Mireille Chabaud

Joëlle Fournier

Lukas Brichet

Iltaf Abdou-Pavy

Leandro Imanishi

Laurent Brottier

Elodie Pirolles

Valérie Hocher

Claudine Franche

Didier Bogusz

Joe ¨lle Fournier

Vale ´rie Hocher

Didier Bogusz

Luis G Wall

Sergio Svistoonoff

Hassen Gherbi

David G Barker
email: david.barker@inra.fr

Chitotetraose activates the fungal-dependent endosymbiotic signaling pathway in actinorhizal plant species

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

A limited number of soil microorganisms have acquired the remarkable capacity to colonize plant roots and form mutually beneficial endosymbiotic associations. The most widespread of these are the obligate biotrophic fungi belonging to the Glomeromycota phylum, able to associate with the majority of terrestrial plants resulting in the formation of highly ramified intracellular root cortical structures known as arbuscules [START_REF] Bonfante | Plants and arbuscular mycorrhizal fungi: an evolutionary-developmental perspective[END_REF][START_REF] Parniske | Arbuscular mycorrhiza: the mother of plant root endosymbioses[END_REF]. Arbuscular mycorrhizal (AM) fungi scavenge vital soil nutrients such as phosphorus, which are then delivered to the host plant via the elaborate symbiotic interface created within arbuscule-containing cortical cells, in exchange for energy-rich metabolites. The AM association is thought to have contributed to the successful establishment of the earliest terrestrial plant species over 400 million years ago [START_REF] Martin | Ancestral alliances: Plant mutualistic symbioses with fungi and bacteria[END_REF].

In addition to this ubiquitous fungal symbiosis, certain dicot species belonging to the Eurosid 1 clade subsequently evolved additional beneficial associations with nitrogen-fixing soil bacteria. Gram-negative rhizobia primarily associate with legume Fabales hosts, whilst grampositive filamentous actinobacteria known as Frankia are able to colonize actinorhizal host species belonging to the Fagales, Cucurbitales and Rosales orders. In the majority of cases the nitrogen-fixing bacteria multiply and differentiate intracellularly within de novo specialized plant organs known as root nodules, thereby providing a valuable source of nitrogen to the host in return for photosynthate-derived resources. Interestingly, initial root colonization by nitrogen-fixing bacteria can be either intra-or intercellular depending upon the host plant [START_REF] Ibañez | Starting points in plant-bacteria nitrogen-fixing symbioses: intercellular invasion of the roots[END_REF]. Whilst intracellular root entry via infection thread compartments constructed within root hairs has been well-documented in model legume hosts such as Medicago truncatula [START_REF] Fournier | Mechanism of infection thread elongation in root hairs of Medicago truncatula and dynamic interplay with associated rhizobial colonization[END_REF][START_REF] Gage | Infection and invasion of roots by symbiotic, nitrogen-fixing rhizobia during nodulation of temperate legumes[END_REF], intercellular modes of colonization in legumes are only poorly understood at the molecular/ cellular levels. Both types of root colonization by Frankia have also been observed for actinorhizal hosts [START_REF] Ibañez | Starting points in plant-bacteria nitrogen-fixing symbioses: intercellular invasion of the roots[END_REF][START_REF] Svistoonoff | Actinorhizal root nodule symbioses: what is signalling telling about the origins of nodulation?[END_REF]. For example, intracellular entry via root hairs appears to be common amongst Fagales species such as Alnus glutinosa or Casuarina glauca, whereas intercellular modes of root colonization appear to be predominant for Rosales species typified by Ceanothus spp, Elaeagnus angustifolia or Discaria trinervis [START_REF] Liu | The infection process and nodule initiation in the Frankia-Ceanothus root nodule symbiosis-a structural and histochemical study[END_REF][START_REF] Miller | The initiation, development and structure of root nodules in Elaeagnus angustifolia L. (Elaeagnaceae)[END_REF][START_REF] Valverde | Time course of nodule development in the Discaria trinervis (Rhamnaceae) Frankia symbiosis[END_REF]. In the case of initial root colonization by AM fungi, experiments performed on both legume and non-legume hosts have demonstrated that AM fungal hyphae traverse atrichoblast (non-root hair) cells of the root epidermis within host-constructed transcellular compartments by a mechanism reminiscent of root hair infection thread-mediated colonization by nitrogen-fixing bacterial symbionts [START_REF] Parniske | Arbuscular mycorrhiza: the mother of plant root endosymbioses[END_REF][START_REF] Martin | Ancestral alliances: Plant mutualistic symbioses with fungi and bacteria[END_REF].

An important parameter for the establishment of both AM and nitrogen-fixing endosymbioses is the capacity for mutual recognition of the respective partners. In particular, it is now well-established that host plant perception of appropriate secreted microbial molecules leads to the activation of a Conserved Symbiotic Signaling Pathway (CSSP) required for subsequent microbe root entry [START_REF] Gutjahr | Cell and developmental biology of arbuscular mycorrhiza symbiosis[END_REF][START_REF] Oldroyd | Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants[END_REF]. A characteristic feature of the CSSP is the triggering of sustained nuclear-associated calcium oscillations which can be conveniently monitored in vivo using fluorescent Ca 2+ reporters [START_REF] Barker | Nuclear Ca 2+ signalling in arbuscular mycorrhizal and actinorhizal endosymbioses: on the trail of novel underground signals[END_REF][START_REF] Miwa | Analysis of calcium spiking using a cameleon calcium sensor reveals that nodulation gene expression is regulated by calcium spike number and the developmental status of the cell[END_REF]. For the large majority of rhizobial-legume associations, decorated lipo-chitooligosaccharides (LCOs) known as nodulation (Nod) factors play this key signaling function, and these LCOs are specifically recognized by host Lysin Motif (LysM) receptor like kinase (RLK) receptor complexes [START_REF] Antolin-Llovera | Knowing your friends and foes-plant receptor-like kinases as initiators of symbiosis or defence[END_REF][START_REF] Zipfel | Plant signalling in symbiosis and immunity[END_REF]. Based on research on legumes such as M. truncatula, there is evidence that chitin-based molecules are also involved in pre-infection AM fungal-host signaling. These include both AM fungal LCOs known as Myc LCOs [START_REF] Maillet | Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza[END_REF] and AM fungal short-chain chitin oligomers (referred to collectively as Myc COs) and typified by chitotetraose (CO4) [START_REF] Genre | Short-chain chitin oligomers from arbuscular mycorrhizal fungi trigger nuclear Ca2+ spiking in Medicago truncatula roots and their production is enhanced by strigolactone[END_REF]. Although the precise roles of these chitinaceous factors remain to be determined, recent studies focused on the monocot rice have confirmed the importance of CO4 in AM fungal-host signaling [START_REF] Sun | Activation of symbiosis signaling by arbuscular mycorrhizal fungi in legumes and rice[END_REF], and identified the LysM RLK OsCERK1 as a putative component of the host perception machinery for these fungal-secreted molecules [START_REF] Carotenuto | The rice LysM receptor-like kinase OsCERK1 is required for the perception of short-chain chitin oligomers in arbuscular mycorrhizal signaling[END_REF].

In contrast with Rhizobium-legume associations, relatively little is known about pre-infection microsymbiont-host signaling during the establishment of actinorhizal symbioses. Although strong evidence suggests that intracellular-colonizing Frankia which nodulate A. glutinosa and C. glauca do secrete symbiotic signaling factors, the precise chemical nature of these molecules has not yet been determined. The use of root hair deformation assays, nuclear-localized Ca 2+ reporters to monitor CSSP activation and the expression of symbiotic reporter genes have together led to the conclusion that these Frankia make use of alternative non-chitinaceous signals [START_REF] Ce ´re ´monie | Structural and functional comparison of Frankia root hair deforming factor and rhizobia Nod factor[END_REF][START_REF] Chabaud | Chitinase-resistant hydrophilic symbiotic factors secreted by Frankia activate both Ca 2+ spiking and NIN gene expression in the actinorhizal plant Casuarina glauca[END_REF][START_REF] Cissoko | Actinorhizal signaling molecules: Frankia root hair deforming factor shares properties with NIN-inducing factor[END_REF]. On the other hand, it is currently unknown whether chitin-based molecules are involved in AM fungal signaling to actinorhizal hosts. In order to investigate this important question, we have made use of the in vivo Ca 2+ reporter approach to evaluate CSSP activation in epidermal root tissues in response to treatment with crude AM fungal exudates as well as potential Myc CO and Myc LCO signaling molecules. The two evolutionarily distant actinorhizal hosts, C. glauca and D. trinervis, were selected as typical examples of either intracellular or intercellular modes of Frankia root colonization. Finally, it is important to underline that, in the absence of either morphological root responses or available AM-related reporter genes for the two actinorhizal hosts, this assay is the only technique currently available for assaying CSSP activation in response to potential AM fungal signaling.

Results presented in this article show that, for both actinorhizal hosts, AM fungal exudates are efficient activators of sustained nuclear Ca 2+ spiking in root atrichoblasts, the cellular targets for AM root entry. Furthermore, sub-micromolar concentrations of CO4 were able to mimic CSSP activation for the two host plants and were significantly more efficient elicitors of Ca 2+ spiking compared to the tested Myc LCOs. Interestingly, in contrast to the observed CSSP activation in AM-targeted atrichoblasts, CO4 treatment did not elicit Ca 2+ spiking in root hairs, the epidermal cells of C. glauca which are not colonized by AM fungi. Finally, although responding robustly to AM fungal signaling with CSSP activation, we show that D. trinervis atrichoblasts are unresponsive to crude Frankia supernatants. Together, these findings underline the cellular specificity of these responses and indicate that actinorhizal host plants represent excellent models for studying endosymbiont-host signaling mechanisms for both intracellular and intercellular colonization.

Materials and methods

Plant materials and growth conditions

Transgenic C. glauca plants expressing the nuclear-localised cameleon reporter gene p35S: NUP-YC2.1 [START_REF] Sieberer | A nuclear-targeted cameleon demonstrates intranuclear Ca 2+ spiking in Medicago truncatula root hairs in response to rhizobial nodulation factors[END_REF] were generated via Agrobacterium tumefaciens transformation and propagated by taking cuttings as previously described [START_REF] Chabaud | Chitinase-resistant hydrophilic symbiotic factors secreted by Frankia activate both Ca 2+ spiking and NIN gene expression in the actinorhizal plant Casuarina glauca[END_REF]. Transgenic composite plants of D. trinervis (also now known as Ochetophila trinervis) expressing the p35S:NUP-YC2.1 reporter in roots were obtained using ARquaI A. rhizogenes-mediated transformation according to the protocol described in [START_REF] Fournier | Cell remodeling and subtilase gene expression in the actinorhizal plant Discaria trinervis highlight host orchestration of intercellular Frankia colonization[END_REF]. Transgenic plants were grown in liquid Broughton and Dillworth (BD) medium [START_REF] Svistoonoff | Infection-specific activation of the Medicago truncatula Enod11 early nodulin gene promoter during actinorhizal root nodulation[END_REF].

Preparation of germinated spore exudates of Rhizophagus irregularis and

Frankia supernatants R. irregularis (Agronutrition, Labège, France) germinated spore exudates (GSEs) were kindly provided by Soizic Rochange and prepared as described in [START_REF] Genre | Short-chain chitin oligomers from arbuscular mycorrhizal fungi trigger nuclear Ca2+ spiking in Medicago truncatula roots and their production is enhanced by strigolactone[END_REF] by incubating 125,000 sterile spores in 40 ml of H 2 O for 7 d in the dark. These GSEs were then concentrated 40-fold and applied to freshly excised root segments. Cell-free supernatants were prepared from "induced" cultures of Frankia strains which nodulate either C. glauca (Frankia CcI3, referred to in this article as F. casuarinae [START_REF] Nouioui | Proposal of a type strain for Frankia alni (Woronin 1866) Von Tubeuf 1895, emended description of Frankia alni, and recognition of Frankia casuarinae sp nov and Frankia elaeagni sp nov[END_REF]) or D. trinervis (Frankia BCU110501, referred to in this article as F. discariae [START_REF] Nouioui | Frankia discariae sp nov.: an infective and effective microsymbiont isolated from the root nodule of Discaria trinervis[END_REF]). For both F. casuarinae (Fci) and F. discariae (Fdi) supernatants the post-fix "i" refers to the induction of the Frankia cultures by addition of the respective host plant exudates 5 days before harvesting as described in [START_REF] Chabaud | Chitinase-resistant hydrophilic symbiotic factors secreted by Frankia activate both Ca 2+ spiking and NIN gene expression in the actinorhizal plant Casuarina glauca[END_REF][START_REF] Clavijo | The Casuarina NIN gene is transcriptionally activated throughout Frankia root infection as well as in response to bacterial diffusible signals[END_REF]. These induced Frankia supernatants were applied to root segments at a 100-fold dilution.

Chitin-based elicitors

Chitotetraose (CO4) was purchased from Megazym (LIBIOS, Pontcharra, France). An aqueous stock solution of 10 -3 M CO4 was diluted to either 10 -6 or 10 -8 M. Sulphated and nonsulphated Myc LCOs were kindly provided by Fabienne Maillet and prepared as described in [START_REF] Genre | Short-chain chitin oligomers from arbuscular mycorrhizal fungi trigger nuclear Ca2+ spiking in Medicago truncatula roots and their production is enhanced by strigolactone[END_REF]. Since Myc LCO solutions were prepared in diluted acetonitrile, comparative experiments with chitotetraose were performed using CO4 diluted with identical acetonitrile concentrations (0.5% acetonitrile for 10 -6 M CO4 and 0.005% acetonitrile for 10 -8 M CO4).

Nuclear Ca 2+ spiking responses in C. glauca and D. trinervis roots expressing the p35S:NUP-YC2.1 reporter

Assays for nuclear Ca 2+ spiking were performed using young lateral root segments (0.5 to 0.8 cm long) freshly excised from hydroponically-grown transgenic plants expressing the p35S: NUP-YC2.1 cameleon reporter. Root explants were placed in a microchamber and treated with 150 μl of the solution to be tested as described in [START_REF] Genre | Short-chain chitin oligomers from arbuscular mycorrhizal fungi trigger nuclear Ca2+ spiking in Medicago truncatula roots and their production is enhanced by strigolactone[END_REF]. Confocal FRET-based ratio imaging for detecting and plotting relative changes of nuclear Ca 2+ levels corresponding to YFP-to-CFP fluorescence intensity changes over time was performed according to [START_REF] Sieberer | A nuclear-targeted cameleon demonstrates intranuclear Ca 2+ spiking in Medicago truncatula root hairs in response to rhizobial nodulation factors[END_REF] using a Leica TCS SP2 AOBS confocal laser-scanning microscope. Imaging was performed on both atrichoblasts and trichoblasts (root hairs) in the case of C. glauca. In contrast, Ca 2+ spiking responses were only studied in D. trinervis atrichoblasts since few root hairs developed on D. trinervis roots under our growth conditions. It should be underlined that despite limited root hair formation on hydroponically-grown D. trinervis roots, efficient intercellular Frankia colonization and nodulation can be observed on these roots [e.g. 25]. Experiments were repeated several times for each treatment using independent roots (detailed in S1 Table ).

Results

Initial outer root colonization by AM fungi is intracellular for both C. glauca and D. trinervis

Late stages of AM fungal colonization have been described for both C. glauca [START_REF] Clavijo | The Casuarina NIN gene is transcriptionally activated throughout Frankia root infection as well as in response to bacterial diffusible signals[END_REF][START_REF] Gherbi | SymRK defines a common genetic basis for plant root endosymbioses with arbuscular mycorrhiza fungi, rhizobia, and Frankia bacteria[END_REF][START_REF] Diagne | Uvitex2B: a rapid and efficient stain for detection of arbuscular mycorrhizal fungi within plant roots[END_REF][START_REF] Svistoonoff | The independent acquisition of plant root nitrogen-fixing symbiosis in Fabids recruited the same genetic pathway for nodule organogenesis[END_REF] and D. trinervis [START_REF] Obertello | Interactions between Frankia BCU110501 (actinorhiza) and Gigaspora rosea (arbuscular mycorrhiza) with Discaria trinervis studied by spot inoculation[END_REF]. In both cases, the presence of intracellular symbiotic arbuscules located within inner root cortical cells, accompanied by axially growing intercellular fungal mycelia is consistent with classical Arum-type root colonization [START_REF] Smith | Mycorrhizal Symbiosis[END_REF]. Since the filamentous nitrogen-fixing Frankia microsymbiont penetrates the root outer tissues of these two actinorhizal hosts via contrasting mechanisms, it was essential to examine the initial stages of AM fungal root entry for both species. The images shown in Fig 1 and the accompanying Gif animations (S1 and S2 Figs) are consistent with AM hyphopodium formation on the root surface, followed by intracellular hyphal root penetration of the outer root tissues for the two host plants. This suggests that the AM fungus crosses the epidermal and outer cortical cell layers via a transcellular apoplastic mechanism analogous to what has already been observed for both legume and nonlegume hosts [START_REF] Bonfante | The Lotus japonicus LjSym4 gene is required for the successful symbiotic infection of root epidermal cells[END_REF][START_REF] Genre | Arbuscular mycorrhizal fungi elicit a novel intracellular apparatus in Medicago truncatula root epidermal cells before infection[END_REF][START_REF] Genre | Prepenetration apparatus assembly precedes and predicts the colonization patterns of arbuscular mycorrhizal fungi within the root cortex of both Medicago truncatula and Daucus carota[END_REF]. Furthermore, initial AM fungal entry for the two actinorhizal hosts occurs exclusively via epidermal atrichoblasts, again similar to earlier findings for other AM hosts [START_REF] Chabaud | Arbuscular mycorrhizal hyphopodia and germinated spore exudates trigger Ca 2+ spiking in the legume and non-legume root epidermis[END_REF]. Thus, despite striking differences in the modes of Frankia root colonization for C. glauca and D. trinervis, it is likely that comparable cellular mechanisms operate during initial root entry of the AM fungal partner.

AM fungal spore exudates trigger sustained Ca 2+ spiking in the root epidermis of actinorhizal host plants

Our next objective was to investigate the capacity of AM fungal signals to activate the CSSP in actinorhizal host epidermal tissues, focusing primarily on AM-targeted atrichoblast cells. For this we exploited transgenic plant tissues expressing the cameleon sensor Nup-YC2.1 in order to detect nuclear-associated Ca 2+ oscillations characteristic of CSSP activation [START_REF] Sieberer | A nuclear-targeted cameleon demonstrates intranuclear Ca 2+ spiking in Medicago truncatula root hairs in response to rhizobial nodulation factors[END_REF]. In the case of C. glauca, stable Nup-YC2.1-expressing lines were already available as described in [START_REF] Chabaud | Chitinase-resistant hydrophilic symbiotic factors secreted by Frankia activate both Ca 2+ spiking and NIN gene expression in the actinorhizal plant Casuarina glauca[END_REF]. For D. trinervis, composite plants expressing Nup-YC2.1 in root tissues were obtained via A. rhizogenes-mediated transformation (see Materials & Methods; [START_REF] Fournier | Cell remodeling and subtilase gene expression in the actinorhizal plant Discaria trinervis highlight host orchestration of intercellular Frankia colonization[END_REF][START_REF] Imanishi | Transformed hairy roots of Discaria trinervis: A valuable tool for studying actinorhizal symbiosis in the context of intercellular infection[END_REF]).

Initially, we examined whether crude exudates of germinated AM spores were able to activate the characteristic symbiotic nuclear Ca 2+ spiking response in atrichoblasts of both actinorhizal host plants. Young lateral root segments were harvested from plants growing in liquid medium and treated with a 40x concentrated solution of a R. irregularis germinated spore exudate (GSE) as described in [START_REF] Genre | Short-chain chitin oligomers from arbuscular mycorrhizal fungi trigger nuclear Ca2+ spiking in Medicago truncatula roots and their production is enhanced by strigolactone[END_REF]. Changes in nuclear Ca 2+ levels within root atrichoblasts were recorded over a 20 min period. Compared with H 2 O controls (Fig 2A and2B), GSE treatment activated sustained nuclear Ca 2+ oscillations (spiking) in 80-90% of root atrichoblasts, whether from C. glauca or D. trinervis roots (Fig 2C and2D). Having established that the CSSP can be activated by AM fungal exudates in the D. trinervis epidermis, we then evaluated responses to a diluted supernatant from the F. discariae strain that nodulates D. trinervis (supernatants from host root exudate-induced cultures were prepared as described in [START_REF] Chabaud | Chitinase-resistant hydrophilic symbiotic factors secreted by Frankia activate both Ca 2+ spiking and NIN gene expression in the actinorhizal plant Casuarina glauca[END_REF]). Strikingly, the F. discariae (Fdi) supernatant was unable to elicit Ca 2+ spiking in the D. trinervis root (Fig 2F), despite the fact that intercellular Frankia root colonization takes place between atrichoblast cells [START_REF] Valverde | Time course of nodule development in the Discaria trinervis (Rhamnaceae) Frankia symbiosis[END_REF][START_REF] Fournier | Cell remodeling and subtilase gene expression in the actinorhizal plant Discaria trinervis highlight host orchestration of intercellular Frankia colonization[END_REF]. This contrasts with the situation for C. glauca, where, as previously shown, supernatants from the induced F. casuarinae strain (Fci) trigger sustained spiking in C. glauca root hairs, the target cells for intracellular colonization [START_REF] Chabaud | Chitinase-resistant hydrophilic symbiotic factors secreted by Frankia activate both Ca 2+ spiking and NIN gene expression in the actinorhizal plant Casuarina glauca[END_REF] (Fig 2E).

CO4 mimics the activation of symbiotic Ca 2+ spiking by fungal GSEs in the two actinorhizal hosts

The short-chain chitin oligomer chitotetraose (CO4) is able to activate the AM-dependent CSSP of both legume and non-legume hosts at sub-micromolar concentrations [START_REF] Genre | Short-chain chitin oligomers from arbuscular mycorrhizal fungi trigger nuclear Ca2+ spiking in Medicago truncatula roots and their production is enhanced by strigolactone[END_REF][START_REF] Sun | Activation of symbiosis signaling by arbuscular mycorrhizal fungi in legumes and rice[END_REF]. We therefore tested the capacity of CO4 to trigger nuclear Ca 2+ spiking in root atrichoblasts of C. glauca and D. trinervis at both 10 -6 M and 10 -8 M. Data presented in Fig ( 3A-3D) show that both concentrations of CO4 elicit pronounced and sustained Ca 2+ spiking responses similar to those observed with AM fungal GSEs (Fig 2). Percentages of responding cells ranged from 75-90% and were marginally higher when CO4 was added at μM levels. In contrast to AM fungi, F. casuarinae initially colonizes C. glauca via root hairs, and as shown above, Frankia supernatants elicit Ca 2+ spiking in these epidermal cells (Fig 2E). For this reason, we also evaluated the reactivity of C. glauca root hairs to exogenous 10 -8 M CO4. Out of the 20 root hairs examined (from three independent root segments), not a single cell responded positively to the Myc CO elicitor (Fig 3E). These findings therefore demonstrate that chitotetraose can mimic the activation of the CSSP by AM fungal GSEs in root atrichoblasts of both actinorhizal host plants, but is not able to trigger nuclear Ca 2+ spiking in C. glauca root hairs.

Myc LCOs are less efficient elicitors of nuclear Ca 2+ spiking in actinorhizal hosts as compared to CO4

In addition to Myc COs such as CO4, other chitin-based molecules have been identified as potential AM fungal symbiotic signals. LCOs, which structurally resemble rhizobial Nod factors, are also present in AM fungal exudates, and have been shown to activate the CSSP in legume hosts [START_REF] Maillet | Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza[END_REF][START_REF] Genre | Short-chain chitin oligomers from arbuscular mycorrhizal fungi trigger nuclear Ca2+ spiking in Medicago truncatula roots and their production is enhanced by strigolactone[END_REF][START_REF] Sun | Activation of symbiosis signaling by arbuscular mycorrhizal fungi in legumes and rice[END_REF]. Myc LCOs are present in both sulphated (S-Myc LCOs) and nonsulphated forms (NS-Myc LCOs) in exudates of R. irregularis [START_REF] Maillet | Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza[END_REF]. For comparative purposes, we therefore evaluated nuclear Ca 2+ spiking initiation in both C. glauca and D. trinervis root atrichoblasts in response to either S-Myc LCOs or NS-Myc LCOs. These experiments, summarized in S1 Table andin However, these data also show that Myc LCOs are significantly less active than CO4 in triggering the CSSP in both actinorhizal hosts, and that S-Myc LCOs are even less active than NS-Myc LCOs. In the case of C. glauca, the difference between CO4 and NS-Myc LCO activity can be best appreciated at the lower 10 -8 M concentration. Note also that, since spiking was only observed in 20% of atrichoblasts at 10 -6 M S-Myc LCO, experiments were not performed at the lower concentration of 10 -8 M. Although a similar overall trend was found for both actinorhizal hosts, the ability to discriminate between CO4 and NS-Myc LCOs appears greater for D. trinervis by comparison with C. glauca (Fig 4). This difference can also be appreciated in the representative Ca 2+ spiking profiles shown in S3 Fig, where 10 -6 M NS-Myc LCO elicits a response with lower spiking periodicity in the D. trinervis epidermis as compared to C. glauca. Taken together, we conclude that chitotetraose is a more efficient elicitor of CSSP activation in the actinorhizal host root epidermis compared to the two types of Myc LCOs evaluated in this study.

Discussion

In this article we have investigated early symbiotic signaling between AM fungi and the two distantly-related actinorhizal host plants, C. glauca and D. trinervis, examples of either intracellular or intercellular Frankia root colonization respectively. Host perception of AM fungal signals was studied for both plants using a nuclear-localized Ca 2+ reporter to monitor the activation of the conserved symbiotic signal transduction pathway (CSSP) in epidermal root tissues. This approach has revealed that AM fungal spore exudates (GSEs) elicit sustained nuclear Ca 2+ spiking in root atrichoblasts of both C. glauca and D. trinervis (Fig 2) when applied at doses equivalent to those used previously for the legume M. truncatula [START_REF] Genre | Short-chain chitin oligomers from arbuscular mycorrhizal fungi trigger nuclear Ca2+ spiking in Medicago truncatula roots and their production is enhanced by strigolactone[END_REF]. This finding is coherent with the observation that, for all three host plants, AM fungal root entry is intracellular, with atrichoblasts as the principal epidermal target (Fig 1).

Earlier studies had shown that short-chain chitin oligomers such as CO4/CO5 are present in AM fungal GSEs and furthermore that 10 -8 M CO4 is sufficient to mimic GSE-elicited Ca 2+ spiking in M. truncatula roots [START_REF] Genre | Short-chain chitin oligomers from arbuscular mycorrhizal fungi trigger nuclear Ca2+ spiking in Medicago truncatula roots and their production is enhanced by strigolactone[END_REF]. We show here that the same concentration of CO4 is able to trigger spiking responses in root atrichoblasts of both C. glauca and D. trinervis which closely resemble those observed following GSE treatment (Fig 3). Our data also reveal that, as for M. truncatula, CO4 is a significantly more active elicitor of the CSSP in roots of both actinorhizal hosts compared to either sulphated or non-sulphated Myc LCOs (Fig 4). These findings now add two actinorhizal hosts to the growing list of AM fungal host plants for which 10 -8 M CO4 is able to elicit nuclear Ca 2+ spiking [START_REF] Genre | Short-chain chitin oligomers from arbuscular mycorrhizal fungi trigger nuclear Ca2+ spiking in Medicago truncatula roots and their production is enhanced by strigolactone[END_REF][START_REF] Sun | Activation of symbiosis signaling by arbuscular mycorrhizal fungi in legumes and rice[END_REF]. Not only are Myc COs active on all the AM hosts examined to date, but in all cases CSSP activation is observed in non-root hair atrichoblasts, the cellular targets for AM colonization. Furthermore, for certain hosts, such as rice [START_REF] Sun | Activation of symbiosis signaling by arbuscular mycorrhizal fungi in legumes and rice[END_REF] and C. glauca (Fig 3) this response is cell-specific by comparison with root hairs (see below). Additional direct evidence favoring the role of Myc COs as signaling molecules in the initial establishment of the AM association has come from the recent finding that AM-defective Oscerk1 rice mutants are also defective in responding to CO4 [START_REF] Carotenuto | The rice LysM receptor-like kinase OsCERK1 is required for the perception of short-chain chitin oligomers in arbuscular mycorrhizal signaling[END_REF], thus suggesting that Myc COs are perceived by a novel rice receptor complex comprising OsCERK1 associated with a second CO-binding LysM-containing membrane protein. Future research now needs to be directed towards identifying the corresponding actinorhizal receptor components capable of recognizing these AM fungal signals and thereby triggering the downstream CSSP. Finally, since LCOs do not appear to be involved in early Frankia-host signaling in the case of C. glauca (see below), the observed low-level responses to Myc LCOs are unlikely to be due to crosstalk with a Frankia-related pathway. In consequence, we interpret this Myc LCO activity as resulting from less efficient perception by the AM-associated receptor compared to Myc COs such as CO4.

As stated earlier, initial root colonization of C. glauca by F. casuarinae occurs intracellularly via root hairs [START_REF] Svistoonoff | Actinorhizal root nodule symbioses: what is signalling telling about the origins of nodulation?[END_REF]. A variety of host bio-assays, including the expression of transgenic Ca 2+ reporters, have together revealed that the symbiotic factors present within the Frankia exudate are unlikely to be either LCOs or COs [START_REF] Chabaud | Chitinase-resistant hydrophilic symbiotic factors secreted by Frankia activate both Ca 2+ spiking and NIN gene expression in the actinorhizal plant Casuarina glauca[END_REF][START_REF] Cissoko | Actinorhizal signaling molecules: Frankia root hair deforming factor shares properties with NIN-inducing factor[END_REF], and hence this raises the question of the nature of the receptors for Frankia signals in C. glauca. As expected, C. glauca root hairs are the principal cellular targets for Frankia symbiotic signals [START_REF] Clavijo | The Casuarina NIN gene is transcriptionally activated throughout Frankia root infection as well as in response to bacterial diffusible signals[END_REF]. The fact that short-chain COs present in AM fungal exudates can only activate the CSSP in root atrichoblasts (Fig 3) leads us to propose that, in the case of C. glauca, receptors responding to either Frankia or AM fungal signals are distinct and specifically localized to the appropriate epidermal target cell. In contrast, the situation differs for D. trinervis, since initial Frankia root colonization occurs intercellularly between adjacent atrichoblasts [START_REF] Valverde | Time course of nodule development in the Discaria trinervis (Rhamnaceae) Frankia symbiosis[END_REF][START_REF] Fournier | Cell remodeling and subtilase gene expression in the actinorhizal plant Discaria trinervis highlight host orchestration of intercellular Frankia colonization[END_REF]. We show that, although both AM fungal exudates and CO4 trigger nuclear Ca 2+ spiking in D. trinervis atrichoblasts, spiking is not observed in these cells in response to F. discariae supernatants (Figs 2 and3). One possible interpretation of these findings is that pre-infection symbiotic signaling leading to host CSSP activation is absent during the initial stages of Frankia colonization of D. trinervis. This question clearly merits further investigation since there is evidence from legumes that CSSP-related signaling may not be required during rhizobial intercellular "crack entry" invasion of certain host species [START_REF] Goormachtig | Switch from intracellular to intercellular invasion during water stress-tolerant legume nodulation[END_REF][START_REF] Madsen | The molecular network governing nodule organogenesis and infection in the model legume Lotus japonicus[END_REF]. In conclusion, the results presented in this article indicate that actinorhizal host plants are particularly valuable model systems for comparative studies of endosymbiont-host signaling mechanisms associated with either intracellular or intercellular root colonization. . Summary of the Ca 2+ spiking responses for each treatment including the number of independent roots and the number of cells observed. For each treatment, cells were assigned to one of the three categories presented in histogram format in Fig 4 (non-responding cells, cells with 1-2 spikes/20 min and cells with more than 2 spikes/20 min). Note that, in the case of CO4 treatment, the numbers in brackets refer to the roots/cells treated with CO4 in the presence of acetonitrile (0.005% for 10 -8 M CO4 and 0.5% for 10 -6 M CO4; see Materials & Methods) and correspond to the cells assigned to the three spiking categories. Atr = atrichoblast; RH = root hair. (DOCX)

S1 Table

Fig 1 .

 1 Fig 1. Rhizophagus irregularis hyphae enter actinorhizal host roots intracellularly via atrichoblasts. Roots of C. glauca (A, B) and D. trinervis (C, D) plants were observed 15d after inoculation with spores of R. irregularis. Asterisks mark AM fungal hyphopodia formed on the root surface at sites of hyphal root entry. Single and double arrowheads indicate transcellular hyphae that cross respectively epidermal and outer cortical cells. The two focal planes were selected to highlight either the surface hyphopodia (A, C) or the transcellular hyphae (B, D). To facilitate appreciation of these data, superimposed images (C and D) are presented as an animated Gif in S1 Fig, as well as an animated Gif of a second site of AM fungal colonization of a D. trinervis root (S2 Fig). Bars: 10 μm. https://doi.org/10.1371/journal.pone.0223149.g001

Fig 2 .

 2 Fig 2. Nuclear Ca 2+ spiking elicited in root epidermal cells of both C. glauca and D. trinervis in response to either AM fungal exudates or Frankia supernatants. Freshly excised root segments of C. glauca and D. trinervis were treated with either an H 2 O control (a, b), 40x concentrated AM fungal germinated spore exudates (GSE) (c, d) or 100x diluted supernatants of the appropriate induced Frankia supernatants (SN-Fci and SN-Fdi: see Materials & Methods) (e, f). Ca 2+ spiking responses were monitored in either atrichoblasts (Atr) or root hairs (RH) over 20 min periods following root treatment. These experiments show that fungal GSEs elicit sustained spiking responses in both C. glauca and D. trinervis atrichoblasts (c, d), the cellular targets for AM colonization. A typical Ca 2+ spiking response elicited in C. glauca root hairs by an induced F. casuarinae supernatant is shown in (e). By comparison, the negative response to induced F. discariae supernatant treatment of D. trinervis atrichoblasts is illustrated in (f). Percentages of positively responding cells are indicated for each treatment with the total number of cells examined in brackets. https://doi.org/10.1371/journal.pone.0223149.g002

  histogram form in Fig 4, reveal that both sulphated and nonsulphated Myc LCOs are capable of triggering Ca 2+ oscillations in both C. glauca and D. trinervis atrichoblasts (illustrated for NS-Myc LCOs in S3 Fig).

Fig 3 .

 3 Fig 3. Chitotetraose (CO4) elicits similar nuclear Ca 2+ spiking to AM fungal GSEs in root atrichoblasts of both actinorhizal hosts. Root segments of both C. glauca and D. trinervis were treated with either 10 -6 M (a, b) or 10 -8 M (c-e) CO4, and Ca 2+ spiking responses monitored in epidermal tissues over 20 min periods. (a-d) Both concentrations of chitotetraose elicited sustained spiking responses in atrichoblasts (Atr) of the two hosts resembling those observed with AM fungal exudates (Fig 2). (e) In contrast, 10 -8 M CO4 failed to trigger Ca 2+ spiking in C. glauca root hairs (RH). Percentages of positively responding cells are indicated for each treatment with the total number of cells monitored in brackets. Note that these figures combine all CO4 treatments (see S1 Table).

  Fig 3. Chitotetraose (CO4) elicits similar nuclear Ca 2+ spiking to AM fungal GSEs in root atrichoblasts of both actinorhizal hosts. Root segments of both C. glauca and D. trinervis were treated with either 10 -6 M (a, b) or 10 -8 M (c-e) CO4, and Ca 2+ spiking responses monitored in epidermal tissues over 20 min periods. (a-d) Both concentrations of chitotetraose elicited sustained spiking responses in atrichoblasts (Atr) of the two hosts resembling those observed with AM fungal exudates (Fig 2). (e) In contrast, 10 -8 M CO4 failed to trigger Ca 2+ spiking in C. glauca root hairs (RH). Percentages of positively responding cells are indicated for each treatment with the total number of cells monitored in brackets. Note that these figures combine all CO4 treatments (see S1 Table).

  Fig 3. Chitotetraose (CO4) elicits similar nuclear Ca 2+ spiking to AM fungal GSEs in root atrichoblasts of both actinorhizal hosts. Root segments of both C. glauca and D. trinervis were treated with either 10 -6 M (a, b) or 10 -8 M (c-e) CO4, and Ca 2+ spiking responses monitored in epidermal tissues over 20 min periods. (a-d) Both concentrations of chitotetraose elicited sustained spiking responses in atrichoblasts (Atr) of the two hosts resembling those observed with AM fungal exudates (Fig 2). (e) In contrast, 10 -8 M CO4 failed to trigger Ca 2+ spiking in C. glauca root hairs (RH). Percentages of positively responding cells are indicated for each treatment with the total number of cells monitored in brackets. Note that these figures combine all CO4 treatments (see S1 Table).

  https://doi.org/10.1371/journal.pone.0223149.g003

Fig 4 .

 4 Fig 4. Chitotetraose is a more efficient elicitor of Ca 2+ spiking in root atrichoblasts of both C. glauca and D. trinervis by comparison with Myc LCOs. Root segments of both actinorhizal host plants were treated with either GSEs (40x concentrated), CO4 (10 -8 & 10 -6 M), non-sulphated (NS)-Myc LCOs (10 -8 & 10 -6 M), or sulphated (S)-Myc LCOs (10 -6 M). For each treatment the dark grey bars indicate the percentage of atrichoblast cells with more than 2 spikes within the 20 min imaging period, the light grey bars 1-2 spikes, and the white bars represent non-spiking cells. See S1 Table for details of the number of cells examined for each condition and note that the data presented in this figure for all the CO4 and Myc-LCO treatments were obtained in the presence of acetonitrile (0.005% for 10 -8 M dilutions and 0.5% for 10 -6 M dilutions). https://doi.org/10.1371/journal.pone.0223149.g004

PLOS ONE | https://doi.org/10.1371/journal.pone.0223149October 10, 2019

Acknowledgments

We would like to thank Soizic Rochange (LRSV, University Paul Sabatier-CNRS, Castanet-Tolosan, France) for kindly providing us with R. irregularis GSEs and Fabienne Maillet (LIPM, CNRS-INRA, Castanet Tolosan, France) for providing both sulphated and non-sulphated Myc LCOs. Confocal imaging was performed using the Imagery Platform of the Federated Research Institute (FRAIB) at Castanet-Tolosan.

Funding: Funding was provided by grants from the French National Research Agency (ANR-12-BSV7-0007-02), which also covered the salaries of IA-P, EP, L Brottier and L Brichet, from the National University of Quilmes, Argentina (0395/07, 1411/ 15), the Argentinian National Council for Scientific & Technical Studies (CONICET; PIP 2271 and Bernardo Houssay, 2011), the French National Research Institute for Sustainable Development (IRD-DPF) and ECOS-SUD (A07B02 and A13B03). LI received a PhD fellowship from CONICET (Argentina). This study is part of the 'TULIP' Laboratory of Excellence (ANR-10-LABX-41). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

All relevant data are within the manuscript and its Supporting Information files.

Supporting information


D M Nguyen

Christophe Raynaud
email: christophe.raynaud@insa-lyon.fr

Mihai Lazar

H Vang

Dominique Planson

Comparison of electrical properties of ohmic contact realized on p-type 4H-SiC

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Due to its wide bandgap, high breakdown field, and high thermal conductivity, silicon carbide is the semiconductor material that allows a spectacular reduction of the resistance of the active layers. These values are divided by a factor of 100 compared to silicon due to doping increasing and size reduction. Nevertheless this theoretical factor is shaded by the still higher contact resistances which are obtained on p-type SiC.

This paper compares the electrical resistivity of ohmic contacts formed on p-type 4H-SiC realized by Aluminum ion implantation followed by a thermal annealing to activate the dopants. The influence of the surface state after annealing is investigated. The annealing is realized at 1650° for 45min on one sample, and at 1700°C for 30 min on the second sample. When annealed at 1700°C, the sample is capped in a graphite layer to avoid Si evaporation [1].

Description of the devices

N + 4H-SiC substrates with n-type epilayers purchased from CREE have been used to realize bipolar diodes and TLM structures. Doping level and thickness of the epilayer are 8.5´10 14 cm -3 and 55 µm. A thick epilayer was used in order to obtain a blocking voltage of 6 kV for these diodes (this aspect is not treated in this paper). The emitter of diodes are realized by multiple Aluminum implantations to achieve a box profile of impurities with a constant level at 4´10 19 cm -3 with a depth of 0.5 µm. The post-implantation anneal have been realized in two different ways. Sample A has been annealed at 1650°C during 45 min. Sample B has been annealed at 1700°C during 30 min. in the same furnace but capped with a graphite layer. Ohmic contacts were formed by deposing Ti/Ni and a Ni/Al on the topside and backside of the wafer for cathode and anode, respectively. An RTA annealing at 1000°C during 2 minutes was performed [2].

Current-Voltage (I-V) measurements have been realized using a Sourcemeter Keithley K2410.

Experimental results

Transmission Line Method (TLM)

TLM measurements have been performed on the two wafers with applied bias varying from -3 to 3 V. From the slope of the current-voltage measurement, the resistance is calculated. With varying the distance between the two ohmic contacts, it is possible to deduce the specific contact resistance rc, and the sheet resistance R□. Results are summarised in Table 1. It appears that capping the sample during the annealing reduces considerably the specific contact resistance. The difference between the values of R□ is a priori due to the difference in the temperature annealing. A higher temperature leads to a better activation of dopants and therefore to a lower resistivity or sheet resistance. TLM measurements have also been performed as a function of temperature in order to verify the contact resistivity temperature dependence. From the For both samples, above room temperature, rc is slowly decreasing with temperature: it is divided by 4-5. The surface state obtained after post-implantation annealing has a more significant impact on the contact resistances. The specific contact resistance is one decade lower for the capped sample B compared to the uncapped sample A. In contrast for the both samples closer values are obtained for the sheet resistance R□, decreasing with temperature due to the ionization of the Aluminum dopants.

Characterization of diodes

In forward bias, the diodes have been tested up to 5 V to extract the series resistance RS, and the specific on-resistance rs = RS´S, where S is the diode area. In both samples, rs depends on the diode area, which is probably due to the current spreading in the thinner metallization. However the average value of rs is a little higher in sample A. Numerical calculations taking into account the incomplete ionization, the carrier mobility indicates that resistance of the emitter and of the substrate can be neglected in regards to the epilayer resistance, Repi ~66 W for AE 250 µm diodes. Neglecting the contact resistance of the backside (due to the great surface of contact), we can estimated the specific contact resistance to be in the range 7´10 -3 -10 -2 W.cm 2 for sample A, which is considerably higher than values given by TLM measurements.

The final paper will discuss about this difference, and will also presents results on diodes realized with graphite capping. Moreover, the temperature dependence of forward characteristics will be also studied.

Figure 1 :

 1 Figure 1 : Temperature dependence of specific contact resistance (left) and sheet resistance (right) obtained by TLM measurements on sample A.

[ 1 ]

 1 E. Oliviero, M. Lazar, H. Vang, C. Dubois, P. Cremillieu, J.L. Leclercq, J. Dazord and D. Planson, Materials Science Forum 556-557, 611 (2007) [2] H. Vang, M. Lazar, P. Brosselard, C. Raynaud, P. Cremillieu, J.-L. Leclercq, J.-M. Bluet, S. Scharnholz, D. Planson, Superlattices and Microstructures 40, 626 (2006)

Table 1 :

 1 Results of TLM measurements: each value is an average of 5 measurements for sample A and 7 measurements for sample B.

	rc (Wcm²)	R□ (kW)

Table 2 :

 2 Results of I-V measurements on the diodes : each value of the specific on-resistance rs is an average of 15-20 measurements for both samples A and B.

	rs (Wcm²)	AE 250 µm	AE 400 µm
	Sample A	1.0318	2.4383
	Sample B (with C-capping)	0.9999	2.3551


MD Olivier Benoit

MD, PhD Magali Svrcek

MD Ben Creavin

MD Morgane Bouquot

MD Alexandre Challine

MD Najim Chafai

MD Clotilde Debove

Thibault Voron

MD, PhD Yann Parc

MD, PhD Jeremie H Lefevre
email: jeremie.lefevre@aphp.fr

MD, PhD ; Thibault Voron

Jérémie H Pr

Lefèvre

Prognostic value of tumor deposits in rectal cancer: A monocentric series of 505 patients

Keywords: rectal cancer, tumor deposits, N1c, nodal staging rectal cancer, tumor deposits, N1c, nodal staging pairing factors Tumor deposit, CRT: Chemoradiotherapy (CAP-50), RT: Short course Radiotherap, VI: Vascular invasion, PNI: Perineural invasion, TTT : Treatment

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Synopsis for table of Contents:

Tumor deposits (TDs) were included in TNM staging in 2010 with creation of N1c category. In this study with 505 patients operated for rectal cancer, specimens with tumor deposits had a metastatic risk comparable to a pN2 stage which may lead to changes in adjuvant treatment.

INTRODUCTION

Rectal cancer accounts for around 30% of all colorectal cancer (CRC) with a significant improvement in prognosis and management seen when neoadjuvant chemoradiotherapy is used. [START_REF] Glynne-Jones | Rectal cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up[END_REF][START_REF] Peeters | The TME trial after a median followup of 6 years: increased local control but no survival benefit in irradiated patients with resectable rectal carcinoma[END_REF][START_REF] Lakkis | Management of rectal cancer: the 2016 French guidelines[END_REF] Adjuvant treatment is guided by pathological prognostic factors including lymph node metastasis (LNM), vascular invasion (VI), perineural invasion (PNI) and tumor deposits (TD). [START_REF] Chen | Analysis of stage and clinical/prognostic factors for colon and rectal cancer from SEER registries: AJCC and collaborative stage data collection system[END_REF] Tumor deposits were first described in 1935 as nodules of tumor cells in pericolic or perirectal fat tissue without lymph node architecture. [START_REF] Gabriel | Lymphatic spread in cancer of the rectum[END_REF] They were first included in AJCC/TNM 5 th classification in 1997 and considered as lymph nodes (LN) if their size was > 3mm in diameter or as a tumor extension when they measured < 3 mm. Then the 6 th edition of TNM staging differentiated round-shaped tumor deposits as lymph nodes and spiculated ones as a tumor extension. Finally the 7 th edition in 2010 created a new pN category (i.e. pN1c) for adenocarcinoma with TD without concomitant LNM (N0). [START_REF] Sobin | cancer IUa[END_REF] N1c category represent 5 to 10 % of rectal cancers [START_REF] Chen | Analysis of stage and clinical/prognostic factors for colon and rectal cancer from SEER registries: AJCC and collaborative stage data collection system[END_REF][START_REF] Wei | The clinicopathologic relevance and prognostic value of tumor deposits and the applicability of N1c category in rectal cancer with preoperative radiotherapy[END_REF] with TDs seen in around 20% of all adenocarcinomas. [START_REF] Nagtegaal | Tumor Deposits in Colorectal Cancer: Improving the Value of Modern Staging-A Systematic Review and Meta-Analysis[END_REF] TDs are associated with poor prognostic markers such as a high T stage (T3-T4 stage) [START_REF] Wei | The clinicopathologic relevance and prognostic value of tumor deposits and the applicability of N1c category in rectal cancer with preoperative radiotherapy[END_REF][START_REF] Bouquot | Prognostic value and characteristics of N1c colorectal cancer[END_REF] and a higher N stage. [START_REF] Goldstein | Pericolonic tumor deposits in patients with T3N+MO colon adenocarcinomas: markers of reduced disease free survival and intra-abdominal metastases and their implications for TNM classification[END_REF][START_REF] Ueno | Clinical significance of extrabowel skipped cancer infiltration in rectal cancer[END_REF] 60% of patients with stage IV disease have TDs [START_REF] Puppa | Pathological assessment of pericolonic tumor deposits in advanced colonic carcinoma: relevance to prognosis and tumor staging[END_REF] with a further 72% of colorectal cancer with PNI having TDs also. [START_REF] Wei | The clinicopathologic relevance and prognostic value of tumor deposits and the applicability of N1c category in rectal cancer with preoperative radiotherapy[END_REF] Nagtegaal et al. previously reported in a meta-analysis that N1c tumors are associated with a worse disease-free survival and overall survival. [START_REF] Nagtegaal | Tumor Deposits in Colorectal Cancer: Improving the Value of Modern Staging-A Systematic Review and Meta-Analysis[END_REF] However, most of these studies were retrospective, based on pathological specimens from pre 2010 prior to the reclassification of the TNM stage. [START_REF] Nagtegaal | Tumor Deposits in Colorectal Cancer: Improving the Value of Modern Staging-A Systematic Review and Meta-Analysis[END_REF] It has been suggested that TDs may have worse prognosis in rectal cancer compared to colon cancer [START_REF] Bouquot | Prognostic value and characteristics of N1c colorectal cancer[END_REF], although there a limited number of studies purely examining rectal cancer alone. Moreover, TDs identification after radiotherapy remains controversial.

Although there are some arguments for lower survival due to TDs after neoadjuvant radiotherapy, [START_REF] Lord | The significance of tumour deposits in rectal cancer after neoadjuvant therapy: a systematic review and meta-analysis[END_REF] small number of studies have reported a poor prognosis in TD positive tumors after neoadjuvant radiotherapy [START_REF] Wei | The clinicopathologic relevance and prognostic value of tumor deposits and the applicability of N1c category in rectal cancer with preoperative radiotherapy[END_REF] while others showed no difference. [START_REF] Song | Is the N1c category of the new American Joint Committee on cancer staging system applicable to patients with rectal cancer who receive preoperative chemoradiotherapy?[END_REF] The aim of this study was to assess TD and N1c stage tumor prognosis in rectal cancer.

MATERIAL AND METHODS

Study population

All consecutive patients who underwent total mesorectum excision (TME) for rectal adenocarcinoma in our Department between 2011 and 2016 were included from a prospective database. In order to select only true rectal cancers and avoid confusion with rectosigmoid cancers, all colorectal anastomosis not diverted by ileostomy were excluded. The primary endpoint of this study was to compare prognosis in tumor deposit positive tumors and N1c patients using the new TNM staging. The secondary endpoint was to assess TDs impact in a case-control setting.

Data collection

Clinical information concerning tumor, neoadjuvant treatment and surgical procedure was retrieved from patient charts. Histological data concerning TNM stage, TDs, vascular invasion, perineural invasion, tumor grade or differentiation were included from standardized pathological reports according to the 7 th edition of AJCC classification. [START_REF] Sobin | cancer IUa[END_REF] TDs were defined as tumor nodules in the fatty tissue of the mesorectum without lymph node (LN) structure.

Local recurrence was defined as pelvic or anastomotic recurrence diagnosed by imaging or endoscopy. In cases with synchronous metastasis, distant recurrence was considered when new lesions appeared, or progression of existing disease occurred. Global recurrence was defined by the occurrence of local and/or metastatic recurrence. According to French and ESMO guidelines [START_REF] Glynne-Jones | Rectal cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up[END_REF][START_REF] Lakkis | Management of rectal cancer: the 2016 French guidelines[END_REF] preoperative radiotherapy was always discussed in multidisciplinary meetings for patients with mid and low rectal cancer. Patients with a cT3, cT4 or cN+ tumor were eligible for neoadjuvant radiotherapy. Patient with locally advanced cancer received either a 50.4 Gy radiotherapy associated with oral Capecitabine (CAP-50 protocol) or a short course protocol with 25 Gy radiotherapy. CAP-50 was preferred when a downsizing of tumor was expected to enhance circumferential resection margin.

Neoadjuvant therapy and surgical procedure

Surgery was performed 6-8 weeks after CAP-50 protocol or 1 week after short course protocol.

A total mesorectum excision (TME) was always performed as previously described. [START_REF] Bennis | Morbidity risk factors after low anterior resection with total mesorectal excision and coloanal anastomosis: a retrospective series of 483 patients[END_REF] Most patients had a primary anastomosis, with intersphincteric resection if needed. In cases of anal sphincter invasion, an abdominoperineal excision was performed. All patients had a low anastomosis and so a diverting stoma to protect the anastomosis. Patients with stage III tumor on pathological exam received adjuvant chemotherapy based on multidisciplinary decision.

Patients with N1c stage were considered as stage III.

Outcomes

Data concerning local recurrence, distant metastasis or death were collected from patient charts. Disease-free survival was defined as the time without local and/or metastatic recurrence and analyzed using the date of local and/or distant metastasis. When patients had initial metastatic disease, recurrence was considered when disease progression was observed.

Case-control study

In the case-control study, patients with synchronous metastatic disease were excluded. In each pN category, patients were matched according to sex, BMI, neoadjuvant radiotherapy, surgical procedure, pT stage, VI and PNI presence. A 2 for 1 pairing among pN0 patients and a 1 for 1 pairing in pN1 and pN2 patients was performed. Continuous variables were expressed as mean +/-standard deviation and compared using Mann-Whitney U test. Nominal variables were expressed with percentage and compared using Chi-square test or Fisher test. Multivariate analysis included variables with statistical difference in univariate analysis with p value <0.1. Survival analysis was performed with Kaplan-Meyer curves and compared with log-rank test.

Statistical analysis

RESULTS

Population

A total of 505 patients were included. Clinical data of the cohort is summarized in table 1.

There were 315 (62.4%) men with a mean age of 63.3±12.7 years. Neoadjuvant radiotherapy was performed in 275 (54.5%) patients with 42 patients receiving a short course protocol.

Abdominoperineal excision was performed in 86 (17%) patients. Pathological examination showed a majority of pT3 (N=253; 50.1%) and pN0 (N=272; 53.9%) tumors. TDs were observed in 99 (19.6%) patients, with 37 (7.3%) patients classified as pN1c. Median follow-up was 32 ± 22 months with a total of 49 (9.7%) deaths occurring during that period. Local recurrence was observed in 35 (6.9%) patients. Among patients without synchronous metastasis (n=455), 103 (22.6%) had metastatic progression. In patients with synchronous metastasis (n=50), 32 (64%) had metastatic progression. Among patients with synchronous metastasis, 5 (10%) had hepatic associated surgery and 1 (0.5%) had latero-aortic lymph node clearance.

Patients characteristics according to the presence of TDs

Comparisons between specimen with and without TDs are detailed in table 1. TD positive specimens were more frequently associated with pT3 and pT4 tumors (p<0.001), LN (p<0.001), 1). In multivariate analysis, TDs were associated with pT3-T4 tumors (p=0.037), synchronous metastasis (p=0.003), LNM (p=0.041), VI (p=0.025) and PNI (p<0.001). We didn't include neoadjuvant radiotherapy in the variable as it is correlated with the T and N stages. Multivariate analysis did not show any statistical association with R1 resections. After exclusion of synchronous metastasis, TD positive tumors were also associated with pT3 and pT4 tumors (p<0.001), LN invasion (p<0.001), VI and PNI (p<0.001).

Relationship between TDs and nodal stage

pN1c tumors were seen in 37 (7.3%) patients and were associated with pT3 and pT4 tumors (p<0,001) and synchronous metastasis (p<0.001). There was significantly more VI (p=0.003) and PNI (p<0.001) in pN1c patients. Among pN1 patients, the presence of TDs was associated with pT3 tumors (p=0.011), VI (p=0.004) and PNI (p=0.001). Within pN2 tumors, there was no difference in pT stage between TD positive and TD negative specimens. There was significantly more PNI involvement but no difference in VI (table 2).

Oncological outcomes

In the overall population, three-year overall survival (OS) was 90.5% and disease-free survival (DFS) was 66.4% (Table 3). pN1c patients had a worse DFS compared to pN0 patients (51.2% vs 79.8%; p<0.001), although there was no significant difference in in 3-year OS (figure 1). pN0 patients were associated with a worse DFS when synchronous metastasis (n=283) was excluded (58.5% vs 82.3%; p=0.001). Among pN1 patients, there was no difference in OS, however, a worse DFS due to TDs was noted (35.2% vs 70.1%; p=0.004). A significantly worse 3-year DFS was seen in TD positive pN1 patients (40.1% vs 74.6%; p=0.007) when synchronous metastasis was excluded (n=101), however, this was not seen in the pN2 patients (37.5% vs 44.7%; p = 0.499). There was no significant difference in 3-year OS (figure 2). Following exclusion of synchronous metastasis (n=71), DFS was comparable (41.1% vs 51.4%; p=0.65). There was no difference in adjuvant therapy among pN1 and pN2 patients due to TDs presence (table 2)

Impact of TD numbers

Patients with ≥4 TDs (N=14) had a worse 3-year DFS, however, this was not statistically significant. (20.6% vs. 44.1%; p=0.098). Furthermore, a worse 3-year OS was seen in patient with ≥4 TDs compared with those with 1-3 TDs (62.2% vs 82.2%; p=0.087). Again, this was not statistically significant. Specimens with one TD versus one positive LN with TDs were examined. A worse DFS was noted in the TD group (40.8% vs 81.3%; p<0.001).

Case-control study

After matching, 22 TD-positive specimens were compared with 44 TD-negative patients among the pN0 population. There was 33 and 20 TD-positive specimens in pN1 and pN2 groups respectively matched with a 1:1 ratio. Patient characteristics after matching are reported in supplementary table 1. pN1c patients had a worse 3-year DFS compared with pN0 patients (58.6% vs 82.4%; p=0.035), with no difference in 3-year OS seen. Worse DFS was observed in the pN1 group although this was not significant (respectively 40.1% vs 64.2%; p=0.153).

There was no difference in DFS among pN2 patients (32.9% vs 46.5%; p=0.858) (figure 3)

Addition of TDs with LN metastasis count

Restaging of N stage was seen when the sum of TDs was added to LN positive patients. 19 patients changed from N1 to N2 and 2 N1c patients changed to N2 stage disease. These 21 new N2 patients had a worse DFS that was not statistically significant to the old N2 classification (28.7% vs 42.7%; p=0.644) and was significantly different to the previous N1 classification (28.7% vs 58.9%; p=0.003).

Neoadjuvant chemoradiotherapy

Among TD positive patients, 50 received CAP-50 neoadjuvant protocol and 36 did not have radiotherapy. There was no difference in 3-year OS (87.1% vs 75%; p=0.447), local recurrence (12.7% vs 3.7%; p=0.188) or DFS (35.7% vs 39.9%; p=0.158) between patient that did and did not receive neoadjuvant chemoradiotherapy.

DISCUSSION

The present study found that TDs were present in nearly 20% of the 505 specimens examined, with N1c status counting for 7.3% of the patients. TDs were statistically associated with larger tumors, pT3-pT4, LN invasion, VI, PNI and, most importantly, synchronous metastasis. TDs had an important impact on DFS among N0 and N1 patients. TD prognosis was comparable to pN2 stage and did not impact N2 DFS which is associated with a poor outcome.

The incidence of TDs in the present study was similar to previous studies [START_REF] Nagtegaal | Tumor Deposits in Colorectal Cancer: Improving the Value of Modern Staging-A Systematic Review and Meta-Analysis[END_REF][START_REF] Bouquot | Prognostic value and characteristics of N1c colorectal cancer[END_REF][START_REF] Gopal | Tumor deposits in rectal adenocarcinoma after neoadjuvant chemoradiation are associated with poor prognosis[END_REF][START_REF] Tong | Is the seventh edition of the UICC/AJCC TNM staging system reasonable for patients with tumor deposits in colorectal cancer?[END_REF], however, it was higher than a recently published review on TDs and N1c [START_REF] Jin | Lymph Node Metastasis in Colorectal Cancer[END_REF]. The results in the present study are supported by the use of standardized pathological reports that systematically mentioned TDs and other poor prognostic factors. Moreover, the present study included only patient operated after 2010 so the use of N1c status was systematically used for specimens with TDs and no lymph node invasion. The presence of TDs is different in rectal cancers and may be more frequent compared to colon cancer (roughly 15-18% in colon cancer). [START_REF] Bouquot | Prognostic value and characteristics of N1c colorectal cancer[END_REF][START_REF] Goldstein | Pericolonic tumor deposits in patients with T3N+MO colon adenocarcinomas: markers of reduced disease free survival and intra-abdominal metastases and their implications for TNM classification[END_REF] However, some reports have shown the proportion to be as high as 30%, with a 29% rate of TDs in right sided colon tumors [START_REF] Shimada | Clinical impact of mesorectal extranodal cancer tissue in rectal cancer: detailed pathological assessment using whole-mount sections[END_REF][START_REF] Jin | The impact of tumor deposits on colonic adenocarcinoma AJCC TNM staging and outcome[END_REF], although these studies only included patients prior to 2010 and discrepancies in TDs definition may explain such a difference. The present study has shown that a number of poor prognostic factors were associated with TDs, notably vascular invasion, perineural invasion and synchronous metastasis. These poor prognostic factors have been demonstrated in previous studies [START_REF] Nagtegaal | Tumor Deposits in Colorectal Cancer: Improving the Value of Modern Staging-A Systematic Review and Meta-Analysis[END_REF][START_REF] Bouquot | Prognostic value and characteristics of N1c colorectal cancer[END_REF][START_REF] Gopal | Tumor deposits in rectal adenocarcinoma after neoadjuvant chemoradiation are associated with poor prognosis[END_REF][START_REF] Shimada | Clinical impact of mesorectal extranodal cancer tissue in rectal cancer: detailed pathological assessment using whole-mount sections[END_REF][START_REF] Wang | Poor prognostic and staging value of tumor deposit in locally advanced rectal cancer with neoadjuvant chemoradiotherapy[END_REF][START_REF] Ueno | Extramural cancer deposits without nodal structure in colorectal cancer: optimal categorization for prognostic staging[END_REF] and suggest the cancer cells ability to disseminate.

The following study has shown that TDs were associated with a worse DFS due to distant metastasis, which is comparable to a pN2 stage. Significantly worse DFS was observed in N0 and N1 patients but not in pN2 patients, probably due to the worse prognosis encountered in pN2 stage disease. A worse DFS among N0 and N1 patients was confirmed after exclusion of patients with synchronous metastasis. After matching on poor prognostic parameters, the present study still observed a significantly worse DFS (24%) due to TDs in pN0 patients (i.e N1c). Furthermore, this study has shown a 24% decrease in DFS due to TDs specifically in N1 patients, however, due to small sample sizes after matching, a non-significant difference in pN1 patients was seen. These studies results differs slightly from previous studies that showed a decrease in DFS and OS among LN-negative patient following chemoradiotherapy [START_REF] Wei | The clinicopathologic relevance and prognostic value of tumor deposits and the applicability of N1c category in rectal cancer with preoperative radiotherapy[END_REF][START_REF] Wang | Poor prognostic and staging value of tumor deposit in locally advanced rectal cancer with neoadjuvant chemoradiotherapy[END_REF] but not in LN-positive patients. [START_REF] Wang | Poor prognostic and staging value of tumor deposit in locally advanced rectal cancer with neoadjuvant chemoradiotherapy[END_REF] While previous data showed a decrease in OS due to TDs [START_REF] Nagtegaal | Tumor Deposits in Colorectal Cancer: Improving the Value of Modern Staging-A Systematic Review and Meta-Analysis[END_REF], the present study did not observe any difference in OS due to TDs. This observation might be explain by a 3-year OS of more than 80% even in N2 patients, although, such a high OS has already been reported in previous studies. [START_REF] Gopal | Tumor deposits in rectal adenocarcinoma after neoadjuvant chemoradiation are associated with poor prognosis[END_REF][START_REF] Shimada | Clinical impact of mesorectal extranodal cancer tissue in rectal cancer: detailed pathological assessment using whole-mount sections[END_REF] Jin et al. have shown an impact of TD numbers on OS with a cut-off of ≥4 TDs. [START_REF] Jin | The impact of tumor deposits on colonic adenocarcinoma AJCC TNM staging and outcome[END_REF] The present study showed a trend towards a worse OS and DFS with the number of TDs, although, this was not significant. While Jin et al only studied N1c patients, the present study included both N1c and LN positive patients which may impact on survival. This may be further supported by the finding in the present study that TDs alone have a poorer prognosis than a positive LN alone, with a 40% decrease in DFS. This can be explained by the fact that the pathway involved in cellular dissemination might be different for each modality. Some authors have demonstrated that TDs are due to twist mutation while LN spread is due to snail mutations. [ N1c status is a peculiar status as it represents patients only with TDs and no LN invasion. N1c patients have a worse DFS of 51.2% in this study. N1c cancers were also associated with larger tumors and poor prognostic factors as previously suggested. [START_REF] Bouquot | Prognostic value and characteristics of N1c colorectal cancer[END_REF] Compared to N0 or N1 patients without TDs, DFS was worse among N1c patients and comparable to N2 disease. It has already been proven that N1c status has a poor impact on DFS and OS in colorectal cancer. [START_REF] Bouquot | Prognostic value and characteristics of N1c colorectal cancer[END_REF][START_REF] Yamano | Prognostic significance of classified extramural tumor deposits and extracapsular lymph node invasion in T3-4 colorectal cancer: a retrospective single-center study[END_REF] The present study has shown that TDs did not impact prognosis following neoadjuvant chemoradiotherapy. However, in rectal cancer, it remains uncertain if N1c status is useful after radiotherapy. While some authors suggest that patients with N1c grade have a poor prognosis after radiotherapy [START_REF] Wei | The clinicopathologic relevance and prognostic value of tumor deposits and the applicability of N1c category in rectal cancer with preoperative radiotherapy[END_REF][START_REF] Wang | Poor prognostic and staging value of tumor deposit in locally advanced rectal cancer with neoadjuvant chemoradiotherapy[END_REF] other studies have failed to show a prognostic difference due to confusion between TDs and residual tumor. [START_REF] Song | Is the N1c category of the new American Joint Committee on cancer staging system applicable to patients with rectal cancer who receive preoperative chemoradiotherapy?[END_REF] There are still a number of difficulties in evaluating TDs prognosis after neoadjuvant treatment. Furthermore, neoadjuvant chemoradiotherapy is indicated for advanced tumors or N positive tumors, that have a high risk for TD presence but TDs are not easily diagnosed on MRI with great difficulties to assess their presence preoperatively.

Distinction between TD and invaded LN remains a challenge and is still debated among pathologists. [START_REF] Rock | Debating deposits: an interobserver variability study of lymph nodes and pericolonic tumor deposits in colonic adenocarcinoma[END_REF] TDs may be confused for a completely replaced LN, venous invasion or even tumor spread. [START_REF] Jin | Lymph Node Metastasis in Colorectal Cancer[END_REF] The present study suggests that TDs must be mentioned in pathological reports and confusion between TDs and LN may lead to a lack of metastatic risk evaluation. Current TNM staging is suboptimal in its definition of TDs. A lot of pathologists do not support practices such as excluding nodules with evidence of underlying EMVI, LI or PNI from the pN1c category and thus downstaging a patient from stage III to stage II if reclassifying them according to the TNM 8 rather than the TNM 7 edition. Currently, French and European guidelines recommended post-operative chemotherapy for patients with stage III and IV rectal cancer. [START_REF] Glynne-Jones | Rectal cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up[END_REF][START_REF] Lakkis | Management of rectal cancer: the 2016 French guidelines[END_REF] However, these recommendations are based on expert consensus and protocols used in colon cancer. Recently, it has been suggested that in colon cancer, a longer duration of adjuvant chemotherapy may improve prognosis for high risk cancer. [START_REF] Lieu | Duration of Oxaliplatin-Containing Adjuvant Therapy for Stage III Colon Cancer: ASCO Clinical Practice Guideline[END_REF] With this in mind, the authors suggest that the presence of TDs should be considered when implementing adjuvant treatment regimens and protocols. Chavali et al. suggest that adjuvant radiotherapy improves prognosis for patients with TDs who did not receive neoadjuvant radiotherapy. [START_REF] Chavali | Radiotherapy for Patients With Resected Tumor Deposit-Positive Colorectal Cancer: A Surveillance, Epidemiology, and End Results-Based Population Study[END_REF] The results in the present study are strengthened by a large sample size and standardized pathological reports. Furthermore, patients were only included after 2010 which corresponded to the introduction of the 7 th edition of TNM staging. However, there are still several limitations.

It is a retrospective study with the majority of patients having N0 disease. TDs were present in almost 20% of the population, however, only 37 patients were classified as N1c which lead to a lack of statistical strength for this sub-group. Due to the small numbers of N1 patients after matching, a significant impact on DFS due to TDs could not be ascertained. Furthermore, the majority of patients had <4 TDs, again limiting the true impact of TD numbers on DFS outcomes.

CONCLUSION

In rectal cancer, TDs are a poor prognostic factor with a higher risk of metastatic recurrence comparable to N2 disease. Adjustments in chemotherapy protocols must be discussed for patients classified as N1c or N1 with TDs. Further large scale, multicentre, prospective studies are required to ascertain the prognostic implications of TDs. 25)

1 1 0 0 1 (3) 1 (3) 1 (5) 1 (5

7

 7 Statistical analysis was performed using SPSS Statistic 20 software (IBM, Armonk, NY; USA).

  .001) and PNI (p<0.001) (table

Figure 1 :Figure 2 :

 12 FIGURES LEGEND

Figure 3 :

 3 Figure 3: Survival curves after matching. A: pN0; B: pN1; C: pN2. TD neg: No TDs; TD

Table 1 :

 1 Patients characteristics. *: in multivariate analysis comparison concerned pT0-1-2 vs pT3-4; **: for multivariate analysis comparison concerned pN+ vs pN-

	Multivariate

Table 2 . Comparison between TD positives and negatives specimen according to LN involvement.

 2

		OS (%)	p	DFS (%)	p	LR (%)	p	DM (%)	p
	Population	90.5		66.4		92.3		68.9
	pN0	94.6		79.8		92.4		83.1
	pN1c	89.7		51.2		87.1		55.1
	pN1	90		58.9		92.4		60
	pN1/TD pos	80.8	0.389	35.2	0.004	93.1	0.737	35	0.003
	pN1/TD neg	93.2		70.1		92.1		72
	pN2	80.6		42.7		94		44.1
	pN2/TD pos	76.4	0.438	37.5	0.499	100	0.234	37.5	0.375
	pN2/TD neg	82.1		44.7		92		46.6
	M0
	TD pos	89.5	0.422	46.7 <0.001	92.7	0.825	47.8	<0.001
	TD neg	92.5		76.5		93.4		76.8
	1 TD	82	0.053	40.8 <0.001	79.1	0.220	47.6	0.001
	N1a/TD ng	95.2		81.3		93.4		81.3
	TD number
	≥4 <4	r o 62.2 0.087 F 86	20.6 44.1	0.098	83.3 93.3	0.444	20.6 45.3	0.96
			P e e
				r
				R
				e v i e w

TD: Tumor deposit; CRT: Chemoradiotherapy (CAP-50); RT: Short course Radiotherap; VI: Vascular invasion; PNI: Perineural invasion; TTT : Treatment.

Table 3 : Three-year survival.

 3

	metastasis free survival

OS: Overall survival; DFS: Disease-free survival; LR: Local recurrence free survival; DM: Distant

table 1 .

 1 Patients characteristics after matching.

	)

John Wiley & Sons, Inc.Journal of Surgical Oncology

Availability of data

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.


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RNA seq reveals different yeast behaviours in terms of nitrogen consumption and fermentative aroma production

  Stéphanie Rollero, Audrey Bloem, Anne Julien Ortiz, Carole Camarasa, Benoit Divol To cite this version: Stéphanie Rollero, Audrey Bloem, Anne Julien Ortiz, Carole Camarasa, Benoit Divol. RNA seq reveals different yeast behaviours in terms of nitrogen consumption and fermentative aroma production. International Conference in Wine Sciences Macrowine 2018, May 2018, Saragosse, Spain. . hal-02961832

Fermentative aromas play an important role in the organoleptic profile of wines. These compounds derive for a significant part from the break-down of nitrogen sources naturally found in grape juice, but their production also depends on the yeast strains driving the fermentation. During sequential inoculation of non-Saccharomyces yeasts with Saccharomyces cerevisiae, multiple interactions between yeasts may occur, including competition for nutrients that directly impacts fermentation performances and aroma production. Two unconventional non-Saccharomyces yeasts (Pichia burtonii and Kluyveromyces marxianus) were selected for displaying a behaviour different from that of S. cerevisiae in terms of nitrogen consumption and fermentative aroma production. The uptake of nitrogen sources was monitored together with fermentation and population kinetics. In parallel, total RNA was extracted at specific time points corresponding to lag phase (LP) and time where half of the maximal population (HMP) was reached. RNA sequencing was used to unravel these strain-specific behaviours at a molecular level.

Depending on the yeast strain and the amino acids considered, 3 behaviours were observed:

 Amino acids consumed continuously during the first 72 hours  Amino acids consumed until the HMP was reached (but not depleted)  Amino acids started to be consumed when the HMP was reached Although P. burtonii overexpressed some nitrogen transporters, it: Was able to consume only 20% of YAN Struggled to ferment (only 25% of sugars consumed)

P. burtonii not adapted to environmental conditions:

• Poor adaptation to osmotic pressure?

• Lack of oxygen?

• Lack of nutrients (others than N)?

In Amino acids within the first 72h  GAP1 (general amino acid permease): up-regulated in the 3 strains


Ying Su

Pauline Seguinot

Isabelle Sanchez

Anne Ortiz Julien

José María Heras

Jean-Roch Mouret

Amparo Querol

Audrey Bloem

José Manuel Guillamón

Carole Camarasa

Anne Ortiz Julien

Nitrogen source utilisation by non-Saccharomyces yeasts in winemaking conditions

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Objectives

Results

Conclusions

Acknowledgement

The strains of this project are provided by Lallemand. The author also wants to thank Lallemand for financial support. YS thanks FEMS for granting the conference bursary. Contact: ying.su@iata.csic.es guillamon@iata.csic.es carole.camarasa@inra.fr

1 .Figure 1 .

 11 Figure 1. Enrichment of proteinogenic amino acids during fermentation with isotopic labelled amino acids.

Figure 3 .

 3 Figure 3. The redistribution of labelled valine in Torulaspora delbrueckii. Labelled compounds were showed in bars with darker colour. The numbers on the top of each bar are the percentage of the labelled compound in the total amount of the compound at each stage. The values in the coloured boxes in the metabolic schema represent the percentage of consumed amino acids that is used in the pathway.

Figure 4 .

 4 Figure 4. The redistribution of labelled leucine in Torulaspora delbrueckii.

Figure 2 .

 2 Figure 2. (A) The proportion of consumed arginine, glutamine and ammonium in total consumed nitrogen sources. T 0 represents the initial concentration supplied in the synthetic must. (B) Portion of nitrogen provided by consumed arginine, glutamine, and ammonium recovered in proteinogenic amino acids. Dark colour is the difference between the previous stage and the current stage, light colour represents the amount which has been consumed (A) or incorporated in the previous stage (B).

Figure 3 .

 3 Figure 3. Fate of arginine during fermentation. Blue bar represents the consumed arginine directly incorporated into proteinogenic arginine. Orange bar represents the consumed arginine were catabolised to form other amino acids. The numbers in blue and orange indicate the ratio (in percentage) of arginine incorporated to proteinogenic arginine or catabolised to the total consumed arginine.


Pauline Seguinot

Anne Ortiz-Julien

Pascale Brial

Carole Camarasa

Exploring the potentialities of Metschnikowia species during sequential wine fermentation

fermentation performances

Currently, in the winemaking sector, there is a renewed interest of the use of non-Saccharomyces species during the fermentation process. Long regarded as spoilage microorganisms, the potential of these yeasts, including Metschnikowia pulcherrima species, to improve the sensory quality of wines is now recognized. Due to their inability to complete the fermentation, M. pulcherrima strains must be used in combination with Saccharomyces cerevisiae, added in sequential or co-inoculation. However, the limited knowledge on their traits prevents to more widely gain the benefits and potentialities of M. pulcherrima during wine fermentation. To fill this shortcoming, this study investigated the specificities and the contribution of M. pulcherrima during sequential fermentation with S. cerevisiae.

Compared with S. cerevisiae pure culture, sequential fermentation has a negative impact on:

Fermentation rate

Compared with S. cerevisiae pure culture, sequential fermentation a positive impact on:

the production of acetate esters (and some higher alcohols, depending on lipids, sugars and nitrogen availability) the production of acetate (decrease) and glycerol ( increase) the production of positive thiols, which increases by a factor up to 6 The comparison between sequential inoculation and pure cultures pointed out the benefits but also the shortcomings of the use of M. pulcherrima in fermentation. Thanks to the Box-Behnken model, the impact and interaction of the parameters were assessed, showing that nitrogen was the most influencing nutrient on the sensory and organoleptic profile of wines. These results pave the way for the design of an improved management of sequential inoculation during winemaking to better exploit the potentialities of M. pulcherrima strains.

production of higher alcohols and acetate esters production of thiols

  the implantation of S. cerevisiae, due to the important consumption of glutamate, glutamine and branched amino acids of M. pucherrima the fermentation rate, resulting in an increased fermentation duration the production of medium chain fatty acids and their esters derivatives

  No production of 3-MHA in S.cerevisiae pure culture Range of production of 3-MHA in sequential fermentation: 0-30 ng/L N it ro g e n (m g /


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Environmental conditions can modulate the production of volatile sulfur compounds (VSC) during wine fermentation

Rafael Jimenez-Lorenzo, Pascale Brial, Marc Perez, Christian Picou, Vincent

Environmental conditions can modulate the production of volatile sulfur compounds (VSC) during wine fermentation

In the increasingly competitive worldwide market of wines, a current challenge is to offer products with style and character to meet the consumer's expectations. In this context, controlling the production of volatile compounds during the fermentation, which may have a positive or negative incidence on the wine aroma, is essential. This project focuses on the understanding of the formation of volatile sulfur compounds (VSCs) deriving from yeast metabolism and described as unpleasant aromas in wines. The yeast metabolic network underlying the synthesis of these molecules remains until now poorly elucidated as well as the incidence on the activity of these pathways of many environmental and technological parameters including pH, sugars, sugar concentration, nitrogen resource composition and availability or SO 2 addition. Exploring the profile of VSCs formation within a collection of 40 yeasts (Saccharomyces and non Saccharomyces species) commonly encountered in wine environment revealed an important variability between strains in their abilities to produce VSCs. Furthermore, using a Box-Behnken experimental design, we were able to show pH, sugar, and SO 2 concentrations modulate in combination the production of VSCs, with a straindependent response.

Materials and Methods

  Farines, Audrey Bloem, Carole Camarasa To cite this version: Rafael Jimenez-Lorenzo, Pascale Brial, Marc Perez, Christian Picou, Vincent Farines, et al.. Environmental conditions can modulate the production of volatile sulfur compounds (VSC) during wine fermentation. 7th conference on Yeasts and Filamentous Fungi, May 2019, Milan, Italy. . hal-

•

  with central point in triplicate • Determination of the effects of parameters on VSC production Simple effect Quadratic effect Interactions Screening of 40 different strains on the basis of VSCs production 22 Saccharomyces 18 Non Saccharomyces VSCs analysis by GC-MS (headspace injection method): 15 different compounds analysed Selection of strains displaying a high capacity to produce VSCs• Substantial differences between strains in their capacity to produce VSCs, with in general, lower productions by non-Saccharomyces strains • Most of the molecules produced by a limited number of strains, apart from methionol, produced by all the studied effect; -: negative effect N : nitrogen; S: sugar; L: lipids Green : p<0.001; blue : p<0.01; orange : p<0.05, white : NA Overall, increase in VSCs production by adding of 45 mg/l SO 2• Extent of the response to SO 2 addition depending on both the strain and the nature of the compound 7 th Conference on PHYSIOLOGY OF YEASTS AND FILAMENTOUS FUNGI MILAN

••

  One parameter is fixed, the others two are represented on x and y axis • The studied aroma is represented on the z Quadratic effect of sugar • Positive impact of pH • Quadratic effect of sugar • No impact of pH • Interaction between pH and SO 2 in the production of MTHTP by the strain MTF914:  Positive SO 2 effect of at high pH  Negative SO 2 effect at low pH Fermentation using synthetic must (Bely et al. 1998) Screening of wine yeast collection to select high VSCs producers -methylthioacetate; DEDS: Diethyl disulfide; E3MTP: 3-Methylthio propanoic acid ethyl ester; MTHTP: 2-methyltetrahidrothiophenone; 3MTPAc : 3-Methylthio propyl acetate; 3MTPA : 3-Methylthio propanoic acid; ME: methionol