Fernandez C 1x

Bousquet-Mélou A 1x
email: anne.bousquet-melou@imbe.fr

Keywords: Mediterranean oak, chemical interactions, regeneration, water stress, plant-plant interactions Declarations

How plant-plant interactions will interact with global change drivers such as increased drought during the regeneration phase is a key question to forecast future vegetation dynamics. Chemical interaction and especially allelopathy and drought have been suggested to affect plant performance synergistically, i.e. that plant under drought stress would be more sensitive to allelochemicals and that exposure to allelopathic interactions could increase drought sensitivity through an inhibition of root development and mycorrhization. In this paper, we tested these hypotheses by using a controlled experiment with Quercus pubescens Mill. as a target species and three cooccurring species plus itself as source species. Allelopathic treatments consisted of annual provision of litter and monthly watering with green leaf aqueous extracts during 2 vegetation seasons starting from oak acorns. During the second vegetation season, a drought stress treatment was added on half of the seedlings. Allelopathy of cooccurring species reduced seedlings dimensions while Q. pubescens treatment increased it. During the second vegetation season, seedling growth rate and physiology were reduced by drought but poorly affected by allelopathic treatment. At the end of the experiment, drought stress and allelopathy from Cotinus coggygria and Pinus halepensis both reduced seedling biomass but had opposite effects on the root/shoot ratio. Drought and allelopathy did not interact significantly and, contrary to our hypothesis, there was a tendency of lower allelopathic effects under drought. Our results suggest that drought and allelopathy could additively alter seedling development, but the opposite effects of allelopathy and drought on the root/shoot ratio call for further experiments testing the interaction between these two factors.

Introduction

Plant regeneration plays a central role in the dynamics of plant populations and communities, particularly early phases including germination, growth and establishment of seedlings [START_REF] Nathan | Spatial patterns of seed dispersal, their determinants and consequences for recruitment[END_REF][START_REF] Nathan | Spatiotemporal dynamics of recruitment in Aleppo pine (Pinus halepensis Miller)[END_REF][START_REF] Grubb | The maintenance of species-richness in plant communities: the importance of the regeneration niche[END_REF]. These demographic stages are influenced by both biotic and abiotic factors [START_REF] Lortie | Rethinking plant community theory[END_REF]). In Mediterranean forests, summer drought is among the most constraining factors for seedling establishment, which often represent an important bottleneck for species regeneration (e.g. Marañón et al. 2004;[START_REF] Pérez-Ramos | Ontogenetic conflicts and rank reversals in two Mediterranean oak species: implications for coexistence[END_REF]. Furthermore, summer drought stress is expected to increase with climate change consequently to globally rising temperatures and changing precipitation patterns (IPCC 2014, Giorgi and[START_REF] Giorgi | Climate change projections for the Mediterranean region[END_REF] which may reinforce the drought limitation for seedling establishment [START_REF] Pérez-Ramos | Quercus ilex recruitment in a drier world: A multi-stage demographic approach[END_REF].

Biotic factors such as plant-plant interactions are also particularly important for plant regeneration. The surrounding vegetation can influence seedling establishment processes by a change of resource availability and microclimate [START_REF] Gómez-Aparicio | Canopy vs soil effects of shrubs facilitating tree seedlings in Mediterranean montane ecosystems[END_REF][START_REF] Prévosto | Influence of neighbouring woody treatments on Mediterranean oak development in an experimental plantation: Better form but weaker growth[END_REF]Gavinet et al. 2016a), but also through chemical interactions [START_REF] Fernandez | Variations in allelochemical composition of leachates of different organs and maturity stages of Pinus halepensis[END_REF][START_REF] Gavinet | Phenolics of the understory shrub Cotinus coggygria influence Mediterranean oak forests diversity and dynamics[END_REF][START_REF] Weir | Biochemical and physiological mechanisms mediated by allelochemicals[END_REF]). Plant-plant chemical interactions, i.e. allelopathy [START_REF] Rice | Allelopathy[END_REF], can be positive -typically through nutrient release -or negative due to the release of phytotoxic compounds. Negative chemical interactions play an important role in Mediterranean ecosystems [START_REF] Vilà | Plant competition in Mediterranean-type vegetation[END_REF] and in particular in forest succession dynamics [START_REF] Alías | Autotoxicity against germination and seedling emergence in Cistus ladanifer[END_REF]Fernandez et al. 2008[START_REF] Fernandez | Allelochemicals of Pinus halepensis as Drivers of Biodiversity in Mediterranean Open Mosaic Habitats During the Colonization Stage of Secondary Succession[END_REF][START_REF] Fernandez | The Impact of Competition and Allelopathy on the Trade-Off between Plant Defense and Growth in Two Contrasting Tree Species[END_REF]. For example, specialized compounds produced by plants (terpenoids and/or phenolic compounds) can delay seed germination, inhibit seedling growth [START_REF] Mallik | Conifer Regeneration Problems in Boreal and Temperate Forests with Ericaceous Understory: Role of Disturbance, Seedbed Limitation, and Keytsone Species Change[END_REF] or affect root symbionts [START_REF] Duponnois | An integrated view of allelochemicals amid multiple stresses[END_REF][START_REF] Mallik | Regeneration of tree species and restoration under contrasted Mediterranean habitats: field and glasshouse experiments[END_REF] which play a key role during summer drought by improving seedling water and nutrient uptake [START_REF] Sardans | Plant-soil interactions in Mediterranean forest and shrublands: impacts of climatic change[END_REF]. Specialized compounds can also indirectly affect seedling development through modified nutrient cycles during the litter decomposition process [START_REF] Chomel | Secondary metabolites of Pinus halepensis alter decomposer organisms and litter decomposition during afforestation of abandoned agricultural zones[END_REF][START_REF] Kuiters | Role of phenolic substances from decomposing forest litter in plant-soil interactions[END_REF][START_REF] Kainulainen | Decomposition of secondary compounds from needle litter of Scots pine grown under elevated CO2 and O3[END_REF][START_REF] Gavinet | Phenolics of the understory shrub Cotinus coggygria influence Mediterranean oak forests diversity and dynamics[END_REF]. Phenolic compounds are mostly soluble and can be released from plant leaves during rainy events or during the litter decomposition process. Terpenoids in contrast are highly volatile and may interact with surrounding plants in their gaseous phase after volatilization, either from living leaves or from litter (Santonja et al. 2019). Allelopathic potential has long been examined through isolation of compounds and laboratory tests on model species such as Lactuca sativa, with the aim of identifying potential natural herbicides. However, the investigation of allelopathic interactions in natural ecosystems, especially forests, require more realistic experiments, testing the effects of natural extracts of plant materials on co-occurring species, taking into account the phenological stages of the interacting species [START_REF] Inderjit | Are laboratory bioassays for allelopathy suitable for prediction of field responses[END_REF].

The interaction between drought and plant-plant interactions has been intensively debated [START_REF] Maestre | Is the change of plant-plant interactions with abiotic stress predictable? A meta-analysis of field results in arid environments[END_REF][START_REF] Maestre | Refining the stress-gradient hypothesis for competition and facilitation in plant communities[END_REF][START_REF] Michalet | Highlighting the multiple drivers of change in interactions along stress gradients[END_REF]) and this debate becomes particularly important when trying to forecast global change impact on plant distribution [START_REF] Michalet | Facilitation in communities: underlying mechanisms, community and ecosystem implications[END_REF]. The interaction between drought and shading has been intensively studied (e.g. [START_REF] Sánchez-Gómez | Performance of seedlings of Mediterranean woody species under experimental gradients of irradiance and water availability: trade-offs and evidence for niche differentiation[END_REF][START_REF] Holmgren | Non-linear effects of drought under shade: reconciling physiological and ecological models in plant communities[END_REF][START_REF] Laanisto | Polytolerance to abiotic stresses: how universal is the shade-drought tolerance trade-off in woody species?[END_REF] but drought and chemical interactions have rarely been studied jointly. Drought could impact plant-plant chemical interactions by increasing the production of allelochemicals by source plants [START_REF] Duponnois | An integrated view of allelochemicals amid multiple stresses[END_REF][START_REF] Pedrol | Allelopathy and abiotic stress In : Allelopathy: A Physiological Process with Ecological Implications Netherlands[END_REF][START_REF] Gatti | Seasonality effect on the allelopathy of cerrado species[END_REF] or fostering the production of compounds with greater phytotoxic activities [START_REF] Oueslati | Barley Autotoxicity as Influenced by Varietal and Seasonal Variation[END_REF][START_REF] Gatti | Seasonality effect on the allelopathy of cerrado species[END_REF]. Plants may be more sensitive to allelochemicals under drought stress [START_REF] Pedrol | Allelopathy and abiotic stress In : Allelopathy: A Physiological Process with Ecological Implications Netherlands[END_REF]) and allelopathy may aggravate drought stress by antagonistic effects on root development: allelopathy is often inhibiting root development (e.g. [START_REF] Gavinet | Phenolics of the understory shrub Cotinus coggygria influence Mediterranean oak forests diversity and dynamics[END_REF], while increased biomass allocation to roots is a well-known morphological response to drought aiming at increasing seedling ability to capture belowground water [START_REF] Dickson | Oak growth, development and carbon metabolism in response to water stress[END_REF][START_REF] Lloret | Seedling survival of Mediterranean shrubland species in relation to root: shoot ratio, seed size and water and nitrogen use[END_REF][START_REF] Poorter | Biomass allocation to leaves, stems and roots: meta-analyses of interspecific variation and environmental control[END_REF].

Quercus pubescens Willd. (downy oak) forests represent more than one million hectares in France. Downy oak often occurs in the transition of several climatic influences and is sensitive to drought at the seedling stage [START_REF] Morin | Changes in leaf phenology of three European oak species in response to experimental climate change[END_REF], which translates into frequent regeneration failure in the Mediterranean [START_REF] Prévosto | Éclaircir est-il suffisant pour favoriser la régénération de taillis de chêne blanc? Retour sur un dispositif expérimental installé il y a 27 ans en forêt domaniale de Lure (Alpes-de-Haute-Provence)[END_REF]Gavinet et al. 2016b). The natural regeneration from seeds of Mediterranean oaks is uncertain, as many obstacles can hinder the process: poor seed supply, lack of an effective dispersal vector, high predation on seeds or difficulties in setting up seedlings (Puerta-Piñero 2010; Pausas et al. 2009). Emergence, survival and growth of oak seedlings are highly variable depending on the microsite (Prévosto et al. 2011;[START_REF] Pérez-Ramos | Quercus ilex recruitment in a drier world: A multi-stage demographic approach[END_REF]). This variability is often related to competition for resources with existing vegetation, but allelopathic interactions may also contribute to the definition of oaks regeneration niche [START_REF] Li | Effects of germination inhibition on the dynamics of Quercus ilex stands[END_REF][START_REF] Gavinet | Phenolics of the understory shrub Cotinus coggygria influence Mediterranean oak forests diversity and dynamics[END_REF]. Downy oak acorns are likely to be dispersed in places surrounded by species with important contents of phenolics or terpenoids and known to exhibit allelopathic potential such as Pinus halepensis (Fernandez et al. 2008), Acer monspessulanum L. or Cotinus coggygria Scop. and Q. pubescens itself. Indeed, in Mediterranean forest, A. monspessulanum is a companion species of downy oak in the tree stratum while the shrub C. coggygria can dominate the understorey in supramediterranean conditions [START_REF] Hashoum | Biotic interactions in a Mediterranean oak forest: role of allelopathy along phenological development of woody species[END_REF]. P. halepensis also coexists with Downy oak in warmer and drier environments (Santonja et al. 2015a). The chemical content of the four source species has been analyzed in several previous studies. Aqueous extracts from needles, roots and litter of P. halepensis contain large amounts of compounds belonging to different functional phytochemical groups likely to induce allelopathic effects (monoterpenes, sesquiterpenes, phenolics; [START_REF] Fernandez | Variations in allelochemical composition of leachates of different organs and maturity stages of Pinus halepensis[END_REF][START_REF] Gavinet | Do litter-mediated plantsoil feedbacks influence Mediterranean oak regeneration? A two-year pot experiment[END_REF]. Allelochemicals from P. halepensis are known to slow down the germination of herbaceous species [START_REF] Fernandez | Potential allelopathic effect of Pinus halepensis in the secondary succession: an experimental approach[END_REF][START_REF] Fernandez | Allelochemicals of Pinus halepensis as Drivers of Biodiversity in Mediterranean Open Mosaic Habitats During the Colonization Stage of Secondary Succession[END_REF][START_REF] Santonja | Allelopathic effects of volatile organic compounds released from Pinus halepensis needles and roots[END_REF] or the growth of tree species such as Q. pubescens especially in early stage of its development [START_REF] Fernandez | The Impact of Competition and Allelopathy on the Trade-Off between Plant Defense and Growth in Two Contrasting Tree Species[END_REF]) but also the growth of P. halepensis via autotoxicity (Fernandez et al. 2008).

Aqueous extracts from green leaves, senescent leaves and litter of A. monspessulanum, C. coggygria and Q.

pubescens contain flavonoids such as quercetin, isorhamnetin and naringenin, compounds with reported allelopathic activities [START_REF] Paszkowski | Biological activity and tentative identification of flavonoid components in velvetleaf (Abutilon theophrasti Medik.) seed coats[END_REF][START_REF] Macias | Allelopathy in the search for natural herbicide models[END_REF]. Notably, allelopathic effects from those aqueous extracts have been reported on the germination and growth of two herbaceous target species occurring in the same community (Linum perenne and Festuca ovina) with senescent leaves extract that inhibited seeds' germination velocity while green leaves extract impacted seedlings growth [START_REF] Hashoum | Biotic interactions in a Mediterranean oak forest: role of allelopathy along phenological development of woody species[END_REF]. Moreover, total phenolics, terpenes and nutrients contents of the litter are also described in [START_REF] Gavinet | Do litter-mediated plantsoil feedbacks influence Mediterranean oak regeneration? A two-year pot experiment[END_REF]Santonja et al. 2015a.

In this study, we performed a greenhouse experiment to test how Q. pubescens seedlings establishment is affected by plant-plant chemical interactions with itself and three co-occurring species (A. monspessulanum, C. coggygria. and P. halepensis), and how these chemical interactions interact with drought. We hypothesized that i) chemical interactions alter oak seedlings development by decreasing root biomass and mycorrhization, ii) exposure to chemical interactions consequently increase seedlings sensitivity to drought and iii) oak seedlings under drought stress are more sensitive to allelochemicals.

. M a t e r i a l a n d M e t h o d s

Plant and soil material

Plants and soils used in this experiment were collected at the Oak Observatory (O 3HP) at the ''Observatoire de Haute Provence'' [START_REF] Hashoum | Biotic interactions in a Mediterranean oak forest: role of allelopathy along phenological development of woody species[END_REF], in a supramediterranean forest site. Acorns of Q. pubescens from different trees were collected in autumn 2014 and visually sorted to eliminate those damaged, infested or aborted and to select acorns of roughly similar size. The selected acorns were layered in a moist substrate consisting of sand and organic substrate (50:50) at 4°C until the radicle appeared. Litter from the four source species used (Q. pubescens, A. monspessulanum, C. coggygria and P. halepensis) was collected in autumn 2014 directly after the fall of the senescent leaves using tarpaulins placed under the trees, separated according to the different species in order to obtain pure litters from each species, and stored in the laboratory in the dark at ambient temperature. Green leaves of the four source species were harvested each month of the growing season (April to October) during all the experiment duration.

The soil used for the experiment was collected in January 2015 and used directly to prepare the substrate for plantations.

Experimental design

The experiment took place at the botanical garden of Mediterranean Institute of Biodiversity and Ecology, Aix-Marseille University, Marseille, South of France. The climate is typically Mediterranean with a mean annual temperature of 14.9°C and mean annual precipitations of 536 mm (1921 -2019), mainly distributed in spring and autumn with a dry summer season. Two hundred acorns were sowed in February 2015 in 6L pots (1 acorn per pot, 18.3 cm wide, 25.5 cm high) filled with a substrate composed of 50% natural soil, 25% peat moss substrate, and 25% perlite. The use of natural soil ensures that soil microorganisms naturally present in Q. pubescens forests of the O3HP site were in the pot.

In order to relate as closely as possible to natural conditions, we considered two of the main release modes of allelochemicals into the environment [START_REF] Rice | Allelopathy[END_REF]: litter decomposition and leachates, i.e. leaching of water-soluble compounds from leaves during rain events. Allelopathic treatments are thus the addition of litter of source species (litter decomposition mode) and aqueous leaf extracts of source species (leachates). The use of aqueous extracts is justified because it is widely recognized that water-soluble compounds are those most often involved in allelopathy [START_REF] Vyvyan | Allelochemicals as leads for new herbicides and agrochemicals[END_REF]. Allelopathic treatments were applied from February 2015 to April 2016 on 40 pots per source species + a control (5 treatments applied on 40 pots each). For each source species, the pot was supplied with 25g of litter in February 2015 and 15g in October 2015 in order to simulate the annual leaf fall, and watered with leaf extracts every month. Depending on the phenological stage of the plant on the O3HP, leaves used for extract preparation were green leaves (vegetation season), senescent leaves (autumn) or litter (winter). The two processes of chemical interactions through leachates and litter have thus been investigated jointly here to mimick natural conditions. The control was supplied with fiberglass cloth used to imitate the physical litter effect but without releasing allelochemicals [START_REF] Sydes | Effects of Tree Leaf Litter on Herbaceous Vegetation in Deciduous Woodland: II An Experimental Investigation[END_REF][START_REF] Gavinet | Do litter-mediated plantsoil feedbacks influence Mediterranean oak regeneration? A two-year pot experiment[END_REF] and watered with water. To determine the quantity of leaf litter supply, we measured the natural leaf litter amount of each species on the forest site in ten 400 cm 2 blocks, then a common average value of 25g per pot was used for all source species. A net was placed on each pot to prevent litter loss or addition. Aqueous extracts were prepared with fresh foliar material macerated in water for 24 hours at ambient temperature. To determine aqueous extracts concentrations on a leaf dry mass basis, a sample of fresh green leaves were dried and weighed to determine the leaf water content of each species.

During the first year (from February 2015 to April 2016), 500 ml of aqueous extracts at 2.5% (weight/volume) were added to each pot of the allelopathic treatment every month. The second year (from April 2016 to October 2016), half of the pots of each treatment were submitted to a water stress treatment by reducing water supply to maintain the pots at 20% of field capacity, while the other half were maintained at field capacity (not stressed).

Soil moisture was maintained through an automatic drip irrigation system. Water-stressed pots submitted to allelopathic treatments were supplied monthly with 125 ml of aqueous extracts at 10%, aiming at keeping the same amount of compounds but less diluted than the allelopathic pots under no-stress treatment. Our design thus mimick a first year with no drought stress followed by a second year with drought stress, keeping the allelopathic treatment constant (Figure 1). Treatments of chemical interactions are referred hereafter by the Genus name of the source species, i.e. Acer, Cotinus, Quercus and Pinus.

Fig1: Schematic drawing of the experimental design used to test biotic interactions between companion species and Q. pubescens under drought. The biotic interactions applied through the decomposition of litter and the supply of aqueous extract are designated by the initial of the genus name (Q: Q. pubescens; C: C. coggygria; A:

A. monspessulanum; P: P. halepensis; GF: glass fiber; FC: field capacity; N15/01, N16/04: number of replicates followed by the corresponding date). In April 2016, each treatment was then divided in two subsamples, one submitted to water stress and the other kept at field capacity.

Plant growth response

Seedling size (height and diameter at ground level) was measured at the end of the first growing season, in November 2015, and at the end of the second growing season, in October 2016. At the end of the second growing season, seedlings were harvested, and root systems were gently washed above a sieve to remove soil particles without losing roots. Seedlings were separated between leaves, stems, coarse and fine roots (diameter < 2mm), oven-dried at 60°C for 4 days and weighed. The total number of leaves was also recorded for each seedling. Cuvette (diameter 18 mm, 2.5 cm 2 surface) relied to an Infrared Analyzer (Synersy, CIRAS 3 PPSystem). Gasexchange measurements allowed direct calculations of net photosynthesis (A, μmol CO2 m -2 s -1 ), stomatal conductance to water vapor (gs, mmol.m -2 .s -1 ) and transpiration (E, μmol H2O m -2 s -1 ). Measurements were performed under 30°C temperature, constant CO2 concentration (400 ppm) and saturating PAR (1000 µmol.m -2 .s -

1

). Five measurements were recorded (one measurement every ten seconds), and the average of the five recordings was used for statistical analysis.

In this experiment, we also evaluated the percentage of ectomycorrhizal colonization. This type of symbiotic association is largely represented in Mediterranean forest species where fungi develop mainly around the root, forming a mycelian mantle from which hyphae develop [START_REF] Duponnois | An integrated view of allelochemicals amid multiple stresses[END_REF]. More, they can modify the sensitivity of plants to allelopathic substances. [START_REF] Gallet | Interactions allelopathiques en milieu forestier[END_REF]. We took root samples from each modality directly after the seedlings were collected, at the end of the experiment. Then these samples were stored in 60% alcohol until the time of analysis. We randomly selected 5 samples per modality for analysis, for a total of 50 samples. To quantify the percentage of ectomycorrhizal colonization, we used the line intersection method [START_REF] Giovannetti | An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots[END_REF]. This semi-quantitative method is based on the control of mycorrhization at all points of intersection between the roots present and a grid (1x1cm) placed under a Petri dish. 5 fragments of 3 cm of roots are therefore analyzed in a Petri dish with a few milliliters of distilled water to prevent drying the roots.

The counting is done under a binocular magnifying glass at a 4x40 magnification with high illumination to ensure good visualization. The mycorrhization rate is obtained as the ration of the number of mycorrhized root intersection points to the total number of root intersection points.

The litter from each pot was collected at the end of the experiment, freeze-dried and weighed to calculate the decomposition rate as follows: (total mass supplied -final mass) / total mass supplied.

Statistical analyses

Allelopathic effects on oak seedling emergence were tested using a generalized linear model with a binomial distribution. Allelopathic effects on early oak growth (length and diameter after 10 months of allelopathic treatments, before drought treatment application) were tested through 1-way ANOVA followed by Tukey posthoc tests. Seedling length and diameter relative growth rate during the second year was computed as RGR = (log(X 2) -log(X1)) / (t2 -t1), with X2 is seedling dimension (height or diameter) at the last measurement date t2 (i.e. November 2015), and X1 the same dimension at the first measurement date t1 (i.e. in October 2016). Given that growth during the winter season is negligible, this value essentially represents the growth rate during the second season, when water stress was applied on half of the seedlings. The effects of allelopathy, drought treatment and their interactions on oak RGR, physiology, biomass, biomass allocation and mycorrhization rates were tested through two-ways ANOVAs. Litter mass loss was calculated as the difference of final litter mass and all litter inputs and expressed in percentage. Conditions of normality and homoscedasticity were checked using the Kolmogorov-Smirnov and Levene tests, respectively, and the data was log-transformed if necessary (seedling length, diameter and RGR).

. R e s u l t s

Allelopathic effects on oak seedlings emergence and early growth

Oak seedling emergence was only marginally influenced by allelopathic treatment (χ2 = 7.3, P= 0.06), with a tendency of higher emergence in control conditions than in the Quercus treatment (Figure 2). Allelopathic treatment influenced seedling length (F=18.9, P < 0.001) and diameter (F=3.6, P = 0.009). Seedlings had a smaller diameter in the Acer, Cotinus and Pinus treatments and a higher length in the Quercus treatment (Figure 2).

Allelopathy and drought effects on Q. pubescens growth and biomass allocation

Seedling RGR during the second growing season was affected by drought for both height and diameter growth (P < 0.001) but not by allelopathy nor by the interaction between these factors (Figure 3), although allelopathy marginally influenced diameter RGR (P = 0.07; Figure 3) with a tendency of better growth under Pinus treatment. Drought and allelopathy affected all biomass and biomass allocation parameters, except fine roots biomass (Figure 4). No significant interaction was detected. Over the total duration of the experiment, the drought treatment caused an average decrease of 27% of total biomass, 39% of aerial biomass, and 20% of root biomass compared to seedlings under non-stressed conditions. Drought also caused a 48% decrease in the number of leaves, which is the most significant morphological effect of this treatment (data not shown). Litter and leachates of Cotinus led to a decrease in the number of leaves, coarse and fine root biomass, total seedling biomass and root/shoot ratio compared to the control. Root biomass and root/shoot ratio were also reduced by the Pinus treatment (Figure 4).

Allelopathy and drought effects on Q. pubescens physiology and mycorrhization rate

Drought reduced stomatal conductance (gs) and photosynthesis (A) by 33.5 and 28.5% respectively compared to the unstressed condition (Figure 5). Allelopathy marginally influenced stomatal conductance (P = 0.05) and influenced seedling photosynthetic rate (P= 0.02), with in both cases seedlings under Quercus treatment having higher values than the control (Figure 5). Mycorrhization rates of Q. pubescens seedlings were generally high, ranging from about 70% to 75%, with no significant differences of mycorrhization rates between drought treatments (P = 0.92), allelopathy treatments (P=0.96) or their interaction (P=0.84) (data not shown).

Allelopathy and drought effects on litter decomposition

Drought and allelopathy had an interactive effect on litter decomposition (FAxD=3.5, P = 0.01; Figure 6). Under well-watered conditions there was a difference in species decomposition rates according to the gradient Quercus < Acer <= Pinus < Cotinus, with the Cotinus litter decomposing particularly fast with a loss of 61% of its mass at the end of the experiment under control conditions. The drought treatment reduced only the decomposition of Cotinus and Pinus litter, leading to a decrease of the difference between species in the drought treatment (Figure 6).

. D i s c u s s i o n

Allelopathy from companion species but not autotoxicity affects Q. pubescens seedlings development

The present results confirm the allelopathic potential of C. coggygria and P. halepensis, as they decreased seedling biomass and in particular root biomass of Q. pubescens. Several studies have found that the root part of a target plant is more sensitive to allelochemicals than the aerial part [START_REF] Ben-Hammouda | Allelopathic effects of barley extracts on germination and seedlings growth of bread and durum wheats[END_REF][START_REF] Turk | Allelopathic effect of black mustard (Brassica nigra L) on germination and growth of wild oat (Avena fatua L)[END_REF][START_REF] Gatti | Allelopathic effects of aqueous extracts of Artistolochia esperanzae O Kuntze on development of Sesamum indicum L seedlings[END_REF]Zhang et al. 2010). This sensitivity could be related to the root uptake role implying a direct contact between roots and allelochemicals in the soil [START_REF] Turk | Allelopathic effect of black mustard (Brassica nigra L) on germination and growth of wild oat (Avena fatua L)[END_REF]. Inhibition of root growth by allelochemicals may be due to changes in DNA synthesis in cells of root apical meristem, alteration of mitochondrial metabolism [START_REF] Abrahim | Effects of four monoterpenes on germination, primary root growth, and mitochondrial respiration of maize[END_REF] or changes in cellular mitotic indices [START_REF] Dayan | Phytotoxicity of quassinoids: physiological responses and structural requirements[END_REF][START_REF] Romagni | Allelopathic effects of volatile cineoles on two weedy plant species[END_REF]. As in this study, an inhibition of root development by C. coggygria leaf aqueous extracts has also been demonstrated on Q. pubescens 2-month-old seedling [START_REF] Gavinet | Phenolics of the understory shrub Cotinus coggygria influence Mediterranean oak forests diversity and dynamics[END_REF]). However, [START_REF] Gavinet | Do litter-mediated plantsoil feedbacks influence Mediterranean oak regeneration? A two-year pot experiment[END_REF] found no effect of C. coggygria and P. halepensis litter on Q. pubescens seedlings development, which suggest that aqueous extracts may have a greater allelopathic effect than litter. The chemical composition and the quantity of allelochemicals in the leaves are highly variable between green leaves and litter leaves [START_REF] Fernandez | Variations in allelochemical composition of leachates of different organs and maturity stages of Pinus halepensis[END_REF][START_REF] Hashoum | Biotic interactions in a Mediterranean oak forest: role of allelopathy along phenological development of woody species[END_REF][START_REF] Silva | Phytotoxicity of volatiles from fresh and dry leaves of two Asteraceae shrubs: Evaluation of seasonal effects South[END_REF]. [START_REF] Nektarios | Allelopathic Effects of Pinus halepensis Needles on Turfgrasses and Biosensor Plants[END_REF] also found that green needle extracts of P.

halepensis had a more pronounced negative effects on herbaceous species growth than litter extracts. The supply of aqueous extracts and litter could also have cumulative effects as the concentration of available allelochemicals in soil water is a direct determinant of the phytotoxic activity of allelochemicals in soil [START_REF] Kobayashi | Factors affecting phytotoxic activity of allelochemicals in soil[END_REF]). Litter decomposition allow to release nutrients and allelochemicals according to initial leaf litter quality [START_REF] Chomel | Secondary metabolites of Pinus halepensis alter decomposer organisms and litter decomposition during afforestation of abandoned agricultural zones[END_REF], 2016, Santona et al. 2015 a, b), and Cotinus and Pinus litter have a high concentration of phenolics and terpenoids, respectively (Santonja et al. 2015a;[START_REF] Gavinet | Do litter-mediated plantsoil feedbacks influence Mediterranean oak regeneration? A two-year pot experiment[END_REF].

We found no evidence of autotoxicity for Q. pubescens. The treatment with litter and aqueous extracts from Q.

pubescens tended to inhibit seedling emergence but increased seedling length. These effects may be due to mechanical litter effects [START_REF] Facelli | Plant litter: its dynamics and effects on plant community structure[END_REF][START_REF] Kostel-Hughes | Effects of leaf litter depth on the emergence and seedling growth of deciduous forest tree species in relation toseed size[END_REF]. Q. pubescens litter had the largest and thickest leaves, which may constitute a greater physical barrier in line with its tendency to decrease seedling emergence compared to the artificial litter treatment. [START_REF] Xiong | Effects of litter accumulation on riparian vegetation: importance of particle size[END_REF] also showed that the negative effects of litter increased with the size of the litter leaves. Similarly, after cutting oak litter leaves, [START_REF] Li | Factors affecting establishment of Quercus liaotungensis Koidz under mature mixed oak forest overstory and in shrubland[END_REF] observed an increase in seedling emergence. The increase in seedling length is consistent with our hypothesis of a physical effect of Quercus litter through light interception [START_REF] Facelli | Plant litter: its dynamics and effects on plant community structure[END_REF], which can accelerate the elongation of the stem. Interestingly, Quercus treatment improved seedling photosynthesis and transpiration. As this species litter present the lowest decomposition rate (Figure 5) and is known to release few nutrients (Santonja et al. 2015b), the positive effects of its own litter may come from improved seedling water status thanks to a lower soil moisture evaporation, as also suggested by [START_REF] Gavinet | Do litter-mediated plantsoil feedbacks influence Mediterranean oak regeneration? A two-year pot experiment[END_REF].

Drought affect Q. pubescens seedlings physiology and growth but do not interact with allelopathy

Water stress induced a progressive decrease in stomatal conductance and photosynthesis, a classic physiological response to drought already documented for different oak species [START_REF] Chaves | How Plants Cope with Water Stress in the Field?[END_REF][START_REF] Vaz | Droughtinduced photosynthetic inhibition and autumn recovery in two Mediterranean oak species (Quercus ilex and Quercus suber)[END_REF][START_REF] Arend | Seasonal photosynthetic responses of European oaks to drought and elevated daytime temperature[END_REF]. The reduced photosynthesis combined with a lower leaf biomass indicates a lower carbon gain for the plant, which participate to explain the lower seedling biomass under drought stress. Seedling biomass reduction by drought was particularly marked for leaves but not for roots, indicating a change in biomass allocation and an increased root/shoot ratio as expected [START_REF] Poorter | Biomass allocation to leaves, stems and roots: meta-analyses of interspecific variation and environmental control[END_REF]. However, contrary to our hypothesis, drought effects were not more important for seedlings submitted to allelopathic treatments. This is particularly surprising as regards the Cotinus and Pinus treatments which strongly reduced root development. These allelopathic treatments, however, caused no change in leaf carbon and water exchange rates, contrary to previous results showing that allelopathy can reduce plant water uptake, stomatal conductance or photosynthesis [START_REF] Barkosky | Caffeic acid-induced changes in plant-water relationships and photosynthesis in leafy spurge Euphorbia esula[END_REF]Zhou and Yu 2006). In our study, seedling length and diameter relative growth during the second year did not differ between allelopathic treatments, whatever the drought stress. This suggest that i) allelopathic effects were restricted to the first phase of our experiment, ii) root biomass reduction that occurred in the first year was not strong enough to induce a reduction of water absorption by roots during subsequent drought. An element of explanation of this latter effect could be that Cotinus and Pinus treatments did not affect rooting depth, which is the main factor controlling seedling water absorption [START_REF] Padilla | Rooting depth and soil moisture control Mediterranean woody seedling survival during drought[END_REF]. Finally, the absence of treatment effect on mycorrhization rate may hide differences in mycorrhization type : for instance, mycorrhizae with longer extrametrical mycelia may help seedlings to sustain their carbon and water exchange and growth rates (García de Jalón et al. 2020) despite lower root biomass.

Our results do not support the hypothesis of [START_REF] Pedrol | Allelopathy and abiotic stress In : Allelopathy: A Physiological Process with Ecological Implications Netherlands[END_REF] that sensitivity to allelochemicals is increased under drought stress. At the contrary, allelopathic effects tended to be less pronounced (although not significantly) under drought stress. A reduced allelopathic effect under drought stress is possible if the lower water availability prevents the transport of soluble allelochemicals to the roots. The dry treatment also reduced litter decomposition rate for Cotinus and Pinus, possibly limiting the release of litters' allelochemicals through volatilization or solubilization. However, it must be highlighted that drought was applied during the second year of oak seedling development only, a period during which allelopathic effects were reduced as indicated by growth and physiological measurements. Similarly, [START_REF] Gavinet | Phenolics of the understory shrub Cotinus coggygria influence Mediterranean oak forests diversity and dynamics[END_REF] showed that Cotinus inhibited root development of young seedling but not that of older saplings. Differences of root biomass measured at the end of the experiment may thus reflect only legacy effects of the first year allelopathic effects. Drought applied during the first year, or on more sensitive target species, may lead to different results. For instance, [START_REF] Hashoum | Mediterranean woody plant specialized metabolites affect germination of Linum perenne at its dry and upper thermal limits[END_REF] showed that drought and allelopathy from Cotinus interact to decrease the germination of the herbaceous Linum perenne L. In addition, under natural conditions drought can be more pronounced that the drought treatment applied here and reach lethal levels for seedlings, particularly under the expected climate change [START_REF] Giorgi | Climate change projections for the Mediterranean region[END_REF]. In that case, an inhibition of oak seedling root development by neighboring allelopathic species such as Cotinus and Pinus could reduce survival probability in case of more pronounced water stress. Finally, we have not investigated whether drought could induce changes in the production of allelochemicals by the source species, which must be considered to assess if allelopathic interactions will change under increased drought stress. However, other studies contradict this view, evidencing at the contrary a lower total phenolics production under water stress (e.g. [START_REF] Karageorgou | The influence of drought, shade and availability of mineral nutrients on exudate phenolics of Dittrichia viscosa[END_REF], meaning that this issue is far from being resolved.

Consequences for oak regeneration in different forest microsites

In a previous study, [START_REF] Hashoum | Biotic interactions in a Mediterranean oak forest: role of allelopathy along phenological development of woody species[END_REF] showed that the dominant tree Q. pubescens and its companion species A. monspessulanum and C. coggygria may limit the growth of understory herbaceous plants in downy oak forests through the production and release of allelochemicals. Here, we evidenced no negative effects of Q. pubescens and A. monspessulanum on Q. pubescens seedlings, suggesting that herb inhibition by allelochemicals could be an advantage for oak regeneration since herbs are usually strong competitors for seedling establishment [START_REF] Gordon | Competition for soil water between annual plants and blue oak (Quercus douglasii) seedlings[END_REF][START_REF] Coll | Competition for water between beech seedlings and surrounding vegetation in different light and vegetation composition conditions[END_REF][START_REF] Benayas | Effects of artificial shading and weed mowing in reforestation of Mediterranean abandoned cropland with contrasting Quercus species[END_REF]Gavinet et al. 2016b). In contrast, C. coggygria affects both herb and oak seedlings, pointing to a possible negative effect of this species on understory diversity and oak regeneration [START_REF] Gavinet | Phenolics of the understory shrub Cotinus coggygria influence Mediterranean oak forests diversity and dynamics[END_REF]. Finally, P. halepensis had negative effect on oak regeneration, but strong autotoxicity have also been evidenced. Oak regeneration is often successful in pine habitats [START_REF] Lookingbill | Spatial pattern of Quercus ilex and Quercus pubescens recruitment in Pinus halepensis dominated woodlands[END_REF], especially under moderate pine cover [START_REF] Gavinet | Hardwood seedling establishment below Aleppo pine depends on thinning intensity in two Mediterranean sites[END_REF], indicating that allelopathy is not a strong limitation fir oak regeneration under pine.

In conclusion, our results suggest a better potential for Quercus pubescens regeneration under its own stands, due to the positive effects of its litter and leachates, but in the absence of Cotinus coggygria in the understory.

Allelopathy does not appear to be stronger under drought stress, but further investigations are needed in particular to elucidate the consequences of the opposite effects of allelopathy and drought on the root/shoot ratio. Finally, the allelopathic treatments tested here were rather limited, particularly during their interaction with drought in the second year. Interactions with drought may be more evident in systems where allelopathy is a stronger driver of plant performance, particularly in the case of strategies adopted by Exotic Invasive Plants Species (e.

Fig2:

  Fig2: Allelopathic treatment effects on Q. pubescens acorn emergence and early seedling growth after the first vegetation season. Different letters represent significant differences (P < 0.05, Tukey post-hoc tests). Data are model predictions of emergence probability and means of growth variables ± standard errors (n=40 acorns per treatment for emergence, n= 27 -32 acorns per treatment for growth).

Fig 3 :

 3 Fig 3: Allelopathy and drought effects on seedling relative growth rate (RGR) in length and diameter during the second growing season. Data are means ± standard errors (n=11-17 seedlings per treatment). Results of two-ways ANOVA testing the effect of allelopathy (FA, DF = 4), drought (FD, DF = 1) and their interaction (FA×FD, DF = 4) are indicated (with the symbols + for P < 0.1, * for P <0.05, ** for P <0.01 and *** for P <0.001).

Fig 4 :

 4 Fig 4: Drought and allelopathy effects on seedling biomass and root/shoot ratio. Allelopathy has been applied during 2 growing seasons and drought during the last growing season. Data are means ± standard errors (n=11-17 seedlings per treatment). Results of two-ways ANOVA testing the effect of allelopathy (FA , DF = 4), drought (FD, DF = 1) and their interaction (FA×FD, DF = 4) are indicated (with the symbols * for P <0.05, ** for P <0.01 and *** for P <0.001).

Fig 5 :

 5 Fig 5: Allelopathy and drought effects on seedling leaf photosynthetic rate (A) and stomatal conductance (gs) during the summer of the second growing season. Data are means ± standard errors (n=10 seedlings per treatment). Results of two-ways ANOVA testing the effect of allelopathy (FA, DF = 4), drought (FD, DF = 1) and their interaction (FA×FD, DF = 4) are indicated (with the symbols + for P < 0.1, * for P <0.05, ** for P <0.01 and *** for P <0.001).

Fig 6 :

 6 Fig 6: Litter mass loss in the different allelopathic and drought treatments. Data are means ± standard errors (n=11-17 pots of surviving seedlings per treatment). Results of two-ways ANOVA testing the effect of allelopathic source species (FA, DF = 3), drought (FD, DF = 1) and their interaction (FA×FD, DF = 3) are indicated (with the symbols * for P <0.05, ** for P <0.01 and *** for P <0.001).

  and Fu S (2010) Allelopathic effects of leaf litter and live roots exudates of Eucalyptus species on crops Allelopathy J 26, 91-100 Zhou YH and Yu JQ (2006) Allelochemicals and photosynthesis In Reigosa, Pedrol and González (eds) Allelopathy: a physiological process with ecological implications Springer, Netherlands, 127-139

  Plant physiological response, mycorrhization rate and litter decomposition CO2 and H2O exchanges were measured four months after the application of water stress, in August 2016, on a subsample of 10 seedlings per treatment and on one leaf per seedling. Each leaf was clipped in a PLC3 Universal

	2.4
	5 Allelopathy treatments		10 treatments: 5 Allelopathy x 2 Water Stress
	N15/01 N16/04 Litter Leachate
	40	23	Q	Q
	40	31	C	C
	40	27	A	A
	40 40	29 32	P GF	P Water	No stress 100% FC
						Stress
	January 2015		February 2015	April 2016	20% FC
	Sowing with litter	Emergence	Budburst
	N=40			N=23-32/40	N= 23-32/40
				Allelopathy treatment: from February 2015 to October 2106
					Water stress treatment: from April 2016 to October 2016

We thank Laura Magaud for help with nursery experiment. This study was funded by the French National Research Agency (ANR) through the SecPriMe2 project (ANR-12-BSV7-0016-01). The O3HP site is supported by the French National Research Infrastructure ANAEE-F

Funding: This study was funded by the French National Research Agency (ANR) through the SecPriMe2 project (ANR-12-BSV7-0016-01). The Oak Observatory at the ''Observatoire de Haute Provence'' (O3HP) site is supported by the French National Research Infrastructure ANAEE-F Availability of data and material: the data will be available upon request to the corresponding author


El-Hadi Zerdazi
email: el-hadi.zerdazi@inserm.fr

Florence Vorspan

Andries T Marees

François Naccache

Jean-Pierre Lepine

Jean-Louis Laplanche

Nathalie Prince

Cynthia Marie-Claire

Frank Bellivier

Stéphane Mouly

Vanessa Bloch

QT length during methadone maintenance treatment: gene x dose interaction

Keywords: Methadone, rs11911509, KCNE1, QT prolongation Electrocardiograph F ST : Fixation index hERG: human Ether-A-Go-Go Related Gene PCA: Principal components analysis QTc: corrected QT interval SNP: Single Nucleotide Polymorphism TdP: Torsade de Pointe

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

INTRODUCTION

Methadone is largely prescribed as maintenance treatment in patients suffering from opiate dependence around the world because it is considered as the most effective treatment in this indication [START_REF] Amato | An overview of systematic reviews of the effectiveness of opiate maintenance therapies: available evidence to inform clinical practice and research[END_REF]. Patients under methadone maintenance treatment have a lower heroin overdose mortality, lower probability of relapse, HIV infection and criminal activities, and higher functioning improvement compared to those that did not received treatment [START_REF] Kleber | Methadone Maintenance 4 Decades Later[END_REF][START_REF] Alford | Acute Pain Management for Patients Receiving Maintenance Methadone or Buprenorphine Therapy[END_REF][START_REF] O'connor | Pharmacologic treatment of heroin-dependent patients[END_REF][START_REF] Addiction | Effective Medical Treatment of Opiate Addiction[END_REF][START_REF] Metzger | Human immunodeficiency virus seroconversion among intravenous drug users in-and out-of-treatment: an 18-month prospective follow-up[END_REF][START_REF] Dole | Long-Term Outcome of Patients Treated with Methadone Maintenance*[END_REF]. Moreover, Methadone appears to be more effective than other opiate maintenance treatments in retaining patients in cares [START_REF] Mattick | Buprenorphine maintenance versus placebo or methadone maintenance for opioid dependence[END_REF].

Nevertheless, methadone may be responsible for severe cardiac side effects, especially QT interval prolongation in the ECG which can occur in up to 37 % of cases [START_REF] Alinejad | A systematic review of the cardiotoxicity of methadone[END_REF][START_REF] Chou | Methadone Overdose and Cardiac Arrhythmia Potential: Findings From a Review of the Evidence for an American Pain Society and College on Problems of Drug Dependence Clinical Practice Guideline[END_REF] and exposes to the occurrence of torsade de pointes (TdP) [START_REF] Romero | Drug-induced torsades de pointes in an underserved urban population. Methadone: is there therapeutic equipoise?[END_REF][START_REF] Alinejad | A systematic review of the cardiotoxicity of methadone[END_REF][START_REF] Krantz | QTc interval screening in methadone treatment[END_REF]. According to several studies, mortality associated with this adverse effect varies from 8 % to 11 % among Methadone users [START_REF] Kao | Trends in Reporting Methadone-Associated Cardiac Arrhythmia, 1997-2011[END_REF][START_REF] Pearson | QT prolongation and torsades de pointes among methadone users: reports to the FDA spontaneous reporting system[END_REF]. This QT prolongation can occur over a wide range of dosages including those recommended for maintenance treatment [START_REF] Pearson | QT prolongation and torsades de pointes among methadone users: reports to the FDA spontaneous reporting system[END_REF], although controversial, a dose-dependence relationship has been reported (Mayet, ). It has also been reported that QT prolongation and TdP risk are more commonly reported in patients taking methadone for opioid dependence than for pain management [START_REF] Kao | Trends in Reporting Methadone-Associated Cardiac Arrhythmia, 1997-2011[END_REF].

One of the mechanisms involved in methadone-associated QT prolongation is the blockade in the human heart of the hERG (human Ether-A-Go-Go Related Gene, KCNH2) potassium channel that mediates the rapid repolarizing I Kr current [START_REF] Katchman | Influence of Opioid Agonists on Cardiac HumanEther-a-go-go-related Gene K+ Currents[END_REF]. Although various opioids agonist are known to block the hERG channel, methadone appears to be among the most potent inhibitor of the hERG repolarizing current at therapeutic range [START_REF] Katchman | Influence of Opioid Agonists on Cardiac HumanEther-a-go-go-related Gene K+ Currents[END_REF]. This may explains why these adverse effects (QT prolongation and TdP) mainly affect patients under methadone compared to morphine, heroin or buprenorphine treatments [START_REF] Kao | Trends in Reporting Methadone-Associated Cardiac Arrhythmia, 1997-2011[END_REF][START_REF] Anchersen | Prevalence and clinical relevance of corrected QT interval prolongation during methadone and buprenorphine treatment: a mortality assessment study[END_REF].

Medication-induced QT interval prolongation implies the interaction of several non-genetic and genetics risk factors [START_REF] Newton-Cheh | Genetic determinants of QT interval variation and sudden cardiac death[END_REF][START_REF] Paulussen | Risk factors for drug-induced long-QT syndrome[END_REF][START_REF] Roden | Drug-Induced Prolongation of the QT Interval[END_REF]. Among identified genetic risk factors, DNA variants on KCNE1 [START_REF] Kääb | A Large Candidate Gene Survey Identifies the KCNE1 D85N Polymorphism as a Possible Modulator of Drug-Induced Torsades de Pointes[END_REF], KCNQ1 [START_REF] Yang | Allelic Variants in Long-QT Disease Genes in Patients With Drug-Associated Torsades de Pointes[END_REF], KCNH2 [START_REF] Yang | Allelic Variants in Long-QT Disease Genes in Patients With Drug-Associated Torsades de Pointes[END_REF], NOS1AP [START_REF] Aberg | Genome-wide association study of antipsychotic-induced QTc interval prolongation[END_REF] and SCN5A [START_REF] Yang | Allelic Variants in Long-QT Disease Genes in Patients With Drug-Associated Torsades de Pointes[END_REF] have been shown to predispose to QT prolongation in case of drug exposure. Moreover, we previously observed a significant association of a KCNH2 single nucleotide variant with increased QT length in methadone-treated patients [START_REF] Hajj | KCNH2 polymorphism and methadone dosage interact to enhance QT duration[END_REF].

The aim of the current study was to test the association of additional candidate genes with corrected QT (QTc) duration among an extended population of methadone users, and to focus on a possible gene x methadone dosage interaction in this association.

METHODS

Subjects

Two hundred and sixteen subjects aged over 18 years, with lifetime opiates dependence in total remission for more than 3 months were recruited in outpatient treatment program. The main objective of this study was to describe gene x environment interactions regarding the dose of methadone prescribed, but also to identify gene x environment interactions regarding QTc enlargement was only a secondary objective.

Steady state was defined as being on methadone maintenance program with a stable dose, and regular follow up for at least three months. Furthermore, subjects with persistent use of intravenous route of administration for any kind of substances were excluded, as well as those with recent history of drug overdose (during the last three months).

Trained interviewers asked patients about their lifetime use of opiates, previous maintenance treatments (methadone and buprenorphine) and other substances use. The Mini International Neuropsychiatric Interview (MINI) [START_REF] Sheehan | The Mini-International Neuropsychiatric Interview (M.I.N.I.): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10[END_REF][START_REF] Lecrubier | The Mini International Neuropsychiatric Interview (MINI). A short diagnostic structured interview: reliability and validity according to the CIDI[END_REF] was used to assess current and lifetime dependence or abuse criteria for opiates and other substances of abuse according to the DSM IV TR classification [START_REF]DSM-IV-TR. 4e édition, texte révisé[END_REF] Concomitantly prescribed medications were also recorded and any described QT prolongation was checked in each patient according to the CredibleMeds.org database (accessed on 06.03th.2016). In this database, medications were gradually classified as with conditional, possible, or confirmed risk of TdP (due to QT lengthening). Every concomitant medication was flanked with a score from 1 to 3 proportional to its risk category, and treatments that were not listed in the database have a 0 score. Finally, we computed for each patient a "Drugs lengthening QT score" by summing the scores of his medications.

No patient reported previous history of syncope, TdP or Long QT syndrome upon enrolment. All subjects signed a free and informed consent and received complete description of the study. The study was approved by the Ethic Committee of Paris, Ile-de-France (CPP VI, May 19 th 2008) and was registered to the clinicaltrials.gov website (NCT00894452).

Electrocardiographs (ECGs)

ECGs were recorded at rest using a 12 leads digital apparatus in the morning before the daily methadone intake in order to be in steady state trough condition. ECGs were blindly analysed by two trained cardiologists. According to their ECGs, we did not include patients with: poor ECG quality, conditions that may lengthen QT interval such as bradycardia or bundle branch block, or for which QT interval calculation was impossible. The QTc interval was then calculated using Bazett's formula and was considered as borderline or prolonged if it exceeded 430 ms for men and 450 ms for women. If ECG abnormalities were detected, a senior cardiologist then followed those patients.

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Single Nucleotide Polymorphisms (SNPs) genotyping

Genomic DNA was extracted from EDTA-treated peripheral blood samples and genotyped on the Illumina Infinium PsychArray24 v1.1 BeadChip, containing 571 054 SNPs.

SNPs selection and genetic quality control:

We selected KCNE1, KCNQ1, KCNH2, NOS1AP and SCN5A genes according to previously published studies that found association of variants on these genes with drug-induced QT interval prolongation [START_REF] Aberg | Genome-wide association study of antipsychotic-induced QTc interval prolongation[END_REF][START_REF] Kääb | A Large Candidate Gene Survey Identifies the KCNE1 D85N Polymorphism as a Possible Modulator of Drug-Induced Torsades de Pointes[END_REF][START_REF] Yang | Allelic Variants in Long-QT Disease Genes in Patients With Drug-Associated Torsades de Pointes[END_REF]. We thus retrieved SNPs that overlap with their genomic coordinates according to the GH37p13 version of the Human genome (version used in the PsychArray). We finally retained only those that passed quality control selection with the following parameters: Minor Allele Frequency ≥ 0.1, Genotyping rate ≥ 0.05, and no significant deviation from Hardy Weinberg Equilibrium (p-value < 0.001).

The per-individual quality control consisted of three steps. First, we removed subjects for which we suspect inbreeding or DNA sample contamination according to their identity by descent calculation and retained only subjects with PI HAT between 0.2 and 0.8. Second, we searched for sex discordances between genetic and reported sex, and third we excluded outliers with excessive missing genotyping (> 0.03) and heterozygosity (> ±2.5 SD) rates [START_REF] Anderson | Data quality control in genetic case-control association studies[END_REF].

Statistical analysis

Our study is an ancillary objective of a larger project, the "Methadose" project, which the main objective is to predict the steady dose for methadone maintenance treatment according to genetic and clinical factors. Thus, we did not perform an a priori power calculation for our main objective, which is testing the association between candidate SNPs and QTc length.

Associations between QTc interval and dichotomic clinical variables were explored with the Wilcoxon-Mann-Whitney U test. For continuous variables, we used the Pearson or the Spearman correlation coefficients, as appropriate.

Genetic associations were tested with linear regression model taking the QTc length as the dependent variable. The same regression was used to test for interaction between the top SNP and methadone dosage, by including also clinical variables associated with QTc length with a 10% entry pvalue. We chose Cohen's f 2 [START_REF] Cohen | Statistical power analysis for the behavioral sciences[END_REF] method for the calculation of the genetic association effect size. This method allows the evaluation of a selected variable effect size within a multivariate regression model.

As a supplementary quality check, the association tests with both clinical variables and the regressions were performed again without two subjects with extreme methadone dosage (above 200 mg per day) to ensure that the association was not only driven by those two outliers.

Gene-based analysis was performed according to Plink reported method [START_REF] Purcell | PLINK: a tool set for whole-genome association and population-based linkage analyses[END_REF] with the following parameters (r 2 =0.5, p=0.2 and max SNP number = 5). Empirical p-value was obtained after 10 000 permutations.

All tests were performed with a two-tailed hypothesis and an alpha risk of 5%. Corrections for multiple testing were applied using the Bonferroni method. Analyses were achieved with Plink v1.90b4. 10 [35,36] and R version 3.2 [START_REF] Core | R: A Language and Environment for Statistical Computing[END_REF].

Population stratification managing

Ethnic diversity in our sample was taken into account by three means. First, Principal components analysis (PCA) was used to correct for population stratification as a covariate in the linear regression model. Principal components were calculated using the whole genotype data available in the PsychArray chip after pruning markers that are in linkage disequilibrium (r 2 > 0.2), that are located in sex chromosomes, and those that mapped to the hot spot region of the major histocompatibility complex.

Second, QTc length as well as association with SNPs and genes were compared between ethnic subgroups observed in our population. To define those groups, the 154 subjects retained for the analysis were merged with 1000 Genomes populations to calculate principal components of the whole sample (1000 Genomes & our cohort). The ethnicity of our subjects was then visually determined (Figure S1). One hundred and forty three subjects (92.9 %) mapped to European population of 1000 Genomes samples, while the rest of the sample mapped to Africans population.

Third, we also calculated the fixation index (F ST ) for all variants on the PsychArray chip that passed quality control filters according to Weir and Cockerham method [START_REF] Weir | Estimating F-Statistics for the Analysis of Population Structure[END_REF]. The F ST was then used to assess the impact of population divergence on the variants especially those retained for the analysis. This analysis was conducted with Plink software.

RESULTS

After selection for available variables of interest, genotyping quality control, 154 individuals were retained for subsequent analysis (Figure 1).

Population characteristics

In our cohort, the average QTc length was 415.2 ± 2.5 ms (range : 350 to 575 ms). Thirty-eight (24.7 %) patients have prolonged or borderline QTc interval (Table I). Patients were predominately men (76.6 % ; sex ratio 3.3), and the QTc interval was significantly longer in women than in men (422.4 ms vs 412.9 ms respectively; W = 1600 ; p = 0.03). Subjects were aged between 20 and 64 years with a mean of 40.9 years (± 0.7) and a median of 42 years. Age was not significantly correlated with QTc length (r Pearson = 0.04 ; p = 0.62)

The methadone dosage varied from 7 to 320 mg per day (mean : 63.8 mg per day ± 3.3 mg per day, median : 60 mg per day). QTc length and methadone dosage were significantly correlated (r Pearson = 0.26 ; p = 1 x 10 -3 ), and we calculated that each additional 10 mg per day of methadone increases the QTc length by 1.8 ms (IC 95% [0.7 ms -3 ms]).

We observed that two subjects (1.3 %) were stabilized with a daily dose of more than 200 mg/day of methadone, and even after removing them from the analysis the correlation between QTc and methadone dosage remained significant (r Pearson = 0.18 ; p = 0.02).

Fifty-three patients (34.4 %) had a prescription of at least one other medication with a known TdP risk. Those patients had nominally longer QTc length but without statistically significant difference (with vs. without, 419.3 ms vs 413 ms; W = 2409; p = 0.31).

We did not find significant difference in QTc length mean between Caucasian and African subjects (415.4 ms vs 411.7 ms; W = 813; p = 0.86). Additional population characteristics and variables associations with QTc length are shown in (Table I).

Genetic associations

Of the 579 selected SNPs, 126 passed quality control (Figure S2). Fourteen SNPs were nominally significantly associated with QTc length (p < 0.05), but only one (rs11911509 on KCNE1) remained significant after Bonferroni multiple testing correction (p = 3.84 x 10 -4 ; p corrected = 0.049, Cohen f 2 effect size = 0.131) (Table II). According to our adjusted model after further correction for ethnicity, the QTc length increases by 13.2 ms (± 3.6 ms) for each additional minor allele (A vs C) of the rs11911509 SNP.

<Table II here>

The distribution of this SNP, rs11911509, was not different in the two ethnic groups observed in our population, defined as patients with European or African ancestry according to 1000 Genomes database (Χ 2 = 0.96 ; df = 2 ; p = 0.62). Moreover, the eleven KCNE1 SNPs were not impacted by population structure as all the maximum F ST over KCNE1 SNPs was of 0.12, especially the F ST of the rs11911509 SNP which was of zero.

Overall comparison of QTc length found a significant difference with respect to rs11911509 SNP genotype (F = 6.46, Df = 2, p = 2.1 x 10 -3 ) (Figure S4). The average QTc length was significantly longer in AA than in AC+CC carriers (AA vs AC+CC : 430.4 ms (±7.2 ms) vs 412.2 ms (± 2.6 ms) ; W = 2119 ; p= 0.01) (Figure 2). We also performed the analysis again after removing the 2 subjects with methadone dosage above 200 mg per day, one is AA carrier and the other is CC carrier. The overall comparison of QTc lengths across genotypes was still significant (F = 4.94 ; Df = 2 ; p = 8.3 x 10 -3 ) (Figure S5). Doing so, the mean QTc length was also significantly longer among AA carriers (AA vs AC+CC : 424.4 ms (±4 ms) vs 412.2 ms (± 2.6 ms); W = 1970 ; p= 0.03).

The gene-based approach found a significant association of KCNE1 with the QTc length using ethnic PCA dimensions as a covariate (number of used SNPs = 5, p empirical = 0.02) (Table S1).

Methadone dosage x rs 11911509 SNP interaction

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To test for interaction, we built a regression model with QTc length as dependant variable. As independent variables we included: (1) the methadone dosage, (2) the rs 11911509 SNP minor allele count, (3) their interaction, (4) independent variables that were associated with QTc length in univariate tests with an entry p-value below 0.1 (gender and antipsychotics co prescription), and ( 5) principal components to correct for ethnic stratification.

We found a significant interaction between methadone dosage and the number of rs11911509 SNP minor allele A (β = 0.18 ± 0.07; p = 0.01) to predict QTc length. This model explains 15 % of the QTc length variance. The comparison of this model with the regression model that did not include the interaction found a significant improvement of the second model (15% vs 12% of QTc explained variance; F = 6.6 ; p = 0.01). Furthermore, The correlation between methadone dosage and QTc length was only significant among AA carriers (r pearson = 0.65 ; p = 4.6 x 10 -4 ) (Figure 3).

DISCUSSION

The main finding of this study is the association of the KCNE1 gene with the QTc length among French methadone users. The association was found both in gene-based approach and at an individual SNP level (rs11911509). Interestingly though, we have found a significant interaction between the rs11911509 genotype & the methadone dosage that could explain a non-negligible variance of the QTc length among patients at steady state of methadone maintenance treatment.

In our patient cohort, the median methadone daily dose was slightly higher than the usually prescribed dose in France (60 mg per day vs 51.3 mg per day) [START_REF] Brisacier | Traitements de substitution aux opiacés[END_REF], but lower than in other non French studies [START_REF] Fanoe | Syncope and QT prolongation among patients treated with methadone for heroin dependence in the city of Copenhagen[END_REF][START_REF] Ehret | Drug-Induced Long QT Syndrome in Injection Drug Users Receiving Methadone: High Frequency in Hospitalized Patients and Risk Factors[END_REF][START_REF] Krantz | Dose-related effects of methadone on QT prolongation in a series of patients with torsade de pointes[END_REF] in which the median daily dose is commonly higher than 70 mg/day. The association between methadone daily dose and QTc prolongation is still debated. Genetic factors might account for the previously reported discrepancies between studies [START_REF] Mayet | Methadone maintenance, QTc and torsade de pointes: Who needs an electrocardiogram and what is the prevalence of QTc prolongation? Drug Alcohol[END_REF][START_REF] Fanoe | Syncope and QT prolongation among patients treated with methadone for heroin dependence in the city of Copenhagen[END_REF][START_REF] Ehret | Drug-Induced Long QT Syndrome in Injection Drug Users Receiving Methadone: High Frequency in Hospitalized Patients and Risk Factors[END_REF][START_REF] Krantz | Dose-related effects of methadone on QT prolongation in a series of patients with torsade de pointes[END_REF][START_REF] Roy | Increased incidence of QT interval prolongation in a population receiving lower doses of methadone maintenance therapy[END_REF][START_REF] Peles | Corrected-QT intervals as related to methadone dose and serum level in methadone maintenance treatment (MMT) patients: a crosssectional study[END_REF]. Here, we observed that the association between methadone dose and QTc length could be genotype dependent. Indeed, it was only significant among rs11911509 AA carriers. We conducted a previous study [START_REF] Hajj | KCNH2 polymorphism and methadone dosage interact to enhance QT duration[END_REF] that did not find a significant association between QT length and other KCNE1 SNPs, but this could be due to lack of power, or because SNPs that were previously tested, i.e., KCNE1 rs1805127, rs1805128 and rs2236609 are not implicated in the interaction of the methadone molecule and the KCNE1 Potassium voltage-gated channel protein. Those SNPs were not in linkage disequilibrium with the significant SNPs rs11911509 that we identified in the current study (r 2 : 0.027 and 0.017 respectively). Furthermore, we herein tested a larger sample of SNPs (126 SNPs versus 3 SNPs in Hajj & al.) and recruited additional patients (154 subjects versus 82 subjects in Hajj & al.) [START_REF] Hajj | KCNH2 polymorphism and methadone dosage interact to enhance QT duration[END_REF], which allowed us to perform a gene-based approach that was not available in our previous methodology.

The rs11911509 SNP that we identified here is an intronic variant of the KCNE1 gene with no known effect according to the Ensembl database [START_REF] Zerbino | [END_REF], and no phenotype has been associated with this SNP to our knowledge. Nevertheless, according to the 1000 Genomes database (accessed via Ensembl), this variant is in a high linkage disequilibrium (r 2 ≥ 0.8) among European populations with several other SNPs (rs4817657, rs4816488, rs4817658, rs35983014, rs9984007, rs11701051, rs62211545, rs8128386, rs8131017 and rs11702790) that were previously associated with phenotypes involving QT prolongation. Thus, it may act as a proxy for other variations that are associated with lengthened

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repolarization.

The KCNE1 gene encodes for a transmembrane domain protein that combines with another protein coded by the KNCQ1 gene to form a Potassium voltage-gated channel, also referred to as Minimal K+ channel (MinK), with a slow activation compared to the homomeric KCNQ1 channels [START_REF] Takumi | Cloning of a membrane protein that induces a slow voltage-gated potassium current[END_REF] and KCNH2 (hREG) channels. Mutations in KCNE1 can cause Long QT syndrome [START_REF] Crotti | Congenital long QT syndrome[END_REF] and is associated with an increased risk of atrial fibrillation [START_REF] Liang | KCNE1 rs1805127 polymorphism increases the risk of atrial fibrillation: a meta-analysis of 10 studies[END_REF]. Most of these mutations were associated with a reduction of the slowly activating potassium current [START_REF] Abbott | KCNE1 and KCNE3: The yin and yang of voltage-gated K+ channel regulation[END_REF], mechanism that partly explains QT prolongation [START_REF] Volders | Probing the contribution of IKs to canine ventricular repolarization: key role for beta-adrenergic receptor stimulation[END_REF]. But it is interesting to notice that some studies also involve KCNE1 mutations in dysfunctions of the hERG potassium channel that mediates the rapid repolarizing current [START_REF] Ohyama | Inhibition of cardiac delayed rectifier K+ currents by an antisense oligodeoxynucleotide against IsK (minK) and overexpression of IsK mutant D77N in neonatal mouse hearts[END_REF][START_REF] Bianchi | Cellular Dysfunction of LQT5-MinK Mutants: Abnormalities of IKs, IKr and Trafficking in Long QT Syndrome[END_REF]. As a reminder, it's well established that methadone QT prolongation involves a potent blockade of the hERG potassium channel [START_REF] Katchman | Influence of Opioid Agonists on Cardiac HumanEther-a-go-go-related Gene K+ Currents[END_REF]. The hypothesis of a double disruption that may lead to an additive effect on QT lengthening can be proposed [START_REF] Geelen | Concomitant Block of the Rapid (I(Kr)) and Slow (I(Ks)) Components of the Delayed Rectifier Potassium Current is Associated With Additional Drug Effects on Lengthening of Cardiac Repolarization[END_REF].

In our cohort, we found that women had a longer QTc interval than men, which is consistent with previous studies showing that women were more prone to drug-induced QTc prolongation. Some authors suggest that a greater polypharmacy and/or intrinsic factors linked to gender may explain such propensity [START_REF] Drici | Is Gender a Risk Factor for Adverse Drug Reactions?[END_REF]. To explore a potential polypharmacy effect, we used as proxy variables the methadone dosage and the Drugs lengthening QT score, and none of them was associated with sex (data not shown). Furthermore, neither allelic nor genotypic distributions of the rs11911509 SNP were different between genders. This suggests that the observed difference in QTc length between sexes may be explained by other not explored factors.

Association of antipsychotics co prescription with the QTc length did not reach significance in our cohort, but this prescription remains a risk factor of interest. Indeed, many studies pointed out the potential for antipsychotics to prolong the QTc interval [START_REF] Drici | Cardiovascular risks of atypical antipsychotic drug treatment[END_REF], particularly in case of methadone maintenance treatment [START_REF] Lee | Survey of methadone-drug interactions among patients of methadone maintenance treatment program in Taiwan[END_REF]. The lack of association that we observed may be due to insufficient statistical power to detect this effect.

A major strength of this study is that patients were included at steady state of methadone maintenance treatment, which excludes the cofounding effect of methadone dosage fluctuations. Moreover, the drugs lengthening QT score allows us to weigh the propensity of each concomitantly prescribed medication to lengthen the QT. This method was previously proposed in a pharmacogenomics GWAS of QT interval [START_REF] Avery | Drug-gene interactions and the search for missing heritability: a cross-sectional pharmacogenomics study of the QT interval[END_REF].

It is important to note, however, that our study has also some limitations. First, the QTc interval was not prospectively assessed. Second, additional transient risk factors associated with prolonged cardiac repolarization such as potassium and magnesium serum concentrations were not available. Third, two subjects had a daily methadone dosage up to 200 mg, which can drive the associations. Nevertheless, after removing these individuals, the correlation between QTc length and Methadone remained significant. Moreover, none of those two individuals was influential (Cook distance < 1) when we performed a diagnosis of our genetic association models (data not shown). Fourth, we had two ethnic populations in our sample. However, we used Eingenstrat method to control for population stratification, which seems sufficient enough to control for type-I error rate inflation especially for SNPs with low F ST on both SNP-based and Gene-based analysis [START_REF] Derks | The Relation Between Inflation in Type-I and Type-II Error Rate and Population Divergence in Genome-Wide Association Analysis of Multi-Ethnic Populations[END_REF]. Furthermore, we found that the effect size of genetic association of rs11911509 SNP with QTc length does not vary much when the analysis was restricted to Caucasian (n = 142 ; Cohen f 2 effect size = 0.135 vs 0.131 on the whole sample), but then the association is not statistically significant, probably due to statistical power reduction. Finally, we did not perform an a priori power calculation for the genetic association, however, the achieved power for the presented results on 154 subjects was of 80 % (α risk of 4 x 10 -4 , and Cohen's f 2 effect size of 1.131).

CONCLUSION

In conclusion, we herein highlighted a gene x environment interaction explaining part of the variance of QT length during methadone maintenance treatment. Our clinical message is that among the rs11911509 SNP AA genotype carriers, where the QTc lengthening after methadone-increased dosage is more significant, QTc monitoring should be mandatory.

As this side effect may potentially life-threatening consequences, and because methadone remains the most prescribed treatment for patients suffering from opiates dependence, further studies including KCNE1 genotyping are needed to better explore the implication of the MinK channel in the pathogenesis of this side effect.

FIGURES Figure 1: Flowchart of patients' selection

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Figure 2 :

 2 Figure 2 : Comparison of Bazett corrected QT length between AA and AC+CC carriers of the rs11911509 SNP genotypes (KCNE1 gene)

  Gossop, Lintzeris, Markides, & Strang, 2011; Fanoe, Hvidt, Ege, & Jensen, 2007; Ehret et al., 2006; Krantz, Kutinsky, Robertson, & Mehler, 2003; Roy et al., 2012; Peles, Bodner, Kreek, Rados, & Adelson, 2007

ACKNOWLEDGMENTS

We would like to show our gratitude to collaborators of the METHADOSE study: Maeva FORTIAS, Cyrille ORIZET, Xavier LAQUEILLE, Philippe COEURU, Pierre POLOMENI, Elisabeth AVRIL, Didier TOUZEAU and Anne-Marie SIMONPOLI. We are grateful to patients for their participation to the study. We express thanks for Pr Eske DERKS, QIMR Berghofer, Translational Neurogenomics Group, for her precious comments about this works.

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ROLE OF FUNDING SOURCES

METHADOSE study, OST07013 was funded by the DRC (Direction de la Recherche Clinique) of the Assistance Publique -Hôpitaux de Paris). This article is protected by copyright. All rights reserved.

CONFLICT OF INTEREST