Ilann Bourgeois
email: ilann.bourgeois@noaa.gov

Jean-Christophe Clément

Nicolas Caillon

Joël Savarino

Foliar uptake of atmospheric nitrate by two dominant subalpine plants: insights from in situ triple-isotope analysis

Keywords: Subalpine plants, atmospheric nitrate, stable isotopes, foliar uptake, nitrogen deposition

The significance of foliar uptake of nitrogen (N) compounds in natural conditions is not well understood, despite growing evidence of its importance to plant nutrition. In subalpine meadows, N-limitation fosters the dominance of specific subalpine plant species, which in turn ensure the provision of essential ecosystems services. Understanding how these plants absorb N and from which sources is important to predict ecological consequences of increasing N deposition.

• Here, we investigate the sources of N to plants from subalpine meadows with distinct landuse history in the French Alps, using the triple isotopes (Δ 17 O, δ 18 O, and δ 15 N) of plant tissue nitrate (NO 3 -). We use this approach to evaluate the significance of foliar uptake of atmospheric NO 3 -(NO 3 atm ).

• The foliar uptake of NO 3 atm accounted for 4-16 % of the leaf NO 3 -content, and contributed more to the leaf NO 3 -pool after peak biomass. Additionally, the gradual 15 N enrichment of NO 3 -from the soil to the leaves reflected the contribution of NO 3 atm assimilation to plants' metabolism.

• The present study confirms that foliar uptake is a potentially important pathway for NO 3 atm into subalpine plants. This is of major significance as N emissions (and deposition) are predicted to increase globally in the future.

Introduction

Monitoring atmospheric nitrogen (N) deposition has greatly improved our understanding of the effects of anthropogenic changes on the environment [START_REF] Vitousek | Human alteration of the global nitrogen cycle: sources and consequences[END_REF][START_REF] Galloway | Transformation of the nitrogen cycle: recent trends, questions, and potential solutions[END_REF][START_REF] Fowler | Effects of global change during the 21st century on the nitrogen cycle[END_REF]. Human activities (e.g., fossil fuel combustion and agriculture) have led to increased emissions of reactive nitrogen species (Nr = NO y , NH x ), which in turn have increased inputs of N to the environment worldwide [START_REF] Galloway | Nitrogen cycles: past, present, and future[END_REF][START_REF] Cui | Centennial-scale analysis of the creation and fate of reactive nitrogen in China (1910-2010)[END_REF]. Some consequences of this altered N cycle include soil acidification, biodiversity loss, intensified N leaching, eutrophication, and public health issues [START_REF] Aber | Nitrogen saturation in northern forest ecosystems[END_REF][START_REF] Galloway | The Nitrogen Cascade[END_REF][START_REF] Ward | Workgroup Report: drinking-water nitrate and health-recent findings and research needs[END_REF][START_REF] Clark | Estimated losses of plant biodiversity in the Accepted Article This article is protected by copyright[END_REF].

Mountain regions are particularly vulnerable to changes in the N cycle: their shallow soils have a low acidification buffering capacity, while the endemic plants have developed N uptake strategies that are optimized for low nutrient environments [START_REF] Bowman | Nitrogen critical loads for alpine vegetation and terrestrial ecosystem response: are we there yet?[END_REF][START_REF] Bassin | Species composition of subalpine grassland is sensitive to nitrogen deposition, but not to ozone, after seven years of treatment[END_REF][START_REF] Boutin | Nitrogen deposition and climate change have increased vascular plant species richness and altered the composition of grazed subalpine grasslands[END_REF]. For example, intermediate stages of N saturation [START_REF] Aber | Nitrogen saturation in northern forest ecosystems[END_REF] have been measured in elevated sites in the Colorado Front Range, USA [START_REF] Williams | Critical loads for inorganic nitrogen deposition in the Colorado Front Range, USA[END_REF][START_REF] Burns | The effects of atmospheric nitrogen deposition in the Rocky Mountains of Colorado and southern Wyoming, USA-a critical review[END_REF][START_REF] Baron | Hindcasting nitrogen deposition to determine an ecological critical load[END_REF], as a result of regionally elevated N deposition [START_REF] Mladenov | Atmospheric deposition as a source of carbon and nutrients to an alpine catchment of the Colorado Rocky Mountains[END_REF][START_REF] Clow | Spatial patterns of atmospheric deposition of nitrogen and sulfur using ion-exchange resin collectors in Rocky Mountain National Park, USA[END_REF][START_REF] Nanus | Spatial and temporal variation in sources of atmospheric nitrogen deposition in the Rocky Mountains using nitrogen isotopes. Atmospheric Accepted Article This article is protected by copyright[END_REF].

In high altitude catchments, the N balance is still poorly understood. The fraction of atmospheric N efficiently retained or lost, and where and how this occurs, remains unknown. While deposition of atmospheric N often exceeds losses to streams via leaching and runoff [START_REF] Baron | Nitrogen fluxes in a high elevation Colorado Rocky Mountain basin[END_REF][START_REF] Mast | Links between N deposition and nitrate export from a high-elevation watershed in the Colorado Front Range[END_REF], the respective importance of potential N sinks (e.g., microbial and abiotic immobilization, plant uptake, denitrification) is not yet constrained [START_REF] Weintraub | Interactive effects of vegetation type and topographic position on nitrogen availability and loss in a temperate montane ecosystem[END_REF]. In addition, atmospheric N dynamics in the mountainous soil/plant continuum are inextricably linked to the complex topography and hydrology of these ecosystems [START_REF] Balestrini | Catchment features controlling nitrogen dynamics in running waters above the tree line (central Italian Alps)[END_REF][START_REF] Weintraub | Interactive effects of vegetation type and topographic position on nitrogen availability and loss in a temperate montane ecosystem[END_REF]

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This article is protected by copyright. All rights reserved. 2017; Bourgeois et al., 2018b). While assessing the fate of deposited N in high altitude catchments is complex, it is necessary to understand nutrient partitioning in these essential ecosystems to preserve freshwater quality, erosion control and their patrimonial value [START_REF] Clément | The effects of snow-N deposition and snowmelt dynamics on soil-N cycling in marginal terraced grasslands in the French Alps[END_REF][START_REF] Thébault | Nitrogen limitation and microbial diversity at the treeline[END_REF].

Several studies have established that reactive atmospheric N species (e.g., NO 2 , HNO 3 , and NH 3 ) may be incorporated directly into plant leaves and used as a nutritional source [START_REF] Vallano | Foliar δ 15 N values as indicators of foliar uptake of atmospheric nitrogen pollution[END_REF] and literature therein). In N-limited soils, N availability to plants largely depends on competing or loss processes such as microbial immobilization [START_REF] Kaye | Competition for nitrogen between plants and soil microorganisms[END_REF] and leaching [START_REF] Hood | Landscape Controls on Organic and Inorganic Nitrogen Leaching across an Alpine/Subalpine Ecotone, Green Lakes Valley, Colorado Front Range[END_REF]. Foliar uptake can provide an alternative N absorption pathway to plants, and its significance needs to be evaluated to better predict how these N-poor ecosystems will respond to a globally increasing N deposition [START_REF] Fowler | The global nitrogen cycle in the twenty-first century[END_REF]. However, an accurate parameterization of this mechanism is absent from most ecological models [START_REF] Sparks | Ecological ramifications of the direct foliar uptake of nitrogen[END_REF]. To our knowledge, no study has quantified the magnitude of foliar uptake in situ (i.e., under natural atmospheric N deposition rates).

Most estimates of foliar uptake are reported as contributing 0-50% of plant N demand [START_REF] Sparks | Ecological ramifications of the direct foliar uptake of nitrogen[END_REF] and literature therein). Yet, these studies were either conducted at the individual plant scale, in the laboratory (i.e., unnatural conditions), or at the ecosystem scale, using imperfect throughfall measurements. An accurate partitioning of N absorption pathways (root vs. foliar) will be key to predicting the impact of increasing atmospheric N deposition on ecosystem productivity.

The dual isotopes (δ 18 O and δ 15 N) of nitrate (NO 3 -) have provided a refined understanding of the N cycle across a variety of terrestrial and aquatic ecosystems [START_REF] Durka | Effects of forest decline on uptake and leaching of deposited nitrate determined from 15 N and 18 O measurements[END_REF][START_REF] Mayer | Sources of nitrate in rivers draining sixteen watersheds in the northeastern US: isotopic constraints[END_REF][START_REF] Wankel | Nitrogen sources and cycling in the San Francisco Bay Estuary: a nitrate dual isotopic composition approach[END_REF][START_REF] Nanus | Evaluating regional patterns in nitrate sources to watersheds in national parks of the Rocky Mountains using nitrate isotopes[END_REF][START_REF] Hastings | Anthropogenic impacts on nitrogen isotopes of ice-core nitrate[END_REF]. [START_REF] Kendall | Tracing anthropogenic inputs of nitrogen to ecosystems[END_REF] characterized the δ 18 O and δ 15 N ranges of NO 3 -from different sources (e.g., nitrification, atmospheric deposition), from which the origin of NO 3 -in a given environmental pool can be determined. However, terrestrial reservoirs of NO 3 -and NH 4 + can undergo a large variety of physical, chemical or biological processes (e.g., mineralization, nitrification, assimilation, volatilization) that impact their isotopic composition, also called fractionation. During the nitrification process, the newly produced NO 3 -is 15 N-depleted compared to the residual NH 4 + pool [START_REF] Mariotti | Experimental determination of nitrogen kinetic isotope fractionation: some principles; illustration for the denitrification and nitrification processes[END_REF]. During the denitrification and biological

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assimilation processes, the residual NO 3 -pools are 15 N-and 18 O-enriched [START_REF] Mariotti | Experimental determination of nitrogen kinetic isotope fractionation: some principles; illustration for the denitrification and nitrification processes[END_REF][START_REF] Mariotti | Nitrogen isotope fractionation associated with nitrate reductase activity and uptake of NO 3 by Pearl Millet[END_REF][START_REF] Granger | Coupled nitrogen and oxygen isotope fractionation of nitrate during assimilation by cultures of marine phytoplankton[END_REF][START_REF] Granger | N and O isotope effects during nitrate assimilation by unicellular prokaryotic and eukaryotic plankton cultures[END_REF]. Although some of these isotopic effects were successfully characterized, understanding the complex dynamics of the N cycle based on δ 18 O and δ 15 N of NO 3 -is not straightforward. Pioneering studies successfully used the dual isotope approach to evaluate the sources of NO 3 -in moss tissues (Liu et al., 2012b(Liu et al., ,a, 2013c,a),a). However, the interpretation of δ 18 O and δ 15 N values of NO 3 -(referred to as δ 18 O-NO 3 -and δ 15 N-NO 3 -in this paper) is much more challenging in vascular plants due to the simultaneous effects of source mixing and biological isotope fractionation and their impacts on plant tissue NO 3 -isotopes (Liu et al., 2013b[START_REF] Liu | Nitrate dynamics in natural plants: insights based on the concentration and natural isotope abundances of tissue nitrate[END_REF]. Recently, the triple isotope (Δ 17 O, δ 18 O, and δ 15 N) composition of NO 3 -has been proposed as a more robust tool to identify NO 3 -sources and infer the biological underlying processes driving the N cycle [START_REF] Michalski | Tracing atmospheric nitrate deposition in a complex semiarid ecosystem using Δ 17[END_REF][START_REF] Costa | Analysis of atmospheric inputs of nitrate to a temperate forest ecosystem from Δ 17 O isotope ratio measurements[END_REF].

Atmospheric NO 3 -(NO 3 - atm ) is characterized by a high Δ 17 O value
ranging from 20 to 35‰ [START_REF] Savarino | Isotopic composition of atmospheric nitrate in a tropical marine boundary layer[END_REF], whereas biologically-derived NO 3 -(NO 3 bio ) exhibits a Δ 17 O value of 0 ‰ [START_REF] Michalski | Tracing atmospheric nitrate deposition in a complex semiarid ecosystem using Δ 17[END_REF]. The contribution of NO 3 atm can thus be quantified through a simple two end-member mixing model. This triple isotopic tracer of NO 3 -unequivocally reveals direct NO 3 atm inputs in environmental pools, and provides a better assessment of biologicallyinduced fractionation of NO 3 -isotopes compared to the traditional dual isotope technique [START_REF] Rose | Drivers of atmospheric nitrate processing and export in forested catchments[END_REF][START_REF] Fang | Microbial denitrification dominates nitrate losses from forest ecosystems[END_REF]. The triple isotopic tracer method has for instance recently been used to monitor NO 3 atm uptake and assimilation by forests and tundra across a variety of ecosystems [START_REF] Liu | Nitrate is an important nitrogen source for Arctic tundra plants[END_REF].

Here, we tested two main hypotheses: i) NO 3 -triple isotopes can be used to quantify the proportion of Alps. We also tested the question of land-use influence on foliar uptake of N. The vegetation at the Lautaret pass is dominated by grassland communities whose diversity and functional traits depend on

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past (terraced vs. unterraced) and current (mowing, grazing) land use [START_REF] Quétier | Plant traits in a state and transition framework as markers of ecosystem response to land-use change[END_REF]. These distinct land-use trajectories have been shown to deeply impact the N turnover rates in subalpine soils [START_REF] Robson | Neglect of mowing and manuring leads to slower nitrogen cycling in subalpine grasslands[END_REF][START_REF] Robson | Land use in subalpine grasslands affects nitrogen cycling via changes in plant community and soil microbial uptake dynamics[END_REF][START_REF] Legay | Comparison of inorganic nitrogen uptake dynamics following snowmelt and at peak biomass in subalpine grasslands[END_REF], mostly because of differences in N availability, microbial communities and abiotic parameters [START_REF] Legay | Influence of plant traits, soil microbial properties, and abiotic parameters on nitrogen turnover of grassland ecosystems[END_REF]. Here, we hypothesized that foliar uptake of NO 3 atm would contribute in larger proportion to plant N nutrition in less fertile grasslands with low N availability.

Material & Methods

Site Description

The Lautaret and Les Cours sub-watersheds are located in the upper Romanche valley of the central French Alps, near the Lautaret pass (45°02'N; 6°20'E, 1650-2000 m a.s.l., Figure S1). The site forms part of the French National Research Infrastructure to study continental ecosystems and their biodiversity (AnaEE -France, https://www.anaee-france.fr) and of the Long-Term Ecological

Research network (LTER, https://www.ilter.network). The Lautaret pass is characterized by a seasonal snow-cover (November to April) and a short vegetative period (May to September, hereafter denoted as "annual"). Temperatures averaged -3.0°C and 13.0°C in January and August 2016, respectively, while multi-year (2004-2010) means were -6.5°C and 13.9°C. The annual cumulative precipitation was 609 mm, significantly lower than the multi-year (2004-2010) mean of 956 mm.

Here, we consider three grasslands featuring three combinations of past (terraced vs. unterraced) and present (mown vs. unmown) land uses [START_REF] Quétier | Plant traits in a state and transition framework as markers of ecosystem response to land-use change[END_REF]. Two grasslands are situated on the terraced slopes of Les Cours watershed (Figure S1); one is mown for hay in August and grazed in autumn (terraced and mown, TM) while the other is not mown but grazed in summer (terraced and unmown, TU). The third grassland is located on the Lautaret watershed (Figure S1) and is unterraced, never mown (unterraced and unmown, UU) and only very lightly grazed in summer. The management practices in the study area remained unchanged since at least 2003, which led to substantial

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differences in plant communities and soil properties between each grassland [START_REF] Legay | Comparison of inorganic nitrogen uptake dynamics following snowmelt and at peak biomass in subalpine grasslands[END_REF].

Generally, the terraced meadows were dominated by species showing exploitative traits (higher specific leaf area and leaf nitrogen content; [START_REF] Quétier | Plant traits in a state and transition framework as markers of ecosystem response to land-use change[END_REF] such as Dactylis glomerata, whereas unterraced grasslands were dominated by conservative species (higher internal N recycling; [START_REF] Quétier | Plant traits in a state and transition framework as markers of ecosystem response to land-use change[END_REF] like Festuca paniculata. These two species have very different functional traits, N uptake strategies, and competition levels with soil microbial biomass [START_REF] Robson | Land use in subalpine grasslands affects nitrogen cycling via changes in plant community and soil microbial uptake dynamics[END_REF][START_REF] Legay | Comparison of inorganic nitrogen uptake dynamics following snowmelt and at peak biomass in subalpine grasslands[END_REF].

Samples collection and analysis

To account for spatial variability, each grassland was subdivided into three plots to provide three plant concentrations in plants were not investigated. From April to October 2016, separate samples for wet and dry deposition were collected every three weeks using an atmospheric deposition collector (WADOS Kroneis GmbH, Austria). Precipitation samples were kept frozen until further chemical analysis. The dry deposition funnel was rinsed with 500 mL of ultrapure water (18.2 MΩ.cm -1 ) and the sample stored at -20°C.

All plants were thoroughly washed with ultrapure water. Aerial parts (i.e., leaves, shoots, and stems)

were then separated from those below ground (i.e., roots), and then frozen. Leaves, shoots, and stems were combined and collectively referred to as "leaves" hereafter. Samples were later freeze-dried (Heto DryWinner, Allerod, Denmark), ground to powder (RETSCH Mixer Mill MM400), and

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homogenized. Dissolved inorganic nitrogen (DIN) was extracted by shaking (300 RPM for 1h) 1.0 g (D. glomerata) and 1.5 g (F. paniculata) dry weight (dw) of powdered leaf and root tissues in 50 mL of ultrapure water. Soils were passed through a 2 mm sieve to remove rocks and roots. Subsamples of sieved soils (10.0 g dw) were shaken (300 RPM for 1h) in 50 mL of ultrapure water to extract the DIN. After centrifugation (4000 RPM for 10 min), all plant and soil samples were filtered using 0.22 μm quartz filters (QMA Whatman). Precipitation samples were left to melt at ambient temperature and filtered using 0.45 μm quartz filters (QMA Whatman).

All samples were subsequently analyzed for concentrations of NH 4 + , NO 3 -, and NO 2 -(the latter being always negligible compared to [NO 3 -]) using a colorimetric technique (Gallery Plus, Thermo Fisher Scientific, Waltham, Massachusetts, USA). The analytical error of ±0.01 mg L -1 was determined from the standard deviation of commercial control solutions (ThermoFisher ® ). Isotopic analyses were conducted on a MAT 253 IRMS using an adapted version of the denitrifier method [START_REF] Kaiser | Triple oxygen isotope analysis of nitrate using the denitrifier method and thermal decomposition of N 2 O[END_REF][START_REF] Morin | Tracing the origin and fate of NO x in the Arctic atmosphere using stable isotopes in nitrate[END_REF]. The analytical errors -calculated as the standard deviation of the residuals from the linear regression between raw data of the reference standards (USGS 32, USGS 34 and other sources has a Δ 17 O of zero [START_REF] Michalski | Tracing atmospheric nitrate deposition in a complex semiarid ecosystem using Δ 17[END_REF]. In this study, a simple mixing model was used to quantify the unprocessed NO 3 atm fraction (ƒ atm ) in samples, according to [START_REF] Michalski | Tracing atmospheric nitrate deposition in a complex semiarid ecosystem using Δ 17[END_REF]:

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(Eq.1)

ƒ atm = Δ 17 O-NO 3 - sample / Δ 17 O-NO 3 - atm
We used a value of Δ 17 O-NO 3 atm = 24.4 ± 2.4 ‰, corresponding to the mean concentration-weighted value of Δ 17 O-NO 3 -found in wet and dry deposition samples collected during the study (Table 1).

To our knowledge, there are two possible sources of NO 3 atm in leaves. One is the direct leaf uptake via stomata or cuticles, and the other is the translocation of deposited NO 3 atm from soil to roots, and then to the leaves. Therefore, we used the following isotope mass-balance approach to estimate the foliar absorption of NO 3 atm :

(Eq.2)

Δ 17 O-NO 3 - leaf = Δ 17 O-NO 3 - atm * ƒ uptake + Δ 17 O-NO 3 - root * ƒ translocation (Eq.3) ƒ uptake + ƒ translocation = 1
Where ƒ uptake and ƒ translocation are the fractions of atmospheric NO 3 atm out of the leaf NO 3 -pool from foliar uptake and translocation, respectively. After substituting Eq.3 into Eq.2, we get:

(Eq.4) ƒ uptake = (Δ 17 O-NO 3 - leaf -Δ 17 O-NO 3 - root ) / (Δ 17 O-NO 3 - atm -Δ 17 O-NO 3 - root )

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biological processes that affect its δ 15 N and δ 18 O values [START_REF] Tsunogai | Tracing the fate of atmospheric nitrate deposited onto a forest ecosystem in Eastern Asia using Δ 17[END_REF][START_REF] Dejwakh | Using 17 O to investigate nitrate sources and sinks in a semi-arid groundwater system[END_REF][START_REF] Riha | High atmospheric nitrate inputs and nitrogen turnover in semi-arid urban catchments[END_REF]):

(Eq.5)

δ 18 O bio = (δ 18 O sample -δ 18 O atm * ƒ atm ) / ƒ bio (Eq.6) δ 15 N bio = (δ 15 N sample -δ 15 N atm * ƒ atm ) / ƒ bio
where δ 15 N atm = -6.0 ± 4.1 and δ 18 O atm = 66.6 ± 5.8 ‰ were inferred from the mean concentrationweighted NO 3 -isotopes in wet and dry deposition samples collected during the study (Table 1).

Statistical analysis

All statistical analyses were conducted using the R software (v3.2.3). A Mann-Whitney test was applied to determine significant differences of mean concentrations and isotopic values of NO 3 -and NH 4 + between plant tissues and species, on an annual (Figure 1) or seasonal (Figure 2) basis.

Differences and correlations were considered significant for p<0.05.

Results

Atmospheric N deposition

Mean and associated standard deviation of Δ 17 O-NO 3 -in wet and dry deposition from May to September 2016 were 23.6 ± 2.0 and 25.1 ± 2.7 ‰, respectively (Table 1). These values are in the same range as previously reported data for Δ 17 O-NO 3 atm [START_REF] Michalski | Tracing atmospheric nitrate deposition in a complex semiarid ecosystem using Δ 17[END_REF][START_REF] Tsunogai | Tracing the fate of atmospheric nitrate deposited onto a forest ecosystem in Eastern Asia using Δ 17[END_REF][START_REF] Tsunogai | Accurate and precise quantification of atmospheric nitrate in streams draining land of various uses by using triple oxygen isotopes as tracers[END_REF][START_REF] Costa | Analysis of atmospheric inputs of nitrate to a temperate forest ecosystem from Δ 17 O isotope ratio measurements[END_REF]. The weighted mean Δ 17 O value for total deposition (wet + dry) was 24.4 ± 2.4 ‰ (Table 1), and was used to quantify the atmospheric component of NO 3 -pools in plants

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(see Material & Methods, Eq. 1). Mean annual total δ 18 O-NO 3 atm (66.6 ± 5.8 ‰) and δ 15 N-NO 3 atm (-6.0 ± 4.1 ‰) were also consistent with a reservoir of exclusively NO 3 atm [START_REF] Kendall | Tracing anthropogenic inputs of nitrogen to ecosystems[END_REF], and these values were used to correct samples from their atmospheric δ 18 O and δ 15 N components (see Material & Methods, Eqs. 5 and 6). Based on these data, the calculated magnitude of the annual NO 3 atm deposition flux was 3-5 kg.N-NO 3 -.ha -1 .yr -1 (Bourgeois et al., personal communication), higher than the N critical load for subalpine ecosystems estimated to be 1-4 kg.N.ha -1 .yr -1 [START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF][START_REF] Nanus | Spatial variation of atmospheric nitrogen deposition and critical loads for aquatic ecosystems in the Greater Yellowstone Area[END_REF].

Soil inorganic N pools N availability in soils differed in the three meadows. Soil NO 3 -concentration in the UU meadow (14.6 ± 19.0 μg.g -1 dw) was significantly lower than in the terraced meadows (58.9 ± 46.9 and 52.3 ± 48.7

μg.g -1 dw in TU and TM, respectively; Table 2). Soil NH 4 + concentration was higher in the unmown meadows (13.0 ± 13.7 μg.g -1 dw in UU and 17.5 ± 29.9 μg.g -1 dw in TU) compared to the mown one (6.2 ± 4.0 in TM), although not significantly. The ratio NO 3 -/NH 4 + was significantly lower in the UU meadow. These results were in line with previous studies at the same site.

Mean and associated standard deviation of Δ 17 O-NO 3 -in soils were low, and significantly higher in the UU meadow (0.9 ± 0.6 ‰) than in the terraced ones (0.5 ± 0.8 and 0.3 ± 0.4 ‰ in TU and TM, respectively; Table 2). While no significant difference was observed in NO 3 -isotopic values between the TU and TM meadows (δ 18 O-NO 3 -= -10.3 ± 6.6 and -10.9 ± 5.1 ‰, and δ 15 N-NO 3 -= -1.3 ± 9.6 and -2.6 ± 6.5 ‰, respectively), δ 18 O-NO 3 -and δ 15 N-NO 3 -were significantly higher (-4.3 ± 6.8 ‰) and lower (-5.1 ± 6.6 ‰), respectively, in the UU meadow.

Plant inorganic N pools

The mean [NO 3 -] in plant tissues varied between 10.0 ± 5.4 and 38.8 ± 50.8 μg.g -1 dw, and no significant difference was found between plant species or between plant tissues, except for D.

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glomerata in TU fields (Figure 1a). This range of [NO 3 -] is similar to the 6-60 μg.g -1 dw range in Arctic vegetation where N is limited [START_REF] Liu | Nitrate is an important nitrogen source for Arctic tundra plants[END_REF]. The mean [NH 4 + ] in plant tissues varied between 1.3 ± 0.8 and 94.0 ± 100.1 μg.g -1 dw and was very low in all plant leaves. D. glomerata roots held significantly higher NH 4 + concentrations compared to F. paniculata (Figure 1b). Positive Δ 17 O-NO 3 -in all tissues of both D. glomerata and F. paniculate evidences direct uptake of unprocessed NO 3 atm (Figure 1c). The mean Δ 17 O-NO 3 -ranged from 1.0 ± 0.7 ‰ in D. glomerata roots to 3.4 ± 3.2 ‰ in F. paniculata leaves. According to Eq.1, the mean NO 3 atm contribution accounted for 4 ± 3 % to 14 ± 13 %, and up to 33 % (when Δ 17 O = 8.1 ‰), of the NO 3 -pool in plant tissues (Figure 1c). F.

paniculata exhibited similar high mean δ 18 O-NO 3 -and δ 15 N-NO 3 -in roots and leaves (Figure 1d and e). In D. glomerata leaves, the mean δ 18 O-NO 3 -and δ 15 N-NO 3 -spanned significantly higher values than in the roots.

The mean [NO 3 -] in leaves was significantly higher after peak biomass in all plants (Figure 2a), whereas the mean [NH 4 + ] did not significantly vary (Figure 2b). The period after peak biomass was also characterized by significantly higher mean Δ 17 O-NO 3 -in leaves and significantly lower mean δ 15 N-NO 3 -in roots compared to the earlier period (Figures 2c and2d). There was no significant difference for δ 18 O-NO 3 -between the two periods (Figure S2).

Discussion

NO 3 -and NO 3 atm uptake

One possible scenario that could explain the presence of NO 3 atm in plant leaves involves direct foliar absorption of gaseous species (e.g., NO 2 , HNO 3 ) and/or the incorporation of NO 3 atm dissolved in precipitation [START_REF] Vallano | Foliar δ 15 N values as indicators of foliar uptake of atmospheric nitrogen pollution[END_REF]. The respective importance of each absorption pathways depends on a large array of leaf properties, among which the leaf wettability (i.e., the capacity to retain water). In both cases, these N compounds enter plant leaves following passive diffusion mechanisms either via the stomata or through the cuticle, depending on the chemical and physical

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characteristics of the pollutant and the leaf, and are eventually transformed into NO 3 - [START_REF] Vallano | Foliar δ 15 N values as indicators of foliar uptake of atmospheric nitrogen pollution[END_REF][START_REF] Sparks | Ecological ramifications of the direct foliar uptake of nitrogen[END_REF]. More important for this study, HNO 3 , NO 3 -, and, hypothetically, NO 2 lead to a similarly 17 O-enriched NO 3 atm when assimilated in leaves [START_REF] Savarino | The NO+O 3 reaction: a triple oxygen isotope perspective on the reaction dynamics and atmospheric implications for the transfer of the ozone isotope anomaly[END_REF], regardless of the absorption pathway. Therefore, we will from now on use the term "foliar uptake of NO 3 atm " to refer to the absorption of NO 3 --producing species through the leaves, with no further consideration of the mechanism involved. This scenario is supported by a recent study of subalpine meadows in the French Pyrenees which highlighted the strong foliar uptake capacity of Poaceae (i.e., F. paniculata and D. glomerata family) in an experiment where δ 15 N-labeled NO 3 -and NH 4 + were sprayed on natural subalpine grasslands [START_REF] Pornon | Contribution of plant species to the high N retention capacity of a subalpine meadow undergoing elevated N deposition and warming[END_REF].

Another possible scenario is the translocation of deposited NO 3 atm from soils to above-ground biomass [START_REF] Pornon | Contribution of plant species to the high N retention capacity of a subalpine meadow undergoing elevated N deposition and warming[END_REF]. Root-to-shoot transport of NO 3 -via the xylem is well-documented for herbaceous species, with NO 3 -being either stored in petioles or reduced in mesophyll cells [START_REF] Hachiya | Interactions between nitrate and ammonium in their uptake, allocation, assimilation, and signaling in plants[END_REF]. During translocation, NO 3 -can also be partly reduced in roots or stored in the vacuole. In our study, year-round positive Δ 17 O-NO 3 -values in D. glomerata and F. paniculata roots suggest that at least part of the NO 3 atm in the plants was acquired via root uptake (Figure 1c).

However, Δ 17 O-NO 3 -in leaves was significantly higher compared to roots after peak biomass (Figure 2c). Such a relationship can only be explained by foliar uptake being the dominant NO 3 atm assimilation pathway. Indeed, the enrichment of NO ), because biological and transport processes do not affect the Δ 17 O value [START_REF] Michalski | Tracing atmospheric nitrate deposition in a complex semiarid ecosystem using Δ 17[END_REF]. We found that neither soils (Table 2) nor roots (Figure 2c)which would be the reservoirs of NO 3 atm in leaves in this scenario -showed a similar increase in exogenous NO 3 -after peak biomass.

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In order to further highlights the importance of foliar uptake of NO 3 atm we introduce the terminology X leaves-roots = X leaves -X roots , where X can be ] leaves-roots (Figure 3a). Foliar uptake of NO 3 atm can therefore be quantified by considering the range of positive NO 3 atm enrichment in leaves compared to roots, using Eq.4. When Δ 17 O leaves-roots is positive (i.e., foliar uptake contributes more NO 3 atm than root translocation), it spans a 0.9-3.8 ‰, corresponding to a ƒ uptake = 4-16 %.

Isotopic evidence of NO 3 -assimilation in roots and leaves Uptake and subsequent transport of NO 3 -in plants is commonly regarded as yielding little, if any, isotopic fractionation due to the lack of bond breakage in these processes [START_REF] Mariotti | Nitrogen isotope fractionation associated with nitrate reductase activity and uptake of NO 3 by Pearl Millet[END_REF][START_REF] Evans | Nitrogen isotope composition of tomato (Lycopersicon esculentum Mill. cv. T-5) grown under ammonium or nitrate nutrition[END_REF]. Conversely, the co-variation of O and N isotopic fractionation can be a good indicator of biological processes such as denitrification [START_REF] Wexler | Isotopic signals of summer denitrification in a northern hardwood forested catchment[END_REF][START_REF] Fang | Microbial denitrification dominates nitrate losses from forest ecosystems[END_REF] and assimilation [START_REF] Granger | Coupled nitrogen and oxygen isotope fractionation of nitrate during assimilation by cultures of marine phytoplankton[END_REF][START_REF] Granger | N and O isotope effects during nitrate assimilation by unicellular prokaryotic and eukaryotic plankton cultures[END_REF]. In plants, variations of δ 18 O-NO 3 -and δ 15 N-NO 3 -are mainly influenced by NO 3 -reduction (NR) into NH 4 + in the protein-making process [START_REF] Tcherkez | Viewpoint : Isotopic fractionation by plant nitrate reductase, twenty Accepted Article This article is protected by copyright. All rights reserved. years later[END_REF].

Laboratory and hydroponic experiments on NR-induced isotopic fractionation showed that δ 18 O-NO 3 and δ 15 N-NO 3 -co-vary along a 1:1 slope [START_REF] Karsh | Eukaryotic assimilatory nitrate reductase fractionates N and O isotopes with a ratio near unity[END_REF][START_REF] Estrada | Stable isotopic composition of perchlorate and nitrate accumulated in plants: hydroponic experiments and field data[END_REF]. These findings were supported by the only field analysis of NR fractionation, where a slope of 1.27 was found between δ 18 O-NO 3 -and δ 15 N-NO 3 -in tree roots (Liu et al., 2013b). Here, the linear regressions between δ 18 O-NO 3 bio and δ 15 N-NO 3 bio (see Eqs.5 and 6) showed similar slopes of 0.76 and 0.71 in species-aggregated roots and leaves, respectively (Figure 4a). These slopes were lower than the expected 1:1 line, suggesting the production and uptake of new NO 3 bio with low δ 18 O and δ 15 N. This was also found by [START_REF] Wexler | Isotopic signals of summer denitrification in a northern hardwood forested catchment[END_REF] who reported weaker in situ denitrification slopes (0.4 -0.7) compared to laboratory experiments (1.0) due to the production of new NO 3 bio (see also [START_REF] Granger | Isotopic overprinting of nitrification on denitrification as a ubiquitous and unifying feature of environmental nitrogen cycling[END_REF]. Here, the results suggest that constant uptake of new NO 3 bio by roots and subsequent translocation to the leaves could account for the observed slopes.

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In addition, the slope of 0.05 for the correlation between Δ 17 O-NO 3 -and δ 18 O-NO 3 bio further underscores the assimilation-driven δ 18 O enrichment of NO 3 bio in roots (Figure 4b). In the leaves, most δ 18 O-NO 3 -values fall outside of the mixing line between NO 3 atm and NO 3 bio when most of the δ 18 O-NO 3 -values in the roots fall on the mixing line (Figure 4b). This suggests that an additional assimilation step occurs in aboveground tissue after transport from the roots. This possible additional NR step in leaves is also supported by the significant correlation between δ 18 O leaves-roots and δ 15 N leaves- roots (0.99) and confirms NO 3 atm metabolic reduction after foliar uptake (Figure 3b).

Different NO 3 -uptake patterns glomerata roots could also stem from a particularly high NH 4 + affinity [START_REF] Grassein | Relationships between functional traits and inorganic nitrogen acquisition among eight contrasting European grass species[END_REF], although no evidence exists that uptake and concentration of NH 4 + are linked. TU meadows had the highest [NH 4 + ] in soils (Table 2) which may have favored NH 4 + uptake by D. glomerata as N availability is a strong driver of N uptake strategy [START_REF] Houlton | A climate-driven switch in plant nitrogen acquisition within tropical forest communities[END_REF][START_REF] Stahl | Dynamic niche sharing in dry acidic grasslands -a 15 Nlabeling experiment[END_REF][START_REF] Wang | Constrained preferences in nitrogen uptake across plant species and environments[END_REF].

For F. paniculata, a more conservative species, the data showed a significantly higher ƒ atm in the roots (Figure 1c), mirroring the lower N cycling rates in UU soils previously documented [START_REF] Robson | Neglect of mowing and manuring leads to slower nitrogen cycling in subalpine grasslands[END_REF][START_REF] Robson | Land use in subalpine grasslands affects nitrogen cycling via changes in plant community and soil microbial uptake dynamics[END_REF][START_REF] Legay | Comparison of inorganic nitrogen uptake dynamics following snowmelt and at peak biomass in subalpine grasslands[END_REF], and the longer residence time of unprocessed NO 3 atm illustrated by higher Δ 17 O-NO 3 -in this meadow (Table 2). For this subalpine plant species growing in nutrient-poor soils (Table 2), uptake of NO 3 atm appeared to be an important source of nutrients, a hypothesis also supported by higher ƒ atm in its leaves compared to D. glomerata (Figure 1c).

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The higher ƒ atm in all plant leaves after peak biomass may indicate a shift in subalpine plants NO 3 uptake pattern: root-based in spring after snowmelt, and leaf-based in summer when competition with microbial immobilization is stronger [START_REF] Legay | Comparison of inorganic nitrogen uptake dynamics following snowmelt and at peak biomass in subalpine grasslands[END_REF] and the foliar surface for atmospheric exchange is at a maximum.

It remains unclear why δ 15 N-NO 3 -in D. glomerata roots was lower than the estimated δ 15 N-NO 3 bio =

5.1 ‰ reported by Bourgeois et al., (2018a) at the same site (Figure 4a andc). Because these low δ 15 N-NO 3 -values span the reported δ 15 N range of NO 3 -produced from NH 4 + atm nitrification (Figure 4a and c), it could indicate that NH 4 + atm , along with NH 4 + mineralized from organic N (NH 4 + org ), is an important substrate for nitrification (Bourgeois et al., 2018a). Alternatively, low δ 15 N-NO 3 -could be linked to the low nitrification rates in subalpine meadows [START_REF] Robson | Neglect of mowing and manuring leads to slower nitrogen cycling in subalpine grasslands[END_REF], leading to the production of δ 15 N-depleted NO 3 -because of isotopic fractionation when the NH 4 + pool is only partially nitrified [START_REF] Mariotti | Experimental determination of nitrogen kinetic isotope fractionation: some principles; illustration for the denitrification and nitrification processes[END_REF]. Conversely, we found 15 N-enriched NO 3 -in F. paniculata roots compared to NO 3 bio (Figure 4a), which could have resulted from several distinct or concomitant sources and processes:

(i) NO 3 -could be derived from manure as suggested by isotopic source partitioning (Figure 4a andc), but this is improbable considering the low occurrence of grazing and the absence of manuring in unterraced grasslands.

(ii) an association of F. paniculata with mycorrhizal fungi [START_REF] Mouhamadou | Differences in fungal communities associated to Festuca paniculata roots in subalpine grasslands[END_REF][START_REF] Binet | Effects of mowing on fungal endophytes and arbuscular mycorrhizal fungi in subalpine grasslands[END_REF] may impact N and O isotopes of NO 3 -. However, the NR capacity of mycorrhizal fungi is expressed only in the presence of NO 3 -and absence of NH 4 + (Liu et al., 2014 and literature therein), which is not the case here. In addition, a review on the isotopic imprint of plant-fungal associations showed that mycorrhizal plants are 15 N-depleted (total N) compared to their fungal symbionts [START_REF] Hobbie | Nitrogen isotopes link mycorrhizal fungi and plants to nitrogen dynamics[END_REF].

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(iii) NO 3 -could stem from the storage of nutrients from previous years in tiller bases [START_REF] Baptist | Carbohydrate and nitrogen stores in Festuca paniculata under mowing explain dominance in subalpine grasslands[END_REF], 15 N-enriched by assimilation. This assumption is well supported by the slow N turnover in plant tissues in subalpine Poaceae [START_REF] Pornon | Contribution of plant species to the high N retention capacity of a subalpine meadow undergoing elevated N deposition and warming[END_REF], and by the year-round elevated δ 15 N and δ 18 O in F. paniculata (Figure 1d ande).

Whereas these results suggest a potentially significant correlation between root uptake of NO 3 atm and NO 3 bio and land use (probably due to different N speciation and availability; Table 2), the correlation between land-use trajectories and foliar uptake of NO 3 atm was not evident in this study. The proportion of NO 3 atm in leaves seemed to increase as the intensity of the land management decreased (along with N availability in soils, Table 2), although this result was not statistically significant (Figure 1c). A similar pattern was observed for δ 18 O-NO 3 -(Figure 1d) and δ 15 N-NO 3 -(Figure 1e).

Additional work will be needed to further investigate this question.

Uncertainties and future work

In this work, isotopic mass balance calculations were limited to two grass species that were assumed to be most representative of plant communities in subalpine environments. Therefore, the variability in the significance of foliar absorption across the wide range of subalpine plant species was not specifically studied here. Since our study site is remote and far from N emissions, it would be interesting to study how foliar uptake varies with shifting abundances of atmospheric Nr species, for instance close to urban centers (with high NO 2 ) or agricultural zones (with high NH 3 ). Similar broadscale studies like that by [START_REF] Liu | Nitrate is an important nitrogen source for Arctic tundra plants[END_REF] would be necessary to assess the variability of foliar NO 3 atm uptake across ecosystems, and to elucidate the drivers of this variability (e.g., water content, N availability, N speciation in the atmosphere and in the soils). There is now compelling evidence that forest canopies exert a strong control on the proportion and speciation of atmospheric N reaching soils [START_REF] Templer | Use of mixed ion exchange resin and the denitrifier method to determine isotopic values of nitrate in atmospheric deposition and canopy throughfall[END_REF][START_REF] Rao | Atmospheric nitrogen inputs and losses along an urbanization gradient from Boston to Harvard Forest[END_REF][START_REF] Guerrieri | Isotopic evidence for the occurrence of biological nitrification and nitrogen deposition processing in forest canopies[END_REF]. Quantifying foliar uptake of atmospheric N from forest canopies would be a significant step toward understanding how elevated N

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deposition affects forested ecosystems. Another important aspect is the need to account for foliar NO 3 atm uptake in N-limited environments subject to global changes. For instance, [START_REF] Mayor | Elevation alters ecosystem properties across temperate treelines globally[END_REF] demonstrated that global temperature increases may lead to a strong N/P decoupling in alpine soils above the tree line, which could be further exacerbated by foliar NO 3 atm uptake, a variable not considered to date.

In the present study, we focused on the foliar uptake of NO 3 atm . However, foliar uptake is also affected by other N species like PAN [START_REF] Sparks | The uptake of gaseous organic nitrogen by leaves: A significant global nitrogen transfer process[END_REF], other organic N compounds [START_REF] Lockwood | Foliar uptake of atmospheric organic nitrates[END_REF] or NH 3 [START_REF] Hanstein | Uptake and utilization of atmospheric ammonia in three native Poaceae species: leaf conductances, composition of apoplastic solution and interactions with root nitrogen supply[END_REF][START_REF] Pornon | Contribution of plant species to the high N retention capacity of a subalpine meadow undergoing elevated N deposition and warming[END_REF]. The 4-16 % range for foliar uptake of NO 3

atm is but one part of the total foliar N uptake. Constraining the foliar uptake of these N species should be another important focus of future studies, especially given that their relative abundance in the atmosphere is changing [START_REF] Li | Increasing importance of deposition of reduced nitrogen in the United States[END_REF].

Conclusion

The present study has advanced the current understanding of the potential NO

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Soils extracts

[NO 3 -] (μg.g -1 dw) [NH 4 + ] (μg.g -1 dw) Δ 17 O (‰) δ 18 O (‰) δ 15 N (‰)
UU meadows (n=18) 14.6 ± 19.0 13.0 ± 13.7 0.9 ± 0.6 -4.3 ± 6.8 -5.1 ± 6.6

TU meadows (n=21) 58.9 ± 46.9 17.5 ± 29.9 0.5 ± 0.8 -10.3 ± 6.6 -1.3 ± 9.6 TM meadows (n=21) 52.3 ± 48.7 6.2 ± 4.0 0.3 ± 0.4 -10.9 ± 5.1 -2.6 ± 6.5

UU, Unterraced-Unmown; TU, Terraced-Unmown; TM, Terraced-Mown land-use trajectories.

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List of Figures

Figure 1 Annual In a) the isotopic ranges of NO 3 -produced rectangles (cf., [START_REF] Kendall | Tracing anthropogenic inputs of nitrogen to ecosystems[END_REF] ). In all panels, the red diamond corresponds to pass as measured by Bourgeois et al. (2018). The dash ect of NO 3 -assimilation on δ 18 O-NO 3 -(enrichment), δ 1 ffect). Measurements located within a mixing range ning a mixture of NO 3 atm and NO 3 bio . Data points locat nt NO 3 -samples that were isotopically fractionated (e.g the regression lines between sample isotopic values we wn). Note that each data point represents the mean valu .

NO 3 -

 3 atm in the nitrate pool of subalpine plant tissues, and ii) NO 3 -triple isotopes can be used to evaluate the significance of foliar N uptake and track its seasonal variability. To verify these hypotheses, we measured the concentration and triple isotopic composition (Δ 17 O, δ 18 O, and δ 15 Nthe concentration of ammonium (NH 4 + ), in precipitation, soils, and different tissues (roots and leaves) of two dominant subalpine plant species at the Lautaret pass in the central French

-

  samples per grassland. A total of 9 individual plants were thus collected per sampling date (3 grasslands × 3 individual plants). Between May and September 2016, we harvested either one tuft of D. glomerata or of F. paniculata from each subplot (including several tillers and the roots) depending on which species dominated the meadow. At the same time, we collected cylindrical soil cores (4.5 cm in diameter and 8 cm in length) from the same locations. Sampling was performed twice a month around midday until peak biomass was reached (from May to July), and continued on a monthly basis afterward (July through September). Potential diurnal variations of inorganic N (NO 3

  USGS 35) and their accepted values -were ± 0.5, 2.1, and 0.3 ‰ for Δ 17 O, δ 18 O, and δ 15 N of NO 3 -, respectively. Slopes and intercepts of the linear regressions were then used to convert raw sample data into calibrated values (VSMOW scale for O isotopes, and N 2 -air scale for N isotopes). Isotopic end-member mixing model While the oxygen isotopic composition of NO 3 bio follows the mass-dependent law δ 17 O ≈ 0.52 x δ 18 O (Miller, 2002), that of NO 3 atm differs as its production pathway involves O 3 , containing an excess of 17 O of 25-35 ‰ (Vicars & Savarino, 2014). As a result, 17 O excess (denoted by Δ 17 O, with Δ 17 O = δ 17 O -0.52 x δ 18 O) of NO 3 atm ranges from 20 to 35 ‰ (Savarino et al., 2013), whereas NO 3 -from

  Data correction for atmospheric influence Mole fractions of NO 3 -(ƒ atm and ƒ bio = 1 -ƒ atm ) can be used to remove the isotopic influence of NO 3 atm on NO 3 sample (with NO 3 sample = NO 3 atm + NO 3 bio ), which allows for a better interpretation of

  were positively correlated: Δ 17 O-NO 3 leaves-roots , and therefore ƒ uptake (Eq.4) increases with higher [NO 3 -

  δ 15 N-NO 3 -in D. glomerata roots (Figure 1b and e) could reflect its exploitative strategy, characterized by fast uptake (i.e., lower δ 15 N-NO 3 -due to frequent turn-over with new NO 3 bio ) and intensive NR rates (i.e., production of in-tissue NH 4 + ). The high [NH 4 + ] in D.

  3 -absorption pathways by roots or leaves and their seasonal variability in subalpine plants, and has stressed the practical use of NO 3 -triple-isotope analyses to further our understanding of the role played by plants in the N biogeochemical cycle. We demonstrated that Δ 17 O-NO 3 -in plant tissues is a powerful tool for constraining the direct foliar uptake of NO 3 atm : two dominant subalpine plant species yielded an estimated foliar uptake of NO 3 atm of 4-16 %. The simultaneous analysis of the triple isotopes of NO 3 in plant tissues also provided a new perspective on plant functional biology, most notably on the NR process. We were able to show that NO 3 -assimilation occurs in both the roots and the leaves of subalpine plants. Our findings also suggest a seasonal variability in the respective contribution of N uptake pathways (roots vs. leaves), with greater assimilation occurring in the leaves after peak biomass, although further studies on this topic are needed. Results from this study bring new insights on the role of vegetation in the N balance in high-altitude catchments. We believe that the isotopic This article is protected by copyright. All rights reserved. to roots (indicating foliar uptake of NO 3 atm ). In (b) NO 3 bio isotopic values that have been corrected for NO 3 atm contribution (see Methods section for details) are shown in red (in black are the uncorrected values). The linear regression slope of 0.99 highlights the additional assimilation step occurring in leaves. Note that each data point corresponds the mean value of three plant individuals per sampling date.

Figure 4

 4 Figure 4 Root (brown) and leaf (green) NO 3 -isotopic values showing (a) δ 18 O (‰) vs. δ 15 N (‰), (b) Δ 17 O (‰) vs. δ 18 O (‰) and (c) Δ 17 O (‰) vs. δ 15 N (‰). The three different plant species are represented by three different symbols (see legend). The plant species/land-use trajectory combinations are represented by three different symbols (see legend). TM refers to Terraced-Mown land-use, TU to Terraced-Unmown land-use, and UU to Unterraced-Unmown land-use. In (a) the isotopic ranges of NO 3 -produced from different ammonium sources are indicated by rectangles (cf., (Kendall et al., 2007)). The colors correspond to: grey = NO 3 -from atmospheric ammonium (NH 4 + atm ), turquoise = NO 3 -from mineralized organic matter (NH 4 + org ), beige = NO 3 -from manure NH 4 + . In (b) and (c), the same grey, turquoise and beige colors are used to delineate the areas of mixing between the atmospheric end-member (i.e., 100% NO 3 atm ), represented by the blue diamond, and the other NO 3 -sources mentioned above (i.e., 100% NO 3 bio ). Note the overlapping mixing ranges in (b). The atmospheric end-member (blue diamond) represents to the mean isotopic values of deposited NO 3 atm at the Lautaret pass (Table1). In all panels, the red diamond corresponds to the isotopic values of NO 3 bio at the Lautaret pass as measured byBourgeois et al. (2018). The dashed arrows indicate the expected isotopic effect of NO 3 -assimilation on δ 18 O-NO 3 -(enrichment), δ 15 N-NO 3 -(enrichment), and Δ 17 O-NO 3 -(no effect). Measurements located within a mixing range correspond to plant NO 3 -samples containing a mixture of NO 3 atm and NO 3 bio . Data points located outside a mixing range correspond to plant NO 3-samples that were isotopically fractionated (e.g., through assimilation). When significant, the regression lines between sample isotopic values were plotted for leaves (green) and roots (brown). Note that each data point represents the mean value of three plant individuals per sampling date.

Figure 2 Figure 3 Figure 4

 234 Figure 1 Annual mean a) [NO 3 -] (μg.g -1 d ƒ atm (%, the proportion of NO 3 atm ), d) δ 18 O paniculata leaves (n = 55 and 33 for conc and 47 for concentration and isotopic data Box plots show the mean, as well as the 2 differences between plant species for the significant differences between plant tissu p<0.001). dw), b) [NH 4 + ] (μg.g -1 dw), c) Δ 17 O (‰) and correspon O (‰) and e) δ 15 N (‰) of NO 3 -in D. glomerata and F centration and isotopic data, respectively) and roots (n a, respectively), sampled between May and September 25 th and 75 th percentiles. Different letters indicate signi same biological tissue (leaves or roots). Asterisks indi ues for a given species (* for p<0.05, ** for p<0.01, ** nding F. = 56 r 2016. ificant icate ** for

  b) and c), the same grey mixing between the atmospheric end-mem and the other NO 3 -sources mentioned abo in b). The atmospheric end-member (range correspond to plan through assimilation). When significant, t plotted for leaves (green) and roots (brow three plant individuals per sampling date.

  showing a) δ 18 O (‰) vs. δ 15 N (‰ vs. δ 15 N (‰). The plant species/land-use trajectory fferent symbols (see legend). TM refers to Terraced-M use, and UU to Unterraced-Unmown land-use. d from different ammonium sources are indicated by he colors correspond to: grey = NO 3 -from atmospheric from mineralized organic matter (NH 4 + org ), beige = NO y, turquoise and beige colors are used to delineate the a mber (i.e., 100% NO 3 atm ), represented by the blue diam ove (i.e., 100% NO 3 bio ). Note the overlapping mixing e diamond) represents to the mean isotopic values of able S1

‰

Table 1

 1 Mean NO 3 -and NH 4 + concentrations and NO 3 -isotopic values, and associated standard deviation, in wet and dry deposition collected at the Lautaret Pass from May to September 2016.Under is shown the inferred isotopic values for this study atmospheric end-member.

	Deposition	[NO 3 -] (mg.L -1 )	[NH 4 (mg.L -1 ) + ]	Δ 17 O (‰)	δ 18 O (‰)	δ 15 N (‰)
	Dry deposition (n=10)	-	-	25.1 ± 2.7	69.1 ± 5.7	-5.1 ± 6.2
	Wet deposition (n=9)	5.5 ± 3.2	0.5 ± 0.2	23.6 ± 2.0	63.8 ± 6.0	-7.1 ± 1.7
	Atmospheric end-member	-	-	24.4 ± 2.4	66.6 ± 5.8	-6.0 ± 4.1

Table 2

 2

	Weighted mean annual concentration of NO 3	-and NH 4	+ (μg.g -1 dw) and concentration-
	weighted isotopic values of NO 3	-(‰) in soil extracts and associated standard deviation.

Acknowledgments

This study was supported by grants from the Labex OSUG@2020 ("Investissements d'avenir" -ANR10 LABX56), the ARC -Environnement Région Rhone-Alpes and the Grenoble-Chambéry DIPEE CNRS. It was conducted at the Lautaret Garden (Station Alpine J. Fourier, Univ. Grenoble Alpes, CNRS, 38000 Grenoble, France), member of AnaEE-France (ANR-11-INBS-0001AnaEE-Services) and of the eLTER-Europe network (Univ. Grenoble Alpes, CNRS, LTSER Zone Atelier Alpes, 38000 Grenoble, France). We would like to thank N. Deschamps, C. Nesti, E. Vince and C.

Arnoldi for helping with field and laboratory work and acknowledge J. Renaud for help with SIG and B. Mouhamadou for insightful discussion about plant-mycorrhizae associations. Finally, we acknowledge H. Angot and J. Peischl for their careful and precious English-editing of this manuscript.

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approach used here could help improve the predictive capabilities of ecosystem models with regard to changing N deposition levels, if used on more plant species and at a larger scale.

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Author contribution

IB, J-CC and JS designed the experiment. IB carried out the field work. IB and NC performed the laboratory analyses. IB analyzed the data. IB, JCC and JS discussed the results. IB wrote the paper, with revisions from JCC and JS. glomerata plant leaves and roots.

Supplementary Figures

List of Figures


Didier F Pisani
email: pisani@unice.fr

Valentin Barquissau

Jean-Claude Chambard

Diane Beuzelin

Rayane A Ghandour

Maude Giroud

Aline Mairal

Sophie Pagnotta

Saverio Cinti

Dominique Langin

Ez-Zoubir Amri
email: amri@unice.fr

Mitochondrial fission is associated with UCP1 activity in human brite/beige adipocytes

Keywords: hMADS, UCP1, DRP1, brite/beige adipocyte, mitochondria

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

Overweight and obesity are a consequence of an energy imbalance, which leads to an increase in white fat mass. Brown adipocytes and their activation are promising targets for the treatment of human obesity [START_REF] Kopecky | Expression of the mitochondrial uncoupling protein gene from the aP2 gene promoter prevents genetic obesity[END_REF][START_REF] Langin | Recruitment of brown fat and conversion of white into brown adipocytes: strategies to fight the metabolic complications of obesity?[END_REF], because these cells show high metabolic activity under stimulated conditions in both rodents and adult humans [START_REF] Cannon | Brown adipose tissue: function and physiological significance[END_REF][START_REF] Chondronikola | Brown Adipose Tissue Improves Whole Body Glucose Homeostasis and Insulin Sensitivity in Humans[END_REF][START_REF] Feldmann | UCP1 ablation induces obesity and abolishes diet-induced thermogenesis in mice exempt from thermal stress by living at thermoneutrality[END_REF][START_REF] Ouellet | Brown adipose tissue oxidative metabolism contributes to energy expenditure during acute cold exposure in humans[END_REF][START_REF] Yoneshiro | Recruited brown adipose tissue as an antiobesity agent in humans[END_REF]. Thermogenic adipocytes are found in discrete brown adipose tissue (BAT) depots as brown adipocytes and are interspersed in white fat depots as 'brite/beige' adipocytes [START_REF] Enerback | Human brown adipose tissue[END_REF][START_REF] Harms | Brown and beige fat: development, function and therapeutic potential[END_REF]. Depending on their anatomical location, thermogenic adipocytes found in humans display a molecular signature of either classical brown or brite/beige fat cells [START_REF] Cypess | Identification and importance of brown adipose tissue in adult humans[END_REF][START_REF] Cypess | Anatomical localization, gene expression profiling and functional characterization of adult human neck brown fat[END_REF][START_REF] Jespersen | A classical brown adipose tissue mRNA signature partly overlaps with brite in the supraclavicular region of adult humans[END_REF][START_REF] Sharp | Human BAT possesses molecular signatures that resemble beige/brite cells[END_REF][START_REF] Van Marken Lichtenbelt | Cold-activated brown adipose tissue in healthy men[END_REF][START_REF] Virtanen | Functional brown adipose tissue in healthy adults[END_REF][START_REF] Wu | Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human[END_REF][START_REF] Zingaretti | The presence of UCP1 demonstrates that metabolically active adipose tissue in the neck of adult humans truly represents brown adipose tissue[END_REF].

Given the abundance of white adipose tissue (WAT) in humans, there is growing clinical interest in understanding how brite adipocytes develop, especially since their emergence has been associated with protection against obesity and metabolic dysfunctions in rodents [START_REF] Cederberg | FOXC2 is a winged helix gene that counteracts obesity, hypertriglyceridemia, and diet-induced insulin resistance[END_REF][START_REF] Cohen | Ablation of PRDM16 and Beige Adipose Causes Metabolic Dysfunction and a Subcutaneous to Visceral Fat Switch[END_REF][START_REF] Leonardsson | Nuclear receptor corepressor RIP140 regulates fat accumulation[END_REF][START_REF] Seale | Prdm16 determines the thermogenic program of subcutaneous white adipose tissue in mice[END_REF]. Brite adipocytes are derived either from progenitors, or for a large proportion of cells, through direct conversion of mature white adipocytes [START_REF] Lee | Cellular origins of cold-induced brown adipocytes in adult mice[END_REF][START_REF] Rosenwald | Bi-directional interconversion of brite and white adipocytes[END_REF][START_REF] Wang | Tracking adipogenesis during white adipose tissue development, expansion and regeneration[END_REF]. This mechanism of conversion highlights the plasticity of adipocytes in response to specific physiological situations [START_REF] Cinti | Transdifferentiation properties of adipocytes in the adipose organ[END_REF][START_REF] Lee | Adipose tissue plasticity from WAT to BAT and in between[END_REF]. Compared to white adipocytes, brown and brite adipocytes possess a higher mitochondrial content and express the uncoupling protein 1 (UCP1), which facilitates a proton leak and the uncoupling of the respiratory chain [START_REF] Nedergaard | UCP1: the only protein able to mediate adaptive non-shivering thermogenesis and metabolic inefficiency[END_REF][START_REF] Shabalina | UCP1 in brite/beige adipose tissue mitochondria is functionally thermogenic[END_REF]. This phenomenon results in a high oxidative capacity and increased energy expenditure, which leads to thermogenesis. Conversely, white adipocytes contain fewer mitochondria but their function is essential for adipocyte differentiation and function [START_REF] Vernochet | Adipose-specific deletion of TFAM increases mitochondrial oxidation and protects mice against obesity and insulin resistance[END_REF]. Mitochondria are dynamic organelles that display morphological changes, such as fusion/fission events, which represent an adaptation to the needs of the cell [START_REF] Liesa | Mitochondrial dynamics in the regulation of nutrient utilization and energy expenditure[END_REF]. The increased mass of mitochondria, important for brown adipocyte function, and involves a coordinated interaction between the nuclear and mitochondrial genomes. Mitochondria continuously undergo fusion and fission events: conditions requiring high mitochondrial ATP synthesis are associated with mitochondrial elongation [START_REF] Liesa | Mitochondrial dynamics in the regulation of nutrient utilization and energy expenditure[END_REF][START_REF] Gomes | During autophagy mitochondria elongate, are spared from degradation and sustain cell viability[END_REF], whereas, bioenergetic stress induces mitochondrial fragmentation, which may lead to apoptosis [START_REF] Liesa | Mitochondrial dynamics in the regulation of nutrient utilization and energy expenditure[END_REF][START_REF] Molina | Mitochondrial networking protects beta-cells from nutrient-induced apoptosis[END_REF]. However, mitochondrial fission may not be deleterious per se in brown adipocytes. Indeed, adrenergic stimulation of rodent brown adipocytes induces substantial changes in the mitochondrial architecture, including a high rate of fragmentation [START_REF] Twig | The interplay between mitochondrial dynamics and mitophagy[END_REF][START_REF] Wikstrom | Hormone-induced mitochondrial fission is utilized by brown adipocytes as an amplification pathway for energy expenditure[END_REF]. This phenomenon favors enhanced mitochondrial uncoupling and energy expenditure. Taken together, these observations highlight the importance of mitochondrial biogenesis and dynamics in the function of brite and brown adipocytes.

The in vivo discrepancies between mice and humans in terms of the amount and location of brite fat cells, as well as the difficulty in routinely obtaining fresh human samples, underline the need to decipher the mechanisms regulating brite adipocyte formation and activity in human cells [START_REF] Beranger | In vitro brown and "brite"/"beige" adipogenesis: human cellular models and molecular aspects[END_REF].

Herein, we have characterized the properties of the mitochondria during the conversion of human white to brite adipocytes using the human Multipotent Adipose Derived Stem Cell (hMADS) model [START_REF] Elabd | Human multipotent adipose-derived stem cells differentiate into functional brown adipocytes[END_REF]. We found that human brite adipocyte mitochondria had an enhanced oxidative capacity and sustained fission, which was driven by DRP1.

Materials and Methods

Reagents

Culture media and buffer solutions were purchased from Lonza Verviers (Verviers, Belgium), fetal bovine serum, insulin and trypsin from Invitrogen (Cergy Pontoise, France), hFGF2 from Peprotech (Neuilly sur Seine, France). Other reagents were from Sigma-Aldrich Chimie (Saint-Quentin Fallavier, France).

hMADS cell culture

The establishment and characterization of hMADS cells has been described [START_REF] Elabd | Human multipotent adipose-derived stem cells differentiate into functional brown adipocytes[END_REF][START_REF] Rodriguez | Adipocyte differentiation of multipotent cells established from human adipose tissue[END_REF][START_REF] Rodriguez | Transplantation of a multipotent cell population from human adipose tissue induces dystrophin expression in the immunocompetent mdx mouse[END_REF][START_REF] Zaragosi | Autocrine fibroblast growth factor 2 signaling is critical for self-renewal of human multipotent adipose-derived stem cells[END_REF]. Cells were used between passages 14 and 25, all experiments were performed at least 3 times and cells were free of viruses and mycoplasma. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 15 mM Hepes and 2.5 ng/ml hFGF2. Cells were triggered for differentiation on the second day post-confluence (designated as day 0) in DMEM/Ham's F12 media supplemented with 10 µg/ml transferrin, 10 nM insulin, 0.2 nM triiodothyronine, 1 µM dexamethasone and 500 µM isobutyl-methylxanthine. Two days later, the medium was changed (dexamethasone and isobutyl-methylxanthine removed) and 100 nM rosiglitazone were added. At day 9, rosiglitazone was withdrawn to enable white adipocyte differentiation. Rosiglitazone (100 nM) or GW7647 (300 nM) were added at day 14 to promote white to brite adipocyte conversion and cells were used at day 18.

Transfection experiments were performed using HiPerfect (QIAGEN, France) at day 14 of differentiation. Cells were incubated with a mixture containing HiPerfect and siRNA (50 nM) in DMEM. Four hours later, it was supplemented with F12 medium containing 20 µg/ml transferrin, 20 nM insulin, and 0.4 nM triiodothyronine. siRNA against human DRP1 is from Ambion (Life Technologies, Courtaboeuf, France) and validated to specifically target DRP1 (ID #: s19560).

Western blot analysis

Proteins were extracted and blotted as previously described [START_REF] Pisani | The omega6-fatty acid, arachidonic acid, regulates the conversion of white to brite adipocyte through a prostaglandin/calcium mediated pathway[END_REF]. Primary antibody incubation was performed overnight at 4°C (anti-UCP1, Calbiochem, #662045, dilution 1:750; and tubulin, Sigma #T5201, dilution 1:2000; anti-DRP1, Cell Signaling #5391, dilution 1:1000; anti-phosphoDRP1(Ser616), Cell Signaling #4494, dilution 1:1000; anti-citrate synthase, Abcam #ab96600, dilution 1:10000) and then detected with HRP-conjugated anti-rabbit or anti-mouse immunoglobulins (Promega, Charbonnières-les-Bains, France). Detection was performed using Chemiluminescent HRP Substrate (Millipore, Molsheim, France). OD band intensities were evaluated using PCBas Software.

For mitochondrial complex quantitation, equal amounts of cell proteins were separated using gradient SDS-PAGE (10-20%) and blotted onto nitrocellulose membranes. Saturated membranes were incubated overnight with a 1:1000 dilution of total OXPHOS human Western blot antibody cocktail (#MS601, Mitosciences) followed by 60 min incubation with HRPconjugated anti-mouse immunoglobulins. Chemiluminescence obtained after addition of Clarity ECL Western blotting substrate (BioRad, Marnes-la-Coquette, France) was detected using a ChemiDoc MP Imaging System (Bio-Rad) and quantified with Image Lab 5.0 software (Bio-Rad).

Immunostaining analysis

Cells were fixed with PAF 4% for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, and then sequentially incubated with primary antibody overnight at 4°C (anti-UCP1, Calbiochem, #662045, dilution 1:100; anti-TIMM23, BD Biosciences #611222, dilution 1:500; anti-cytochrome C, SantaCruzBT #sc-13560, dilution 1:100) and with the relevant secondary antibody coupled to Alexa-488 or Alexa-594 (Invitrogen, dilution 1:500) for 30min at RT. Cells were finally mounted and visualized with an Axiovert microscope (Carl Zeiss, Le Pecq, France) under oil immersion and pictures were captured and treated with AxioVision software (Carl Zeiss). The mitochondrial network was analyzed using Fiji software [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF].

Isolation and analysis of RNA

These procedures were carried out according to MIQE recommendations [START_REF] Bustin | The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments[END_REF]. Total RNA was extracted using TRI-Reagent (Euromedex, Souffelweyersheim, France) according to the manufacturer's instructions. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was conducted as described previously [START_REF] Pisani | Differentiation of Human Adipose-Derived Stem Cells into "Brite" (Brown-in-White) Adipocytes[END_REF]. The expression of selected genes was normalized to that of TATA-box binding protein (TBP) and 36B4 housekeeping genes and then quantified using the method. Primer sequences are available upon request.

Mitochondrial DNA quantification

DNA was extracted using a DNA extraction kit (Macherey-Nagel EURL, France). 2 ng of the total DNA were used for qPCR analysis, and the mitochondrial DNA content was calculated from the ratio of the DNA of the NADH dehydrogenase subunit 1 gene (mitochondrial gene) to that of lipoprotein lipase gene (a nuclear gene) as previously described [START_REF] Bordicchia | Cardiac natriuretic peptides act via p38 MAPK to induce the brown fat thermogenic program in mouse and human adipocytes[END_REF].

Oxygen consumption analysis

For respiration analysis, hMADS cells were seeded in a 24 multi-well plate (Seahorse) and differentiated as described previously [START_REF] Loft | Browning of human adipocytes requires KLF11 and reprogramming of PPARgamma superenhancers[END_REF]. The oxygen consumption rate (OCR) of 18-day-old differentiated cells was determined using an XF24 Extracellular Flux Analyzer (Seahorse Bioscience). Uncoupled and maximal OCR were determined using oligomycin (1.2 µM) and FCCP (1 µM). Rotenone and Antimycin-A (2 µM each) were used to inhibit mitochondrial respiration. All parameters were calculated as described previously [START_REF] Brand | Assessing mitochondrial dysfunction in cells[END_REF].

Electron microscopy

hMADS adipocytes were fixed in 1.6% glutaraldehyde in 0.1 M phosphate buffer. They were rinsed with cacodylate buffer, then post-fixed in osmium tetroxide (1%) and reduced with potassium ferrycyanide for 1 h. After a water wash, cells were dehydrated with several incubations in increasing concentrations of ethanol and embedded in Epon resin. Eighty nanometer sections were contrasted with uranyl acetate and lead citrate and then observed with an electron microscope (JEOL JEM 1400) operating at 100 kV. Electron microscopy pictures displaying omental adipose tissue of patients with pheocromocytoma were described in a previous study [START_REF] Frontini | Whiteto-brown transdifferentiation of omental adipocytes in patients affected by pheochromocytoma[END_REF].

Statistical analysis

Data were expressed as mean values ± SEM. They were analyzed using InStat3 software (GraphPad Software, CA, USA) by a one-way ANOVA followed by a Student-Newman-Keuls post-test, or a Student's t-test to assess any statistical differences between experimental groups.

Differences were considered statistically significant when p < 0.05.

Results

hMADS brite adipocytes display increased mitochondriogenesis and mitochondrial activity

hMADS cells are able to differentiate into white adipocytes and then convert into functional brite adipocytes when they are treated with rosiglitazone or GW7647, specific agonists of and respectively [START_REF] Pisani | Differentiation of Human Adipose-Derived Stem Cells into "Brite" (Brown-in-White) Adipocytes[END_REF][START_REF] Barquissau | White-to-brite conversion in human adipocytes promotes metabolic reprogramming towards fatty acid anabolic and catabolic pathways[END_REF]. The expression of genes encoding UCP1 (Figure 1a-b) and other classical brown/brite adipocyte markers, such as carnitine-palmitoyltransferase 1B (CPT1B) and cell death-inducing DFFA-like effector A (CIDEA) (Figure 1c) was significantly higher in hMADS brite adipocytes upon or activation. Likewise, the expression of genes encoding mitochondrial membrane components and respiratory chain subunits displayed similar trends or significant increases the activated hMADS brite adipocytes (Figure 1c). Mitochondrial DNA content, a marker of mitochondriogenesis, was higher in brite compared to white adipocytes (Figure 2a). Levels of the respiratory chain complex proteins were increased (Figure 2b, Supplementary Figure 1a). Specifically, complex II, III and V protein contents were higher in brite adipocytes, while no significant difference was found for complexes I and IV (Figure 2b).

These results suggested an increase in mitochondrial activity in hMADS brite adipocytes.

Oxygen consumption analysis (Supplementary Figure 1b) demonstrated that hMADS brite adipocytes displayed higher basal and maximal mitochondrial respiration compared to white fat cells (Figure 2c), as expected given the increase in uncoupled respiration typical of UCP1 activity (Figure 2c). This increased uncoupling respiration was mainly due to the higher expression of UCP1 in brite fat cells. Expression of other protonophoric proteins (i.e. UCP2, UCP3, SLC25A4, SLC25A5 and SLC25A6) was low and/or unchanged in white and brite adipocytes (data not shown and see references [START_REF] Pisani | Differentiation of Human Adipose-Derived Stem Cells into "Brite" (Brown-in-White) Adipocytes[END_REF][START_REF] Loft | Browning of human adipocytes requires KLF11 and reprogramming of PPARgamma superenhancers[END_REF]).

hMADS brite adipocytes display UCP1 + round-shaped mitochondria

Mitochondria are able to modulate their morphology from a tubular (fused) to a round (fragmented) shape. These morphological adaptations relate to mitochondrial division and, in various situations, correlate with changes in activity or stress. A recent study performed in rodent brown adipocytes showed that the mitochondria acquired a round shape upon UCP1 activation [START_REF] Wikstrom | Hormone-induced mitochondrial fission is utilized by brown adipocytes as an amplification pathway for energy expenditure[END_REF]. Thus, the mitochondrial morphology was analyzed by transmission electron microscopy.

Undifferentiated hMADS cells contained a majority of long tubular mitochondria, while hMADS white adipocytes displayed a mix of tubular and partially fragmented mitochondria (Figure 3a and 3b). In contrast, brite adipocytes exhibited round-shaped mitochondria, suggesting sustained fission activity (Figure 3c and3d). Moreover, mitochondria of brite adipocytes displayed laminar cristae (i.e. arranged from side to side of the organelle) (Figure 3c and3d) compared to the white ones, which were shorter and linear (Figure 3a and 3b) [START_REF] Frontini | Distribution and development of brown adipocytes in the murine and human adipose organ[END_REF]. Analysis of each condition showed that 81 ± 11 % and 89 ± 6 % (mean ± sem) of the mitochondria displayed a round-shaped morphology in brite adipocytes treated with rosiglitazone and GW7647, respectively. In contrast, only 22 ± 6 % exhibited this morphology in white adipocytes.

This mitochondrial morphology looked like the one described in vivo in murine activated brown adipocytes [START_REF] Wikstrom | Hormone-induced mitochondrial fission is utilized by brown adipocytes as an amplification pathway for energy expenditure[END_REF]. To determine the relevance of these results in vivo in humans, we analyzed omental WAT of patients with pheocromocytoma by transmission electron microscopy [START_REF] Frontini | Whiteto-brown transdifferentiation of omental adipocytes in patients affected by pheochromocytoma[END_REF]. In these patients, WAT surrounding the tumor displayed activated brite adipocytes due to the high catecholamine secretion by the tumorigenic adrenal gland [START_REF] Frontini | Whiteto-brown transdifferentiation of omental adipocytes in patients affected by pheochromocytoma[END_REF]. Interestingly, mitochondria of these adipocytes displayed the same fragmented morphology and laminar cristae found in hMADS brite adipocytes, which highlights the relevance of this in vitro model (Figure 3e).

We performed immunostaining experiments in hMADS adipocytes, to assess the co-localization of UCP1 and TIMM23 (an inner mitochondrial membrane protein used as a mitochondrial marker). Interestingly, UCP1-positive mitochondria displayed a fragmented state while tubular structures were observed in UCP1-negative mitochondria (Figure 4a). These observations were confirmed using another mitochondrial marker, cytochrome c (Figure 4b). Analysis of the mitochondrial morphology in UCP1-positive and UCP1-negative cells showed that up to 90% of UCP1-positive adipocytes only contained mitochondria with a round-shaped morphology, while the remaining were of both phenotype (Figure 4d and Supplementary Figure 2). In contrast, 60%

of the mitochondria in UCP1-negative adipocytes adopted a tubular network, and less than 5% displayed a fragmented phenotype (Figure 4d and Supplementary Figure 2).

hMADS brite adipocytes display increased DRP1 activation

To determine the mechanisms responsible for fragmentation of brite/beige adipocyte mitochondria, we first analyzed the expression levels of genes involved in mitochondrial fission and fusion. mRNA expression analysis using datasets from omics analyses, available from the Gene Expression Omnibus (GEO) [START_REF] Edgar | Gene Expression Omnibus: NCBI gene expression and hybridization array data repository[END_REF] under accession numbers GSE71293 [START_REF] Barquissau | White-to-brite conversion in human adipocytes promotes metabolic reprogramming towards fatty acid anabolic and catabolic pathways[END_REF] and GSE59703 [START_REF] Loft | Browning of human adipocytes requires KLF11 and reprogramming of PPARgamma superenhancers[END_REF], demonstrated no significant induction of fission gene expression, such as DNM1L (also named DRP1) or FIS1, nor were fusion and mitophagy genes such as OPA1, MFN2, PINK and PARK2 induced (Supplementary Figure 3a and3b).

As DRP1 was recently shown to control the fission of mitochondria in rodent brown adipocytes [START_REF] Wikstrom | Hormone-induced mitochondrial fission is utilized by brown adipocytes as an amplification pathway for energy expenditure[END_REF], we assessed the phosphorylation of DRP1 on serine 616 (known to activate the human protein) in human white and brite adipocytes. Western blotting experiments clearly showed an increase in DRP1 phosphorylation in both rosiglitazone and GW7647-derived brite adipocytes compared to white fat cells (Figure 4d and Supplementary Figure 4).

DRP1 knock-down impaired mitochondrial uncoupling respiration in brite adipocytes

To investigate the involvement of DRP1 in human brite adipocyte metabolism, we decreased DRP1 expression using a siRNA approach. hMADS cells were transfected at day 14 of differentiation with 50 nM of DRP1-targeted siRNA, or with a relevant siRNA control, and subsequently either converted into brite adipocytes or not. DRP1 protein levels decreased by 50% (Figure 5a and Supplementary Figure 5a), without affecting cell mitochondriogenesis per se, as citrate synthase and chain respiratory complex level were not affected (Figure 5b-c and Supplementary Figure 6). Additionally, DRP1 knock-down did not reduce adipogenesis and white to brite adipocyte conversion, as was demonstrated by the absence of significant changes in UCP1 mRNA and protein levels (Figure 5c, Supplementary Figure 6) in siDRP1-compared to siCTRL-treated cells. Similarly, CPT1B and perilipin 1 and 5 mRNA levels were not affected (Figure 5c). We then analyzed respiration in DRP1 knock-down cells (Figure 6a). hMADS brite adipocytes transfected with DRP1 siRNA displayed lower basal (Figure 6b) and uncoupled (Figure 6c) respiration compared to cells transfected with control siRNA. However, maximal respiration (Figure 6d) and the spare respiratory capacity (Figure 6e) were not significantly affected. Thus, a decrease in DRP1 expression disrupted mitochondrial UCP1-dependent oxygen consumption.

In brite adipocytes, UCP1 uncoupling activity is dependent on mitochondrial fatty acid transport, as fatty acids are both oxidized as a substrate [START_REF] Calderon-Dominguez | Fatty acid metabolism and the basis of brown adipose tissue function[END_REF] and required for UCP1 activation [START_REF] Shabalina | Within brown-fat cells, UCP1-mediated fatty acid-induced uncoupling is independent of fatty acid metabolism[END_REF].

Inhibition of CPT1B activity, the major fatty acid transporter of adipocyte mitochondria, by acute Etomoxir treatment (50 µM, 2 hours), reduced fatty acid oxidation and the UCP1 uncoupling activity in hMADS brite adipocytes (Figure 7a and [START_REF] Barquissau | White-to-brite conversion in human adipocytes promotes metabolic reprogramming towards fatty acid anabolic and catabolic pathways[END_REF][START_REF] Calderon-Dominguez | Carnitine Palmitoyltransferase 1 Increases Lipolysis, UCP1 Protein Expression and Mitochondrial Activity in Brown Adipocytes[END_REF]). CPT1B inhibition did not affect expression of mitochondrial proteins such as citrate synthase, UCP1 and respiratory chain complex proteins (Figure 7b and Supplementary Figure 6a and6b). Interestingly, the treatment of brite adipocytes with Etomoxir decreased DRP1 phosphorylation (Figure 7c and Supplementary Figure 6c). These results highlight the relationship between the DRP1 and UCP1 activities in hMADS brite adipocytes.

Discussion

Brown and brite adipocytes have drawn considerable attention because of their role in the control of energy balance and metabolism in humans. In vivo and in vitro studies in rodents have shed light on the mechanisms involved in differentiation, function and activation of these thermogenic-competent adipocytes. In this work, we have characterized the mitochondrial phenotype of human brite adipocytes using hMADS cells, which recapitulate as an in vitro model for the conversion of white into brite adipocytes [START_REF] Elabd | Human multipotent adipose-derived stem cells differentiate into functional brown adipocytes[END_REF][START_REF] Pisani | Differentiation of Human Adipose-Derived Stem Cells into "Brite" (Brown-in-White) Adipocytes[END_REF][START_REF] Barquissau | White-to-brite conversion in human adipocytes promotes metabolic reprogramming towards fatty acid anabolic and catabolic pathways[END_REF][START_REF] Ghandour | IPreceptor and PPARs trigger the conversion of human white to brite adipocyte induced by carbaprostacyclin[END_REF], which has been described in vivo in mouse white fat depots [START_REF] Rosenwald | Bi-directional interconversion of brite and white adipocytes[END_REF][START_REF] Barbatelli | The emergence of cold-induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown adipocyte transdifferentiation[END_REF].

The shift from a white-to brite-adipocyte phenotype involves profound changes in the mitochondrial metabolic state [START_REF] Shabalina | UCP1 in brite/beige adipose tissue mitochondria is functionally thermogenic[END_REF]. Expression and activation of UCP1 were followed by the increase of oxygen consumption, which was required to maintain ATP synthesis while uncoupling. In addition to higher uncoupling, this increased oxidative capacity in brite adipocytes relied upon active mitochondriogenesis (characterized by a higher mitochondrial DNA content), enhanced respiratory chain activity (characterized by increased maximal respiration and protein levels) and a modification in substrate preference towards fatty acids (increased levels of CPT1B, increased complex II respiratory component and fatty acid oxidation) [START_REF] Barquissau | White-to-brite conversion in human adipocytes promotes metabolic reprogramming towards fatty acid anabolic and catabolic pathways[END_REF]. Interestingly, the phenotypic modifications in brite adipocytes not only compensated for a decrease in ATP synthesis due to UCP1 uncoupling, but they also endowed brite fat cells with sensitivity to specific stimuli. This latter observation was clearly characterized by the enhanced spare respiratory capacity in brite adipocytes. This parameter illustrated the capacity for substrate supply and electron transport to respond to an increase in energy demand [START_REF] Brand | Assessing mitochondrial dysfunction in cells[END_REF]. White adipocytes displayed a very low spare respiratory capacity and thus a poor ability to increase their mitochondrial activity. In contrast, the brite adipocytes were able to sharply increase their oxygen consumption and mitochondrial activity in order to respond to specific stimulation, and thus to increase uncoupling and perform thermogenesis as expected.

In addition to the increased mitochondrial content, previous studies have documented an association between mitochondrial fragmentation and energy expenditure [START_REF] Liesa | Mitochondrial dynamics in the regulation of nutrient utilization and energy expenditure[END_REF][START_REF] Molina | Mitochondrial networking protects beta-cells from nutrient-induced apoptosis[END_REF], which has been confirmed recently in mouse-derived brown adipocytes [START_REF] Wikstrom | Hormone-induced mitochondrial fission is utilized by brown adipocytes as an amplification pathway for energy expenditure[END_REF]. In these cells, mitochondrial fragmentation enhanced mitochondrial uncoupling and energy expenditure and was triggered by the DRP1 activity through its phosphorylation at Ser600. Herein, we demonstrated that mitochondrial fragmentation was associated with UCP1 expression in human adipocytes, as UCP1-positive mitochondria preferentially displayed a round shape, while UCP1-negative mitochondria formed tubular elongated structures. At this stage, hMADS cells treated by PPAR agonists expressed UCP1 and displayed an active brite phenotype, characterized by an increase in uncoupled respiration and oxidative phenotype [START_REF] Barquissau | White-to-brite conversion in human adipocytes promotes metabolic reprogramming towards fatty acid anabolic and catabolic pathways[END_REF]. We cannot rule out an effect of PPAR pathway activation on mitochondria fragmentation, due to the simultaneous occurrence of PPAR activation, UCP1 expression and activation in hMADS cells. Nevertheless, fragmented mitochondria were not found in undifferentiated hMADS cells treated by rosiglitazone, excluding an effect of PPAR activation on fission events (data not shown).

The fragmentation of mitochondria was associated with increased phosphorylation of DRP1 at Ser616, the equivalent of murine Ser600, which is known to promote mitochondrial fission by CDK1/cyclin B [START_REF] Knott | Impairing the mitochondrial fission and fusion balance: a new mechanism of neurodegeneration[END_REF]. Interestingly, in hMADS brite adipocytes and mouse brown adipocytes [START_REF] Wikstrom | Hormone-induced mitochondrial fission is utilized by brown adipocytes as an amplification pathway for energy expenditure[END_REF],

DRP1 regulates UCP1 activity as a decrease in DRP1 expression specifically disrupted mitochondrial uncoupling and associated basal respiration. Of note, fission did not modulate the total mitochondrial oxidative capacity of the cells, since DRP1 knock-down did not significantly change maximal respiration. Our results focused on Ser616 phosphorylation of DRP1, but we cannot exclude other modifications. Indeed, DRP1 can be modulated by numerous posttranslational modifications, such as Ser637 phosphorylation, which inhibits DRP1 activity [START_REF] Chang | Cyclic AMP-dependent protein kinase phosphorylation of Drp1 regulates its GTPase activity and mitochondrial morphology[END_REF],

and N-acetyl-glucosamine glycosylation, which potentiates DRP1 activity by inhibition of Ser637 phosphorylation [START_REF] Gawlowski | Modulation of dynamin-related protein 1 (DRP1) function by increased O-linked-beta-N-acetylglucosamine modification (O-GlcNAc) in cardiac myocytes[END_REF].

Our work is consistent with findings in mice, and confirms that mitochondrial fission in human adipocytes may be considered as a physiological adaptation, instead of a purely deleterious mechanism. In rodents, an increase of DRP1 phosphorylation was found in activated BAT and the blocking of mitochondrial fission mildly reduced uncoupled respiration [START_REF] Wikstrom | Hormone-induced mitochondrial fission is utilized by brown adipocytes as an amplification pathway for energy expenditure[END_REF]. Herein, we have shown in human brite fat cells that DRP1 phosphorylation correlated with UCP1 expression and that DRP1 knock-down abrogated the increase of uncoupled respiration, which returned to the level found in white adipocytes. DRP1 regulated the activity of brite and brown adipocytes, but did not appear to control the acquisition of the thermogenic phenotype. Indeed, in rodents, it has been demonstrated that mitochondrial fragmentation was only observed after catecholamine stimulation [START_REF] Wikstrom | Hormone-induced mitochondrial fission is utilized by brown adipocytes as an amplification pathway for energy expenditure[END_REF]. Similarly, we have shown that DRP1 knock-down did not modify UCP1 expression but only uncoupled respiration. Thus, the link between UCP1 activity and DRP1

activation is yet to be characterized.

As mitochondria morphology is controlled by cycles of fission and fusion events [START_REF] Twig | The interplay between mitochondrial dynamics and mitophagy[END_REF], and a fragmented morphology might be due to decreased fusion events in addition to increased fission. In contrast to results reported in rodents [START_REF] Wikstrom | Hormone-induced mitochondrial fission is utilized by brown adipocytes as an amplification pathway for energy expenditure[END_REF], our data are not in favor of inhibition of fusion events in this process, as several key components associated with fusion did not display any variation at either RNA or protein levels (data not shown). Nevertheless, further studies are required to characterize the mitochondrial fusion mechanism more precisely in hMADS adipocytes.

We also showed that mitochondrial fragmentation was accompanied by morphological changes in cristae during white to brite adipocyte conversion. Indeed, cristae within white adipocyte mitochondria tended to be linear and short while the cristae of brite adipocyte mitochondria displayed a longer, laminar and curved shape. Given the link between cristae structure and ATP synthase dimerization, these changes in the curvature of the cristae could be related to reduced coupled respiration in favor of uncoupled respiration [START_REF] Wikstrom | Hormone-induced mitochondrial fission is utilized by brown adipocytes as an amplification pathway for energy expenditure[END_REF][START_REF] Paumard | The ATP synthase is involved in generating mitochondrial cristae morphology[END_REF].

Conclusion

In conclusion, our data demonstrated that profound mitochondrial modifications occurred during human white to brite adipocyte conversion, and that the mitochondrial fission process described in mouse brown adipocytes also occurs in humans. blots are displayed in Supplementary Figure 5b and6.

CONFLICT OF INTEREST. The authors declare having no conflict of interest.

Figure 1 .Figure 2 .

 12 Figure 1. Characterization of white to brite adipocyte conversion. hMADS cells were

Figure 3 .

 3 Figure 3. Electron microscopy of white and brite adipocyte mitochondria. (a)

Figure 4 .

 4 Figure 4. Characterization of UCP1 + mitochondrial morphology. (a) and (b) hMADS cells

Figure 5 .

 5 Figure 5. DRP1 knock-down does not modify brite adipocyte formation. hMADS cells were

Figure 6 .

 6 Figure 6. DRP1 knock-down disrupted oxygen consumption linked to UCP1. hMADS

Figure 7 .

 7 Figure 7. CPT1B inhibition disrupts UCP1 activity and DRP1 phosphorylation. hMADS

ACKNOWLEDGEMENTS. This work was supported by CNRS. The authors acknowledge the CCMA (Centre Commun de Microscopie Appliquée, Université de Nice Sophia Antipolis, Microscopy and Imaging platform Côte d'Azur, MICA) and the Cytomed Platform from IRCAN.

DL is a member of Institut Universitaire de France. We thank Dr. Abby Cuttriss for editing the manuscript (http://univ-cotedazur.fr/services/visibilite-internationnale/en).