-8055, Torin1 and Torin2, WYE-354 and WYE-132. It was remarkable that KU-0063794 and WYE-354 and their improved derived molecules, AZD-8055 and WYE-132, followed the same relative potency than in mammalian cells, i.e., AZD-8055 > KU-0063794 and WYE-132 > WYE354 (Table 1) [START_REF] Montane | ATP-competitive mTOR kinase inhibitors delay plant growth by triggering early differentiation of meristematic cells but no developmental patterning change[END_REF]. We encountered solubility problems with Torin1 in our culture conditions, and among the drugs we could get and test at that time; AZD-8055 (Fig. 1D) and WYE-132 quickly became our favorites to fully inhibit Arabidopsis WT seedlings growth because of high reproducibility of responses and of temporal stability of dose-dependent inhibition. Furthermore, we demonstrated that the plant growth inhibition by AZD-8055 and WYE-132 is TOR-dependent by showing induced haploinsufficiency to TOR as the TOR/tor heterozygote mutants are hypersensitive to both inhibitors compared to WT (Fig. 1D) [START_REF] Montane | ATP-competitive mTOR kinase inhibitors delay plant growth by triggering early differentiation of meristematic cells but no developmental patterning change[END_REF]. Maximal efficacy was shown through dose-response curves showing complete inhibition of root growth, as opposed to rapamycin in plants or algae (Fig. 1C-F). asTORis were potent in most photosynthetic eukaryotes as they strongly inhibit growth of a large variety of plants (Arabidopsis, potato, tomato, rice; Lotus, millet, Nicotiana), and proliferation in both green algae and diatoms (C. reinhardtii, P. tricornutum) [START_REF] Deng | The TOR Pathway Is Involved in Adventitious Root Formation in Arabidopsis and Potato[END_REF][START_REF] Dong | Expression profiling and functional analysis reveals that TOR is a key player in regulating photosynthesis and phytohormone signaling pathways in Arabidopsis[END_REF][START_REF] Dong | Genome-wide analyses of genes encoding FK506-binding proteins reveal their involvement in abiotic stress responses in apple[END_REF][START_REF] Imamura | TOR (target of rapamycin) is a key regulator of triacylglycerol accumulation in microalgae[END_REF][START_REF] Montane | ATP-competitive mTOR kinase inhibitors delay plant growth by triggering early differentiation of meristematic cells but no developmental patterning change[END_REF][START_REF] Prioretti | The inhibition of TOR in the model diatom Phaeodactylum tricornutum promotes a get-fat growth regime[END_REF][START_REF] Song | The crosstalk between Target of Rapamycin (TOR) and Jasmonic Acid (JA) signaling existing in Arabidopsis and cotton[END_REF]. Table 1 and Fig. 2 show the concentration range of the main asTORis used to inhibit proliferation, to study TOR functions and that helped find new TOR targets in photosynthetic eukaryotes: KU-0063794, AZD-8055, Torin1 and Torin2 [START_REF] Deng | The TOR Pathway Is Involved in Adventitious Root Formation in Arabidopsis and Potato[END_REF][START_REF] Dong | Expression profiling and functional analysis reveals that TOR is a key player in regulating photosynthesis and phytohormone signaling pathways in Arabidopsis[END_REF][START_REF] Kravchenko | Mutations in the Arabidopsis Lst8 and Raptor genes encoding partners of the TOR complex, or inhibition of TOR activity decrease abscisic acid (ABA) synthesis[END_REF][START_REF] Li | TOR-inhibitor insensitive-1 (TRIN1) regulates cotyledons greening in Arabidopsis[END_REF][START_REF] Li | Differential TOR activation and cell proliferation in Arabidopsis root and shoot apexes[END_REF][START_REF] Mohammed | Converging Light, Energy and Hormonal Signaling Control Meristem Activity, Leaf Initiation, and Growth[END_REF][START_REF] Montane | ATP-competitive mTOR kinase inhibitors delay plant growth by triggering early differentiation of meristematic cells but no developmental patterning change[END_REF][START_REF] Mubeen | Target of Rapamycin Inhibition in Chlamydomonas reinhardtii Triggers de-novo Amino Acid Synthesis by Enhancing Nitrogen Assimilation[END_REF][START_REF] Ouibrahim | Potyviruses differ in their requirement for TOR signalling[END_REF][START_REF] Pfeiffer | Integration of light and metabolic signals for stem cell activation at the shoot apical meristem[END_REF][START_REF] Prioretti | The inhibition of TOR in the model diatom Phaeodactylum tricornutum promotes a get-fat growth regime[END_REF][START_REF] Schepetilnikov | TOR and S6K1 promote translation reinitiation of uORF-containing mRNAs via phosphorylation of eIF3h[END_REF][START_REF] Schepetilnikov | Viral factor TAV recruits TOR/S6K1 signalling to activate reinitiation after long ORF translation[END_REF][START_REF] Schepetilnikov | GTPase ROP2 binds and promotes activation of target of rapamycin, TOR, in response to auxin[END_REF]Wang et al., 2018b;[START_REF] Werth | Investigating the effect of target of rapamycin kinase inhibition on the Chlamydomonas reinhardtii phosphoproteome: from known homologs to new targets[END_REF]. Some authors tried to establish a scale of "strength" of these inhibitors in plants starting from values of IC50 kinase in vitro [START_REF] Deng | Target of Rapamycin Is a Key Player for Auxin Signaling Transduction in Arabidopsis[END_REF]Xiong et al., 2017a). Here, as we have discussed above, we would like to stress that knowledge from the biochemical and animal fields should be taken in consideration when using such inhibitors. Indeed, as shown above for KU-0063794, Torin1 and AZD-8055 (Table 1), using the IC50 of in vitro kinase to predict IC50 of proliferation hardly works in mammals and plants. This might hide other characteristics of a drug in vivo as well as its speciation in culture medium. As reported above, the potency of Torin1 in animal cells was higher than that of KU-0063794 but the selectivity was the opposite. Regarding the use of Torins and in particular Torin2 as an asTORi, we have mentioned above that it was described as a pan-PIKK family inhibitor as it exhibited potent biochemical and cellular activity against PIKKs, including ATM and ATR [START_REF] Liu | Characterization of Torin2, an ATP-competitive inhibitor of mTOR, ATM, and ATR[END_REF]. Indeed, mammalian mTOR is involved in the network of responses between DNA damage and cell cycle control, including the activity of the ATM/ATR-CHK1/CHK2-p53 axis [START_REF] Silvera | mTORC1 and -2 Coordinate Transcriptional and Translational Reprogramming in Resistance to DNA Damage and Replicative Stress in Breast Cancer Cells[END_REF]. Since ATM and ATR are conserved in plants and algae and regarding the known role of Arabidopsis ATM in regulation of meristem activity and DNA-damage responses [START_REF] Fulcher | Hypersensitivity to DNA damage in plant stem cell niches[END_REF][START_REF] Hisanaga | The ATM-dependent DNA damage response acts as an upstream trigger for compensation in the fas1 mutation during Arabidopsis leaf development[END_REF][START_REF] Ricaud | ATM-mediated transcriptional and developmental responses to gamma-rays in Arabidopsis[END_REF], care should be taken when analyzing data with Torin2 in plants, especially when high concentrations are used as mentioned above. For our part, we therefore avoid using Torins in order to selectively inhibit the TOR pathway and checked that selective ATP competitive ATM inhibitors did not inhibit growth and so could be used as a selectivity control toward TOR inhibitors [START_REF] Montane | ATP-competitive mTOR kinase inhibitors delay plant growth by triggering early differentiation of meristematic cells but no developmental patterning change[END_REF]. The specificity of a cellular response towards TOR inhibition can also be confirmed in plant and algae through comparing the effect of different selective asTORis such as AZD-8055 and WYE-132 [START_REF] Barrada | A TOR-YAK1 signaling axis controls cell cycle, meristem activity and plant growth in Arabidopsis[END_REF][START_REF] Prioretti | The inhibition of TOR in the model diatom Phaeodactylum tricornutum promotes a get-fat growth regime[END_REF] [START_REF] Prioretti | The inhibition of TOR in the model diatom Phaeodactylum tricornutum promotes a get-fat growth regime[END_REF]but it has to be done at doses leading to similar growth inhibition. Furthermore, we should keep in mind that we should not expect identical molecular and cellular effects of rapamycin and asTORis in plants and algae as rapamycin does not inhibit all TORC1 activity, and might also not inhibit other potential new plant/algae TOR complexes (see above). Taking into account the conservation of the minimal core TORC1 in plants, and the specificity of plant targets that only start to emerge, we think that other plant TOR target sites than the sole S6K T449 will need to be developed to accurately record the level of plant TOR inhibition. Indeed, the mammalian S6K1 T389 equivalent of plant S6K T449 corresponds to a poor TOR target [START_REF] Kang | mTORC1 phosphorylation sites encode their sensitivity to starvation and rapamycin[END_REF].

Pharmacogenetic screens in plant and algae

Selective active site inhibitors have several advantages to other methods of kinase inhibition for genetic screens. First, they allow avoiding possible drawbacks associated with transgenic lines.

Second, they allow to control the time of and the level of inhibition on a given organism, organ, tissue or cell by following the kinetics of any measurable parameter and its dose-dependent evolution, an important aspect in case of essential genes like TOR. Inhibiting TOR from a known WT physiological context is definitely different than comparing loss of function or overexpressing functions.

Furthermore, being cytostatic also in plants [START_REF] Montane | ATP-competitive mTOR kinase inhibitors delay plant growth by triggering early differentiation of meristematic cells but no developmental patterning change[END_REF], these inhibitors offer the possibility to rescue hypersensitive plants and to more accurately follow reciprocal changes or point out any differential behavior of targets when inhibition is relaxed. To date, few screens of mutants resistant or hypersensitive to TOR inhibitors have been reported in plants and algae yet they revealed important aspect of TOR signaling [START_REF] Barrada | A TOR-YAK1 signaling axis controls cell cycle, meristem activity and plant growth in Arabidopsis[END_REF][START_REF] Couso | Synergism between Inositol Polyphosphates and TOR Kinase Signaling in Nutrient Sensing, Growth Control, and Lipid Metabolism in Chlamydomonas[END_REF][START_REF] Crespo | Inhibition of target of rapamycin signaling by rapamycin in the unicellular green alga Chlamydomonas reinhardtii[END_REF]Li et al., 2015). Recently, the vip1-1 C. reinhardtii mutant hypersensitive to the first saturating concentration of 500 nM rapamycin inhibiting growth to the 50% plateau value was isolated [START_REF] Couso | Synergism between Inositol Polyphosphates and TOR Kinase Signaling in Nutrient Sensing, Growth Control, and Lipid Metabolism in Chlamydomonas[END_REF]. This mutant which was also hypersensitive to the single concentration tested of 500 nM AZD-8055 or Torin1 revealed an interaction between TOR and inositol polyphosphate intracellular signaling. In Arabidopsis, a first screen of mutants that show no chlorosis of cotyledons induced by 2 µM AZD-8055 allowed to select 9 mutants among which a new ABI4 (ABA-INSENSITIVE 4) allele, revealing a new role of TOR in abscisic acid (ABA) signaling [START_REF] Li | TOR-inhibitor insensitive-1 (TRIN1) regulates cotyledons greening in Arabidopsis[END_REF]. Interestingly, in our culture conditions we never observed AZD-8055-induced chlorosis at doses up to 10 μM [START_REF] Montane | ATP-competitive mTOR kinase inhibitors delay plant growth by triggering early differentiation of meristematic cells but no developmental patterning change[END_REF], meaning that growth conditions have to be considered consistently with the role of TOR in response to nutrients and stress. We recently screened EMS Arabidopsis mutants resistant to a concentration of 1µM AZD-8055 inhibiting 90% of root lengthening and discovered that the homolog of yeast YAK1 (Yet Another Kinase 1) and human DYRK1A (Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A) was a TOR-inhibition dependent downstream repressor of plant cell proliferation [START_REF] Barrada | A TOR-YAK1 signaling axis controls cell cycle, meristem activity and plant growth in Arabidopsis[END_REF]. We also showed that pINDY, an-ATP competitive inhibitor of DYRK1A, mimics Arabidopsis yak1 loss-of-function mutations. This offers a new way to study TOR-YAK1 axis in plants. In addition, we isolated other mutants sensitive or resistant to AZD-8055 concentrations leading to other levels of growth inhibition which are now under study. Hence, together with studies of known mutants of components of the plant/algae TORC1 pathway, new screening approaches might actively help deciphering TOR pathways in photosynthetic organisms.

In addition to being proof-of-concept that the pharmacogenetic screens can help identify new functions of TOR in plant and algae, these studies remind the importance to carefully design the conditions of the screen but also of further studies. For instance, the haploinsufficiency phenotype of TOR/tor heterozygotes discussed above is a nice illustration of the importance of the "right" dose of an inhibitor to compare physiological context of two genetic backgrounds [START_REF] Montane | ATP-competitive mTOR kinase inhibitors delay plant growth by triggering early differentiation of meristematic cells but no developmental patterning change[END_REF].

The TOR/tor heterozygotes were clearly hypersensitive to AZD-8055 concentrations between 0.1 and 1 µM, but grow the same as the WT at doses below 0.03 µM and above the maximal inhibitory concentrations of 3µM, that almost completely inhibit root growth (Fig. 1 D). For instance, to compare YAK1-and TOR-expression patterns by GUS staining in roots which growth was similarly inhibited by AZD-8055, we used twice lower AZD-8055 concentration for the GUS knock-in TOR/tor-1 heterozygous line [START_REF] Menand | Expression and disruption of the Arabidopsis TOR (target of rapamycin) gene[END_REF] than for a pYAK1::YAK1-GUS homozygous line [START_REF] Barrada | A TOR-YAK1 signaling axis controls cell cycle, meristem activity and plant growth in Arabidopsis[END_REF]. Therefore, the dose-dependent effect of asTORis on the processes analyzed should be preliminary determined prior to design specific genetic screen. We would like to finish this section with guidelines that might help plan experiments with TOR inhibitors in plants and algae, with an emphasis on pharmacogenetic screening.

Guidelines for plant physiology studies and genetic screens with asTORis :

-Clarify the question you want to answer to choose the best developmental or growth stage of the plant or algae to study or to screen, -carefully define growing conditions as growth can be widely influenced and disturbed by even subtle changes, -choose selective drug(s) which has(ve) been best characterized, consider any possible drawback, -check the vehicle drug harmlessness on cell growth and keep its concentration constant whatever the drug concentration used; usually DMSO 0.1% works well in mammalian cells, plants and algae [START_REF] Montane | ATP-competitive mTOR kinase inhibitors delay plant growth by triggering early differentiation of meristematic cells but no developmental patterning change[END_REF][START_REF] Prioretti | The inhibition of TOR in the model diatom Phaeodactylum tricornutum promotes a get-fat growth regime[END_REF]Thoreen et al., 2009), -choose clear-cut parameter (s) to quantify effect of the drug, -establish a dose-response curve in a log 10 way from ca. pM including concentrations active in other species (see text and Table1), -check stability of inhibition over time, -check the reversibility of the drug effect if the drug is cytostatic (asTORis), -confirm selectivity of the effect with other asTORis, -choose a concentration related to the question you ask within the dose-response range, to avoid off target at too high doses, -after selection of a mutant of interest, check its dose-response curve.

If you think you have discovered a direct target of TOR:

-check the drug-dependent effect on different organs or tissues to avoid bulk response that can mask discrete tissue responses, -design peptide(s) encompassing the putative phosphorylation site(s) to demonstrate which one is TOR-dependent, -express the WT and mutated form (s) in phosphorylated amino acid of the new target in a knockout mutant and compare dose response curves.

Conclusion

If rapamycin has opened the study of TOR functions in plant and algae as in yeast and mammals, it should be used cautiously in plants for which overexpression of FKBP12 is required. The use of rapamycin has fewer drawbacks in some algae, like Chlamydomonas reinhardtii, which is naturally sensitive to rapamycin. However, its good potency masks incomplete efficacy in many if not all species studied till now. The requirement of FKBP12 partner to inhibit TOR with rapamycin might also disturb signaling responses due to the cellular role of FKBP12 and putative off targets of rapamycin itself might also interfere. However, in any case, we should keep in mind that rapamycin does not inhibit all TORC1 activity and does not inhibit other TOR complexes potentially present in plants and algae. Conversely, ATP-competitive TOR inhibitors more efficiently inhibit proliferation and growth than rapamycin in algae and plants, as in animals, and are therefore very good tools to study the TOR pathway in photosynthetic eukaryotes. However, we should in return take care of information's about concentration range and singularity from chemical and animal researchers who developed and experienced them. Their use has already helped decipher TOR pathway effectors in plants and algae and we guess they will certainly be of great help in the future. Table 1. Concentrations of rapamycin and different asTORis inhibiting TOR kinase activity (IC50), mammalian cells proliferation (IC50), WT plant root and leaf growth, and WT green alga and diatom proliferation. Values were obtained with different in vitro kinase assays as well as different proliferation assays described in the references. Cell lines are embryonic or cancerous. Range of values was from different mammalian cell lines in the same article. Concentration unit is nM. Note that rapamycin never fully inhibits non-cancerous mammalian cell proliferation, growth of roots and leaves of A. thaliana seedlings or of the unicellular green alga C. reinhardtii. heterozygote, which has higher sensitivity to this asTORi due to haploinsufficiency of TOR. In all species, the efficacy of growth inhibition by rapamycin is never maximal (ca. 50%) while it is maximal by AZD-8055. From [START_REF] Giubellino | Combined Inhibition of mTORC1 and mTORC2 Signaling Pathways Is a Promising Therapeutic Option in Inhibiting Pheochromocytoma Tumor Growth: In Vitro and In Vivo Studies in Female Athymic Nude Mice[END_REF] (A, B), [START_REF] Deng | Target of Rapamycin Is a Key Player for Auxin Signaling Transduction in Arabidopsis[END_REF] (C), [START_REF] Montane | ATP-competitive mTOR kinase inhibitors delay plant growth by triggering early differentiation of meristematic cells but no developmental patterning change[END_REF]) (D), [START_REF] Juppner | The target of rapamycin kinase affects biomass accumulation and cell cycle progression by altering carbon/nitrogen balance in synchronized Chlamydomonas reinhardtii cells[END_REF]) (E) and (Imamura et al., 2016) (F). Figures and images are Figure 2: Formula of main asTORis discussed in this review.

Tables

For original publication of each inhibitor, see text and Table 1. Drawing was done with the ACD/ChemSketch freeware. targets, including ULK1 S758, 4E-BP1-T37/46, but also GRB10 S150 and PRAS40 S183, are resistant to rapamycin but not to asTORis or complete starvation. From data of [START_REF] Kang | mTORC1 phosphorylation sites encode their sensitivity to starvation and rapamycin[END_REF].

ULK1 S758

  reproduced with permission of Oxford Academic (A, B), Wiley Online Library (E) and Taylor & Francis (F) or were originally published under the Creative Commons Attribution License (C, D).

Figure 3 :

 3 Figure 3: The quality of mTORC1 substrates determines their sensitivity to rapamyin, asTORis

Acknowledgements

Work on TOR in our laboratory was supported by Agence Nationale de la Recherche (ANR) grants SIGNAUXBioNRJ (ANR-15-CE05-0021-03), TRANSLATOR (ANR-11-BSV6-0010) and DECORATORS (ANR-14-CE19-0007). We apologize to our colleagues whose work could not be included due to space limitations. No conflict of interest declared.

Supplementary data

AZD-8055 (Chresta et al., [START_REF] Guertin | Defining the role of mTOR in cancer[END_REF][START_REF] Huang | Rapamycins -Mechanism of action and cellular resistance[END_REF][START_REF] Mukhopadhyay | The Enigma of Rapamycin Dosage[END_REF]; c (Montane and Menand, 2013); d [START_REF] Ren | Target of rapamycin signaling regulates metabolism, growth, and life span in Arabidopsis[END_REF], note that growth IC50 of lines overexpressing yeast FKBP12 is observed with ca. 500-1,000 nM rapamycin, e Leaves of 3 weeks old plants grown on soil and under long days (16h) were rubbed with drops of AZD-8055 and grown for 6 days before scoring growth inhibition [START_REF] Ouibrahim | Potyviruses differ in their requirement for TOR signalling[END_REF]. f [START_REF] Crespo | Inhibition of target of rapamycin signaling by rapamycin in the unicellular green alga Chlamydomonas reinhardtii[END_REF][START_REF] Juppner | The target of rapamycin kinase affects biomass accumulation and cell cycle progression by altering carbon/nitrogen balance in synchronized Chlamydomonas reinhardtii cells[END_REF][START_REF] Roustan | Quantitative Phosphoproteomic and System-Level Analysis of TOR Inhibition Unravel Distinct Organellar Acclimation in Chlamydomonas reinhardtii[END_REF]; g [START_REF] Prioretti | The inhibition of TOR in the model diatom Phaeodactylum tricornutum promotes a get-fat growth regime[END_REF]. E-F). Note that mouse embryonic fibroblasts (non-cancerous) are also partially inhibited by rapamycin but completely by Torin1 (Thoreen et al., 2009). Plant dose response to rapamycin is shown for Wild Type (WT) and plant overexpressing A. thaliana (At)-, Human (Hs)-, or yeast (BP12)-FKBP12 (C). For A. thaliana, dose response with AZD-8055 is shown for the WT and the TOR/tor-1

Figures legends


Yonghua Li-Beisson
email: yonghua.li@cea.fr

Hajime Wada
email: hwada@bio.c.u-tokyo.ac.jp

Plant and algal lipids set sail for new horizons

Plant and algal lipids set sail for new horizons

Yonghua Li-Beisson, Hajime

Progress in methodology and research focus

In the past 50 years, plant lipid research has undergone dramatic modernization with the focus shifting and building on from structure and biochemistry to metabolism and function. Thus, the major approaches that are now used to study lipids have shifted from biochemistry to genetics, genomics, and lipidomics. Notably, with the advent of in situ imaging of lipid metabolites and genome editing tools, studies of lipids have Chromatography-tandem Mass Spectrometry (UPLC-MS/MS), for example, has allowed the identification and quantification of not only major structural lipids but also of minor and transitory lipid-based signaling molecules. Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) has enabled visualization of lipid metabolites in situ revealing unexpected heterogeneity in lipid organization and highlights complex subcellular organization and differential gene expression of proteins involved in lipid metabolism. Genome editing tools have enabled researchers to go beyond hypotheses and provide functional evidence for predicted genes or hypothetical pathways, and will continue to do so. As a result, lipids are increasingly found at the frontiers of several research areas including cell cycle, circadian rhythms, chloroplast metabolism, cellular signal transduction processes, autophagy and development, to name just a few.

Model organisms

Research into lipids that is geared towards either industrial applications or uncovering structural diversity and physiological or evolutionary roles, has been facilitated by the ever-expanding choice of model species. The group of Prof. Chris Somerville at Michigan State University (MSU) pioneered the use of Arabidopsis thaliana in lipid research in the 80s'. Since then, a myriad of knowledge and discoveries in the area of plant lipids has been attained and subsequently expanded to crop speciesnotably rapeseed and soybean, with Camelina as an emerging oil crop species -with the goal of increasing oil content in seeds, which has societal and economic value. With this Special Focus Issue, we intend to provide a primer to spark interest on plant and algal lipids. To this end, the review articles covering some of the major areas of plant and algal lipids that feature in this issue should provide a starting point from which to explore the latest trends in the study of plant and algal lipids. This is by no means a comprehensive overview, as other exciting research areas discussed at the ISPL2018 meeting (lipid catabolism, lipid droplet biology, membrane topology and micro-domains, surface lipids, sphingolipids, etc) are not covered here due to space limitations. We therefore encourage students and early career researchers to go beyond what is presented here.

Finally, as demonstrated by the collection of papers included in this Special Focus Issue, it is clear that lipids are at the heart of many fundamental biological processes, and many more probably remain to be evidenced. We hope you enjoy reading the

  been studied for centuries for their practical use in industrial applications ranging from food, feed and biofuel, and for their important roles in plant physiology and development. The International Symposium on Plant Lipids (ISPL), initiated in 1974 by Professor Terry Galliard, provides an important platform for communication, knowledge sharing and networking amongst the growing, international plant lipid community. Since its debut in 1974, ISPL runs once every two years and the ISPL-2018 was the 23 rd symposium, which was chaired by Professor Ikuo Nishida and took place in the impressive Osanbashi Hall, a replica of a gigantic wooden ship overlooking Yokohama Bay, Japan. ISPL-2018 welcomed over 240 delegates from over 22 countries including Ph.D. students, early career scientists, professors, government scientists and company R & D representatives. This Special Focus Issue on "Lipid metabolism in plants and algae" in Plant and Cell Physiology showcases recent developments in the area as discussed during the ISPL-2018.

  another level of complexity and importance in plant and algal physiology and development. The accessibility of Ultra Performance Liquid

  Microalgae have long been subjected to research on lipids due to them being a rich source of genes for the synthesis/ modification/ metabolism of very long chain (VLC)polyunsaturated fatty acids (PUFA), and for their superior biomass productivity, and therefore great potential as a feedstock for biofuel and bio-products. The current trend in microalgae lipid research is apparent by the application of state-of-the-art genome editing and synthetic biology tools. The three flagship algal species being intensively studied at many levels include Chlamydomonas reinhardtii, Nannochloropsis spp. and Pheaodactylum tricornutum. In addition, lipid research has also been investigated in the liverwort Physcomitrella patens, which occupies a unique position in the evolutionary tree and possessing features of both water-living that of land plants. Taken together, lessons learnt through the use of multiple photosynthetic organisms will continue to give rise to unprecedented insights into the structural divergence and evolutionary significance of lipid function in plant and algal development.In this issueHere we feature four invited reviews and six research papers aimed at providing both a glimpse of what was discussed during the last ISPL meeting and highlighting the current trends in plant lipid research. The invited reviews in this Spotlight issue include: i) a description of distinct lipid profiles present within the model moss Physcomitrella patens, which show unique features similar to algae or land plants (Resemann et al. 2019), consistent with its special evolutionary position between the two clades; ii) Lavell and Benning's (2019) latest view on several aspects of plant lipid metabolism, highlighting its complexity and the importance of lipid transport especially in the context of subcellular organization and regulation of glycerolipid metabolism; iii) a review by Kong et al. (2019) on the versatile range of molecular genetic tools that have been developed for the model green microalga Chlamydomonas reinhardtii, including examples of their use in manipulating algal oil accumulation. And finally, iv), Zhou et al. (2019) summarize current efforts in engineering new lipid traits into oilseed crop Brassica species via biotechnological means. The research papers featured here highlight the many ways in which plant and algae lipids affect growth and development at the cellular and whole-organism level. They include a report on the role of VLC-PUFAs in conferring stress tolerance in the green alga Lobospheaera incisa (Kugler et al. 2019). Fujii et al. (2019) describe a role for galactolipids during etioplast-to-chloroplast differentiation. June et al. (2019) report the function of malonyl CoA-acyl-carrier protein malonyltransferase in cell division and oil storage. Warakanont et al. (2019) identified an algal lipase LIP4, orthologous to Arabidopsis Sugar-dependent 1 (SDP1), and report its involvement in triacylglycerol degradation in the green microalga Chlamydomonas reinhardtii. Finally, Chevalier et al. (2019) describe the importance of interplay between jasmonic acid and phosphate signaling on the regulation of glycerolipid metabolism in crop Crambe abyssinica for wax ester production through metabolic engineering and genetic crosses.

  featured articles in this issue, and come away with a stronger interest in lipids, and knowledge of what lies at the frontier of plant and algal lipid research and what is yet to come. Funding Y. Li-Beisson thanks the financial support of ANR Signaux-BioNRJ, and H. Wada acknowledges the financial support of the Japan Society for promotion of Science (KAKENHI no. 17H03692).

  Group photo ISPL-2018 (with permission from Dr. Ikuo Nishida). 327x219mm (300 x 300 DPI)

Plant & Cell Physiology

Acknowledgements

The authors thank Professor Hitoshi Sakakibara, editor-in-chief of Plant and Cell Physiology (PCP) and the Japanese Society of Plant Physiologists for their financial support of the ISPL-2018, and we give our sincere thanks to Dr. Liliana M. Costa, PCP Managing Editor, for her helpful advice and efficient handling of the manuscripts received.