Gene •••

WP2: The effect of genetic type and feed restriction on the urine metabolome of growing rabbits

Mette Skou Hedemann (AU) lt is weil known that feed restriction has good effects on the health and productive performance of growing rabbits: lt reduces the risk of digestive diseases and improves feed efficiency. Th e purpose of the present experiment was to study the biological basis of feed efficiency on ad libitum or restricted feeding in two rabbit lines selected for different purpose: the Prat Line that is se lected for litter size at weaning and the Caldes Line that is selected for post-weaning growth rate. Th e metabolome is influenced by genetic as weil as environmental factors (e.g. diet, physical acttvity and medication) and consequently metabolomics is a valuable tool to study genetics and dietary interventions and the interaction between them. A total of 28 rabbits (14 Prat and 14 Caldes) were included in the experiment at weaning (32 d). The animais were fed a standard diet ( 16.401! cru de protein, 15.2% fibre, 4% fat) and water was always available. When rabbits were 53 d old, th ey were moved to metabolic cages and fed ad libitum until they were 63 d old. Then, they vere subjected to a 3-days period of feed restriction at 90-80 and 80-70% of maintenance requ irements in Caldes and Prat lines, respectively. This period was followed by a 4-days recovery period with ad libitum feeding and then, a new 3-days period of feed restriction at 60-50 and 50-40 % of maintenance requirements in Caldes and Prat lines, respectively, until they reached 73 da ys of age. Urine samples were collected on d 63 and 73 of lite. The samples were prepared for nontargeted LC-MS metabolomics and data were acquired on a UHPLC (Dionex, Sunnyvale, CA, USA) coupled to an Impact HD mass spectrometer (Bruker Daltonics, Bremen, Germany). The data were preprocessed using the XCMS R package and Principal Components Analys is (PCA) was performed using LatentiX 2.12. The PCA scores plots of the data in both positive and negatrve mode showed a clear separation between initial samples, with rabbits fed ad libitum , and samples taken at the end of the trial, just after the animais had been subjected to a strong fed restri cti on (40-50% of maintenance requirements) whereas there was no separation between the breeding lines. Several of the metabolites responsible for the separation between ad libitum and res ri cted feeding are a result of the microflora metabolism (e.g. p-cresol sulfate, hippuric acid, kymuren ic acid, and xanthurenic acid) furthermore the excretion of vitamins, betaines, and feed associated metabolites were changed as a result of the feeding regimen . ln conclusion , there was an effect of feed restriction on the urine metabolome of growing rabbits whereas no effect of genetic type was observed.

WP2: Variability of feed efficiency in growing pigs: towards predictive biomarkers

Florence Gondret (INRA) lmproving food efficiency (FE) is a major challenge for the pork industry, for both eco environmental reasons. lt is therefore essential to identify, on the one hand, the mechanisms involved in the inter-individual variability of FE, and on the other han d, f biomarkers of this trait. This study aims to identify genes whose expression levels • tissues contributed significantly to differences in FE. Different transcriptomic data se i • loin muscle or in the blood were obtained in pigs from a divergent selection on residual feed i ake.

The pigs were from the 6th to 8th generation of selection, weighed between 15 and 11•5 g, and were either feed-restricted or fed ad libitum with standard or fiber-rich diets (Vincent e . • .2015• . Meriot et al., 2016;;Gondret et al., 2017;Reis Furtado Campos et al, 2014). For each an• al, the gain to feed (G:F) and gain to feed energy (G:Fe) were calculated, and their genetic al es for residual feed intake (RFI) were obtained. ln muscle, the expression of 22 400 an notated o ecu lar probes was measured on 71 pigs. ln the blood, 6 000 annotated molecular probes were co monly found across the different datasets (n = 84 pigs). To identify the most important pred ictors of FE, Feed-a-Gene 3rd AM-24 1 h to 26 1 h April 2018-Newcastle, UK

tl• •

Random Forests (Breiman, 2001) were implemented with the Salford Predictive Modeler (V8.0) software. ln addition, sub-networks of co-expression of genes were elicited using WGCNA package (Langfelder and Horvath, 2007) implemented under R (V3.4.2) software, and related to G:F, G:Fe or RFI. ln the muscle, a subset of about fifty genes whose levels of expression explained respectively, 67% of the variability in F:G and 65% of the variability in RFI, was proposed. These genes participated in various biological processes such as the regulation of apoptosis (PSEN1 , CSRNP3 and HBXIP), inflammatory response {TLR1, SERPINA1 and CD40), regulation of glycogenogenesis (IGF2, PPARGC1A and ADIPOQ), intracellular transport (FRAS1, XP01, YWHAZ, STX7) or growth regulation (IGF2, PTK28, STAT5A, AVPR1A, ADAM10, ADAM17, RPS6KB1, PROX1 and NTN1 ). ln the blood, the expression levels of about fifty other genes whose expression levels were assessed during the post-weaning or early growth phases made it possible to predict (R 2 = 0.86) the G:F during the subsequent growth period. ln conclusion, these results confirmed the integrative nature of FE, because the proposed biomarkers were involved bath in productive and non-productive functions .

WP3: Representation of the transit and digestion of nutrients : transposition from a pig to a broiler model

Sonia Roger (INRA)

Regarding the context of animal production, monogastric livestock systems have to be more sustainable. For this, an increase in the efficiency of feed utilization is necessary. This requires a better understanding and prediction of the nutritional value of the feeds. Many experimental data provide the digestibility of nutrients in various conditions. However, very few studies describe the digestive mechanisms along the digestive tract and for ali the nutrients contained in the diet. However, it seems very important to be able to identify the reasons why a diet is digested more efficiently than another and what the causes of differences in digestibility are in the digestive system. Consequently, a modeling approach seems a good way to integrate information available, be able to represent the digestive processes and predict the digestibility. The work performed aimed the development of a madel representing the transit, hydrolysis, fermentation and absorption/excretion of the main nutrients along the digestive tract. A similar structure was adopted for pig and poultry and only selected madel parameters were adapted for each species. Parameters for pigs come from existing models and were then modified for the broiler based on biological knowledge and literature data. The transposition of a digestive madel for pigs to a broiler has been done step by step to preserve the genericity and make only those modifications that are really needed. Modifications were driven either for biological reasons or to improve the predictive capacity of the madel. For now, a generic madel has been developed for growing pigs and broilers.

WP3: An mechanisms perturbation

Gyërgy Këvér (KU)

interactive tool to simulate feed use and animal response to environmental Models are useful tools to predict animal response in a certain scenario, however, they need specifie platform to run. ln our project the aim was to develop software that is available for the users without knowledge on specifie statistical or modelling software. The Decision Support Software (DSS) is going to integrate 4 different mathematical models simulating the digestive mechanisms, the post absorptive nutrient partitioning, the animal's response to an unknown environmental perturbation, and the individual variance of a herd or a flock. The poster presents the concept and the potential functions of DSS developed in WP3. Based upon implementation of _ Feed-a-Gene 3rd AM-24 1 h to 26 1 h April 2018-Newcastle, UK


a fluorescence Fv'/Fm': Chlorophyll a fluorescence under light conditions

Keywords: Changement climatique, Sécheresse extrême, Mortalité des arbres, Perte de conductance hydraulique, Capacité à récupérer Climate change, Drought, Tree mortality, Hydraulic failure, Recovery capacity Fluorescein diacetate Fm: Maximal fluorescence Fv: Variable fluorescence Fv/Fm: Chlorophyll

A successful completion of any type of project requires the help from a large number of persons.

Therefore, I would like to thank all the technicians who taught me a lot about the techniques and the devices. I convey my sincere gratitude to Pierre who spent countless hours in front of the Micro-CT and who is probably tired of me for making last minutes changes. I would also like to thank Julien for his patience in teaching me about all the different techniques

Introduction

Forests, including woodlands and savannas, cover 30% of the world land's surface [START_REF] Fao | Global forest resources assessment 2005-progress towards sustainable forest management[END_REF]) and provide societies with ecosystem services such as biodiversity [START_REF] Ayres | Assessing the consequences of global change for forest disturbance from herbivores and pathogens[END_REF], timber production, watershed protection [START_REF] Allen | A global overview of drought and heat-induced tree mortality reveals emerging climate change risks for forests[END_REF] but also with carbon storage and its associated atmospheric feedbacks [START_REF] Reichstein | Climate extremes and the carbon cycle[END_REF]. In addition, forests also contribute to aesthetic and spiritual benefits which contribute greatly to the well-being of societies [START_REF] Allen | A global overview of drought and heat-induced tree mortality reveals emerging climate change risks for forests[END_REF].

Forests are composed by trees that live at the interface between the atmosphere and soil.

Even if their physical organization allows them to widely explore the environment they evolve in, i.e. branches and leaves assimilate carbon dioxide from the atmosphere while underground roots absorb water and mineral from the soil, trees are motionless and are therefore highly influenced by the weather where they grow. Because water requirements for a single tree could reach up to 50L per day (for conifers) and up to 500L per day (for angiosperms) [START_REF] Sperry | Safety and efficiency conflicts in hydraulic architecture: Scaling from tissues to trees[END_REF], drought is one of the main limiting factors for forests survival and composition (Anderegg et al. 2012b). In fact, numerous studies have been carried out focused on the tolerance of trees to drought, showing some variability both between species (Torres-Ruiz et al. 2017b) and within species [START_REF] Stojnić | Variation in xylem vulnerability to embolism in European beech from geographically marginal populations[END_REF]. During the last decades, human-induced climate change has increased the frequency of heat waves and drought events which has induced important tree mortality events worldwide (Anderegg et al. 2012b). More recently, the Intergovernmental Panel on Climate Change (IPCC) has reported that the increasing concentration of atmospheric carbon dioxide (CO2), mainly caused by humans activities, could lead to an increase in the mean global temperature by about 2.6 to 4.8°C according to the representative concentration pathways (RCP) 8.5 scenario (Intergovernmental Panel on Climate Change 2014). Although higher concentrations of CO2 could lead to a better efficiency of photosynthesis [START_REF] Ainsworth | The response of photosynthesis and stomatal conductance to rising [CO2]: Mechanisms and environmental interactions[END_REF], the rising temperatures associated with the expected changes in the precipitation patterns would increase the high evaporative demand and the plant transpiration rate which would provoke an increase in xylem sap tension and, consequently, exacerbate the risk of hydraulic failure by cavitation. As the percentage of cavitated vessels increases, the hydraulic conductance of the xylem decreases until the flow of water stops and provokes the desiccation of the plant tissues, the cell death and, finally, the death of the tree [START_REF] Mcdowell | Mechanisms of plants survival and mortality during drought: why do some plants survive while others succumb to drought?[END_REF]. These changing conditions would exacerbate the occurrence of droughtinduced tree mortality events [START_REF] Keenan | Increase in forest water-use efficiency as atmospheric carbon dioxide concentrations rise[END_REF][START_REF] Duan | Elevated [CO2] does not ameliorate the negative effects of elevated temperature on drought-induced mortality in Eucalyptus radiata seedlings[END_REF]) and consequently forests dieback [START_REF] Hosking | Mountain beech (nothofagus solandri var. cliffortioides) decline in the kaweka range, north island, new zealand[END_REF][START_REF] Lwanga | Localized tree mortality following the drought of 1999 at Ngogo, Kibale National Park, Uganda[END_REF][START_REF] Landmann | Impacts of drought and heat on forest. Synthesis of available knowledge, with emphasis on the 2003 event in Europe[END_REF].

Despite the fact that xylem hydraulic failure is considered to be the main cause of tree mortality under severe drought conditions, it is still unclear when we could consider that a tree is dead, and therefore, is not able to recover and resprout anymore.

Addressing this question is especially relevant to implement models predicting the effect of the expected increase in the frequency and intensity of drought events on trees [START_REF] Trenberth | Global warming and changes in drought[END_REF]) since, so far, it is not possible to predict accurately when a tree would die from drought.

Therefore, to make a significant step forward in our predictions about the variations in the composition of the forests due to climate change, it is crucial to identify those physiological traits able to define the threshold between a living and a dead tree.

Bibliography Synthesis 1. Climate change, global warming and its consequences

According to the United Nations Framework Convention on Climate Change, climate change is defined as "a change of climate which is attributed directly or indirectly to human activity that alters the composition of the global atmosphere and which is in addition to natural climate variability observed over comparable time periods." In general, climate change is assimilated to global warming which is primarily caused by the accumulation of greenhouse gases (GHGs) in the atmosphere. Indeed, the atmospheric GHGs concentration in 2011 got to levels that were never reached in the last 800,000 years (Intergovernmental Panel on Climate Change, 2014) [Figure 1].

Even if there are natural sinks for carbon, like forests, the IPCC report showed that 40% of the CO2 emissions remained in the atmosphere since 1750 (Intergovernmental Panel on Climate Change 2014). This accumulation of CO2 in the atmosphere is directly related with the temperature changes [Figure 2] as the global mean surface temperature increases in a range of 0.8°c to 2.5°c per trillion tons of carbon emitted as CO2 (Intergovernmental Panel on Climate Change 2014). Therefore, according to the RCP 8.5 scenario (worst-case scenario), the temperature could increase in between +1.5°c in southern Australia and +11°c in the North Pole by 2100 [Figure 3(a)] (Intergovernmental Panel on Climate Change 2014). Also, changes in the water cycle are expected to occur induced by the global warming. Thus, the IPCC reports that changes in average precipitation will not be uniform worldwide (Intergovernmental Panel on Climate Change 2014), with wet regions getting wetter and dry regions getting dryer [START_REF] Trenberth | Global warming and changes in drought[END_REF] [Figure 3 (b)]. It has also been reported The worst-case scenario (RCP8.5) evinced that the temperature elevation could reach between +1.5°c and +11°c in 2100. For precipitations, RCP8.5 scenario show that mid-latitude and subtropical regions will experiment lower means precipitations than actual while in wet regions precipitations will increase. (Intergovernmental Panel on Climate Change, 2014) that changes in the precipitation patterns will not only increase the frequency of the drought events, but also their intensity and duration [START_REF] Trenberth | Global warming and changes in drought[END_REF]. Even though it exists different definitions for drought, meteorological drought seems to be the more common one [START_REF] Wilhite | Chapter 2: Understanding the Drought Phenomenon: The Role of Definitions[END_REF] and is defined, as mentioned by the IPCC in its fourth assessment report, as "in general terms, drought is a 'prolonged absence or marked deficiency of precipitation', a 'deficiency of precipitation that results in water shortage for some activity or for some group' or a 'period of abnormally dry weather sufficiently prolonged for the lack of precipitation to cause a serious hydrological imbalance'." (IPCC 2007).

Drought impact on plant physiology and forests ecosystems

Drought impact in plant water transport capacity

In plants, water moves from the roots to the leaves through the xylem that is a specialized tissue for water transport [START_REF] Kirkham | Structure and Properties of Water[END_REF][START_REF] Mcelrone | Water Uptake and Transport in Vascular Plants[END_REF]. Once water is absorbed by the plant through the roots, it can move over long distances within the plant due to the cohesion-tension (C-T) theory developed by Dixon and Joly in 1894. The C-T theory is based on the cohesion between water molecules [START_REF] Milburn | Properties of water[END_REF][START_REF] Kirkham | Structure and Properties of Water[END_REF] and the pull of the continuous water column in the xylem induced by the plant transpiration, that is what finally causes the ascent of the sap [START_REF] Meinzer | Water transport in trees: current perspectives, new insights and some controversies[END_REF][START_REF] Brown | The Theory of the Rise of Sap in Trees: Some Historical and Conceptual Remarks[END_REF]. Therefore, transpiration creates a tension gradient within the plant that allows water to circulate throughout the xylem under tension [Figure 4].

During a drought event, the combination of lower precipitations and high temperatures increases the evaporative demand from the atmosphere which induces higher evapotranspiration (ET) rates in plants [START_REF] Duan | Elevated [CO2] does not ameliorate the negative effects of elevated temperature on drought-induced mortality in Eucalyptus radiata seedlings[END_REF] and, therefore, increases the tension on the xylem sap [START_REF] Choat | Triggers of tree mortality under drought[END_REF]).

Drought events and forest die-off

Numerous episodes of drought-induced forest mortality have been observed in the last decades [START_REF] Allen | A global overview of drought and heat-induced tree mortality reveals emerging climate change risks for forests[END_REF]. For example, important widespread drought-induced tree mortality events were reported in Africa such as in the tropical moist forest of Uganda [START_REF] Lwanga | Localized tree mortality following the drought of 1999 at Ngogo, Kibale National Park, Uganda[END_REF], in New Zealand with the mortality of Nothofagus forests [START_REF] Hosking | Mountain beech (nothofagus solandri var. cliffortioides) decline in the kaweka range, north island, new zealand[END_REF] and in France during the heat wave and drought during the summer 2003 [START_REF] Landmann | Impacts of drought and heat on forest. Synthesis of available knowledge, with emphasis on the 2003 event in Europe[END_REF].

As those events are likely to get more frequent in a near future [START_REF] Trenberth | Global warming and changes in drought[END_REF], it is important to highlight the role of drought in determining the composition and the structure of forests globally [START_REF] Allen | A global overview of drought and heat-induced tree mortality reveals emerging climate change risks for forests[END_REF]. Indeed, an important amount of forests worldwide is located in areas where the risk for drought is expected to increase in the next decades. Thus, e.g., the progressive water loss during the California drought (2012)(2013)(2014)(2015) led to the loss of 102 million trees [START_REF] Asner | Progressive forest canopy water loss during the 2012-2015 California drought[END_REF]). Contrary to California, which can be considered as a water-limited region, some non-water limited areas experienced similar consequences induced by drought. This is the case for the tropical northern area of Australia, where 6% of the mangrove vegetation died from a drought event combining high temperatures and low precipitations back in late 2015 and early 2016 [START_REF] Duke | Large-scale dieback of mangroves in Australia's Gulf of Carpentaria: A severe ecosystem response, coincidental with an unusually extreme weather event[END_REF]. Therefore, because forests seem to be sensitive to climate change and play an important role in carbon balance that, at the same time, helps regulating the climate [Figure 5] [START_REF] Reichstein | Climate extremes and the carbon cycle[END_REF], it is crucial to prevent and predict the occurrence of drought-induced tree mortality events. For this, it is necessary to understand not only the mechanisms driving tree mortality but also to determine a physiological trait that could be used as a proxy for determining when a tree is dead and not able to recover or resprout anymore.

3. Drought-induced tree mortality

Causes of plants death under drought conditions

Vegetation mortality could be induced by multiple factors such as recurrence of climate stress, insects pests and diseases [START_REF] Franklin | Tree Death as an Ecological Process[END_REF][START_REF] Miao | Vegetation Responses to Extreme Hydrological Events: Sequence Matters[END_REF]. Studies in plant mortality frequently agree in that water limitation is one of the main causes of plants death. In fact, it has been shown that when plants undergo recurrent exposure to drought, their growth decreases significantly, and their risk to die increases (Pederson 1998, Suarez and[START_REF] Suarez | Factors predisposing episodic drought-induced tree mortality in Nothofagus -site , climatic sensitivity and[END_REF]). Also, it seems that plants are likely to die when exposed to a prolonged drought periods with high air temperatures and vapor pressure deficit (VPD) conditions [START_REF] Swetnam | Mesoscale Disturbance and Ecological Response to Decadal Climatic Variability in the American Southwest[END_REF][START_REF] Breashears | Tree die-off in response to global change-type drought: mortality insights from a decade of plant water potential measurements[END_REF][START_REF] Bigler | Drought as an inciting mortality factor in scots pine stands of the Valais, Switzerland[END_REF]. Drought-induced mortality is related to drought intensity and duration [START_REF] Mcdowell | Mechanisms of plants survival and mortality during drought: why do some plants survive while others succumb to drought?[END_REF] [Figure 6]. Thus, there are two hypotheses explaining tree mortality depending on the intensity and duration of the drought event. On one hand, the first hypothesis relates tree mortality with the xylem hydraulic failure that occurs when plants are exposed to intense drought conditions. On the other hand, tree death seems to be induced by a depleting in carbon reserves, i.e. carbon starvation [START_REF] Mcdowell | Mechanisms of plants survival and mortality during drought: why do some plants survive while others succumb to drought?[END_REF], induced by the partial closure of the stomata that occurs under prolonged mild drought conditions [START_REF] Mcdowell | Mechanisms of plants survival and mortality during drought: why do some plants survive while others succumb to drought?[END_REF][START_REF] Mcdowell | The mechanisms of carbon starvation: how , when , or[END_REF]. However, even if hydraulic failure and carbon starvation are not Once the stomata are closed, plant continues losing water due to cuticular conductance. Under waterstress conditions, cavitation events occur reducing the xylem hydraulic conductance. The CAVITRON technique along with the optical method are used to generate vulnerability curves (in red) on which it is possible to determine the P50 and the P88 which correspond respectively to the xylem water potential at which 50% or 88% of hydraulic conductance are lost. (Choat et al. 2018 modified) mutually exclusive mechanisms (McDowell 2011b), many studies agrees on that hydraulic failure is one of the main causes of tree mortality under severe drought [START_REF] Urli | Xylem embolism threshold for catastrophic hydraulic failure in angiosperm trees[END_REF] or that "one primary cause of productivity loss and plant mortality during drought is hydraulic failure" [START_REF] Choat | Global convergence in the vulnerability of forests to drought[END_REF]. Therefore, because this study will be centered on the effect of intense drought-event on trees, a deeper review of the physiological processes and mechanisms related with intense drought induced tree mortality will be established.

Vulnerability to cavitation

Vulnerability to cavitation is extremely variable across species and biomes [START_REF] Delzon | Mechanism of water-stress induced cavitation in conifers: Bordered pit structure and function support the hypothesis of seal capillary-seeding[END_REF][START_REF] Choat | Global convergence in the vulnerability of forests to drought[END_REF]. In fact, different strategies have been developed throughout evolution to preserve the integrity of the plant vascular system, being all them mostly defined by two different constraints: the ability to maintain relatively high water potential by limiting water losses under drought conditions [START_REF] Blackman | Toward an index of desiccation time to tree mortality under drought[END_REF], Martin-StPaul et al. 2017); and the physical limits of the xylem vessels to avoid the entry of air into the xylem conduits, i.e., the vulnerability to cavitation. Xylem vulnerability to cavitation is usually evaluated by constructing vulnerability curves (VC). These curves represent how the percentage loss of hydraulic conductance (PLC)

induced by cavitation varies with the xylem tension, i.e. the xylem water potential [Figure 8].

Valuable information could be extract from such curves as, e.g., the xylem tension inducing 50% loss of hydraulic conductance (P50 value), that is commonly used when comparing the vulnerability to cavitation between species. In conifers, this P50 value also represents a threshold value for xylem tension above which plants cannot recover anymore from drought [START_REF] Brodribb | Hydraulic Failure Defines the Recovery and Point of Death in Water-Stressed Conifers[END_REF].

In angiosperms however, the xylem tension above which plants cannot recover anymore is that representing 88% loss in hydraulic conductance and is called the P88 (Urli et al. 2013).

4. Tree mortality, current methods to evaluate it and ongoing questions

Death in plant physiology

In plant physiology, one study clearly defined tree death: "Death is defined as thermodynamic equilibrium between the organism and the environment, in which plants no longer have energy gradients to drive metabolism or regenerate." (McDowell 2011a). It was pointed out lately that tree death from drought is still poorly define and that the definition given by McDowell (2011a) remained limited in utility as it does not provide with proxies that can help to identify clearly if a tree is dead or not (Anderegg et al. 2012a). Therefore, Anderegg et al (2012a), proposed that tree death can be considered as "a complete system failure due to lack of water resources".

Despite these definitions of tree death, in practice, it is difficult to determine if a tree is dead or not. In fact, even if Anderegg et al. (2012a), settled a definition for tree death, they pointed out the fact that their definition was based on the ability of the tree to recover or not from drought events. They also highlighted the fact that studies needed to clearly define what tree mortality is and provide with criteria that could permit the irrevocable identification of dead trees.

Therefore, with the lack of accuracy in the tree death definition, it seems difficult to determine accurately when a tree is in total failure or at which point its gradients are so low that they cannot maintain the metabolism of the plant anymore. For this reason, most of the studies on tree mortality are based on methods evaluating the ability of the tree to recover or on the identification of living tissues by direct observation.

Evaluating tree mortality

In many studies, trees are considered as dead when they cannot recover from droughtinduced cavitation and are unable to grow the next vegetative season [START_REF] Lloret | Canopy recovery after drought dieback in holm-oak Mediterranean forests of Catalonia (NE Spain)[END_REF][START_REF] Rice | Patterns of tree dieback in Queensland, Australia: The importance of drought stress and the role of resistance to cavitation[END_REF][START_REF] Brodribb | Hydraulic Failure Defines the Recovery and Point of Death in Water-Stressed Conifers[END_REF], Anderegg et al. 2012b, Barigah et al. 2013b). Thus, for conifers, it has been shown that when the percentage of loss in conductance (PLC) is higher than 50%, trees cannot recover anymore. In angiosperms however, the percentage of embolism leading to a significant reduction of the recovery capacity of the trees is around 88% [START_REF] Urli | Xylem embolism threshold for catastrophic hydraulic failure in angiosperm trees[END_REF]).

Because of this major difference between angiosperms and gymnosperms and even if the P50 and P88 are commonly accepted as indicator to identify tree death and as measurable indexes for tree mortality [START_REF] Sperry | what plant hydraulics can tell us about responses to climate-change droughts[END_REF], it is not clear what are the changes occurring at tissue level that make plants being not able to recover anymore. Thus, based on those observations, trees are currently considered as dead from hydraulic failure when they are not able to recover from embolism and/or resprout the next year. Even if this method of tree death identification based on the tree ability to recover is generally accepted by the scientific community, it does not allow to identify the main drivers for tree death. Also, it is necessary to wait until the next growing season to verify if plants are able to recover from drought and this could be an important limiting factor in many studies.

Another common method to determine if a tree is dead from drought can be by direct observation of the color of the tissues beneath the bark. Thus, O'Brien et al. (2014) considered a tree as dead "when no green tissue was observable under the bark on the stem" and confirmed their observations by "re-watering some seedlings" and evaluating which plants were able to recover the year after. This direct observation method is generally accepted but it does not provide us with enough physiological information for establishing a clear threshold between living and dead tree. FDA absorbs blue wavelength (<500nm) and restitute a green fluorescence with a peak at 520nm. (https://www.thermofisher.com/order/catalog/product/F1303)

5. Developing new methods to identify and predict tree mortality 5.1. Detecting living cells: a staining process

In order to determine which living tissues are affected by drought, it is necessary to identify where living cells are and how the amount changes during the dehydration of the tissues. For this, Fluorescein Diacetate (FDA), a cell-permanent esterase substrate able to stain the cytoplasm of living cells [START_REF] Truernit | A simple way to identify non-viable cells within living plant tissue using confocal microscopy[END_REF]) could be used. In fact, FDA was first used to stain animal cells [START_REF] Rotman | Membrane properties of living mammalian cells as studied by enzymatic hydrolysis of fluorogenic esters[END_REF] and then used in plants cells [START_REF] Widholm | The use of FDA and phenosafranine for determining viability of cultured plant cells[END_REF]. Briefly, FDA is a non-polar molecule that enters the living cells where the esterase (active only in living cells)

cleave the acetate residues and form fluorescein molecule, a non-lipophilic molecule that cannot cross the cell membranes and, therefore, accumulates in the living cells [START_REF] Widholm | The use of FDA and phenosafranine for determining viability of cultured plant cells[END_REF] Thus, one of the aims of the study is to develop a staining protocol using FDA that can be used for monitoring the changes in the number of living cells in the different plant tissues during drought. Although qualitative, the goal is to evaluate whether the decrease in the amount of living cells in given plant tissues can be used as a proxy for setting a threshold for plant death (i.e. lack of recovery).

Predicting tree mortality by screening membrane failure

When soil water availability decreases, plant mineral uptake decreases as well and reduces the concentration of key minerals for the cell membrane stability, as potassium or sodium [START_REF] Wang | The critical role of potassium in plant stress response[END_REF]. Such reductions decline the cell membrane stability while the accumulation of reactive oxygen species (ROS) increases. Indeed, normally ROS are destroyed by the antioxidant capacity of the plant. However, under water-stress conditions, the antioxidant capacity of the plant is depleted while the production of ROS by the photosystems (PS) I and II increases [START_REF] Guadagno | Dead or alive? Using membrane failure and chlorophyll fluorescence to predict mortality from drought[END_REF]. Thus, the accumulation of ROS in the cell can certainly leads to the disruption of membranes [START_REF] Petrov | ROS-mediated abiotic stress-induced programmed cell death in plants[END_REF]. Therefore, due to the disruption of membranes, under water-stress conditions, the cells' electrolyte leakage should increase, and this trait could be followed using the electrolytes leakage protocol developed by [START_REF] Zhang | An improved conductivity method for the measurement of frost hardiness[END_REF] and [START_REF] Sutinen | Seasonal differences in freezing stress resistance of needles of Pinus nigra and Pinus resinosa: evaluation of the electrolyte leakage method[END_REF].

In addition, [START_REF] Guadagno | Dead or alive? Using membrane failure and chlorophyll fluorescence to predict mortality from drought[END_REF] also defined plant mortality as a "threshold in membrane disruption for which plants cannot recover" what could suggest that electrolyte leakage could work Electrolyte leakage is measured as electroconductivity increase (%). Fv'/Fm' represents the maximum efficiency of photosystem II under light conditions. We notice a decrease in Fv'/Fm' as the percentage of electroconductivity increases. Therefore, because electroconductivity is linked with membrane stability, which decreases under water-stress condition, it is possible to conclude that under drought conditions, cells damages increase along with the loss of efficiency of the photosystem II. [START_REF] Guadagno | Dead or alive? Using membrane failure and chlorophyll fluorescence to predict mortality from drought[END_REF] as a proxy for point of death. Therefore, the aim is to set a threshold in percentage of electrolyte leakage allowing the identification of tree's death.

It is also known that the variable fluorescence (Fv) is linked with the presence of intact photosystems [START_REF] Franck | Resolution of the Photosystem I and Photosystem II contributions to chlorophyll fluorescence of intact leaves at room temperature[END_REF]. Thus, under drought stress conditions, the accumulation of ROS can lead to membrane failure and the photosystems located in the membrane would suffer from the membrane disruption. Hence, Fv should be impacted resulting in a variation of the chlorophyll a fluorescence (Fv/Fm) with water-stress. Therefore, the chlorophyll a fluorescence in light conditions (Fv'/Fm'), which set the maximum efficiency of the PS II, should also be impacted.

Considering this, it has been recently reported a link between electrolyte leakage and Fv'/Fm' [START_REF] Guadagno | Dead or alive? Using membrane failure and chlorophyll fluorescence to predict mortality from drought[END_REF]. Thus, the maximum efficiency of the PS II in light conditions, represented by the Fv'/Fm' ratio, decreased along with the increasing percentage of electroconductivity (that is to say, electrolyte leakage) [Figure 12]. Unlike electrolyte leakage that is a destructive technique, the variable fluorescence technique uses a fluorometer which can be used on intact plants and is therefore more appropriate to monitor drought impact on plants. By combining those two techniques, monitoring electrolyte leakage and looking at loss of variable fluorescence, it could be expected to predict at which physiological threshold a tree dies from drought.

Determining tree capacity to recover

The Linear Variable Differential Transformer (LVDT) technology converts a rectilinear motion into an electric signal [START_REF] Hunter | LVDT : Linear Variable Differential Transformer[END_REF] and is one of the most common methods for monitoring the variation in stem or trunk diameter to evaluate tree growth along the day [START_REF] Ameglio | PepiPIAF: a new generation of biosensors for stress detections in perennial plants[END_REF][START_REF] Adam | Systeme pepipiaf : systeme de surveillance et d'expertise de la croissance des plantes et des arbres[END_REF]. Indeed, under non-stressing conditions, stem diameter increases each day due to the secondary growth of the tree [START_REF] Simonneau | Diurnal changes in stem diameter and plant water content in peach trees[END_REF] [Figure 13]. On a daily basis, trunk and stems tends to shrink during the day due to the transpiration of the plant and to expand during the night once the stomata are closed and the transpiration rate is almost zero [START_REF] Daudet | Experimental analysis of the role of water and carbon in tree stem diameter variations[END_REF].

Under drought conditions, the stems or trunk will shrink and these variations in stem diameter along the day will significantly decrease until showing any change. The hypothesis is that depending on the duration of the water stress and once rehydrated, plant would show again some variation in stem diameter if they are able to recover from stress. If no changes in diameter are observed after re-watering the plants, the plant can be then considered as dead from drought.

Purpose of the master project

In general, there is a lack of studies focusing on the physiological mechanisms behind tree mortality per se and the response of plant to water stress (McDowell 2011a ;[START_REF] Guadagno | Dead or alive? Using membrane failure and chlorophyll fluorescence to predict mortality from drought[END_REF]. Considering this, the aim of this master project is to evaluate and develop new techniques that, combined with classical methods used in plant physiology [Figure 14], will enable us to:

(i)
Identify key physiological traits that could work as proxies for tree mortality.

(ii) Determine the main changes occurring at the plant tissue level explaining the lack of recovery after drought.

(iii) Establish a physiological threshold for plant death based on physiological measurements and not only in the capacity of recovery of plant the following growing season.

Materials and methods

Plant material, growth conditions and experimental design

The To expose the plants to a severe event of water stress, irrigation was withheld in set of four to six individuals that were removed from the pots, their roots washed with water and the whole individuals were air dried to facilitate dehydration. Plants were let to dehydrate until reaching severe levels of water stress that correspond with high levels of PLC, and then rehydrated at field Percent loss conductivity (%)

Water Potential (MPa)

The relationship between PLC and the water xylem pressure induced by centrifugation can be described by the following sigmoidal equation [START_REF] Pammenter | A mathematical and statistical analysis of the curves illustrating vulnerability of xylem to cavitation[END_REF]:

𝑃𝐿𝐶 = ( / (
)

)
where a is the slope of the curve at the inflection point, and P50 represents the pressure at which 50% loss of conductivity occurred. Barigah et al. 2013b). When the stem water potential reached values below -8MPa, the terminal part of the stem was directly used to evaluate the stem water potential since, probably due to most hydraulic disconnection of the leaves from the stem vessels, it was not possible to carry out the measurements in the leaves anymore. ΨL was measured daily on three healthy leaves directly cut from the plant until their water potential could not be measured anymore using the Scholandertype pressure chamber. Both leaves and stem samples were cut from the plant and immediately stored in a vial containing a piece of wet paper inside to saturate the air and prevent dehydration until carrying out the measurements.

Xylem water potential measurements

Stem diameter variations

Stem diameter variations were measured continuously by Linear Variable Differential Transformer (LVDT) sensors set up on each plant before exposing them to water stress. The needle which correspond to the moving part of the measurement sensor was applied on stem with glue [Figure 18]. Sensors were connected to a data logger (Model CR1000, Campbell Scientific LTD, Logan, Utah, USA) and recorded an average of stem diameter variation (in mV) every 10 minutes.

Relative Water Content

Stem relative water content (S-RWC) and leaf relative water content (L-RWC) were measured every 1 to 3 days according [START_REF] Barrs | A Re-Examination of the Relative Turgidity Techniques for Estimating Water Deficits in Leaves[END_REF]:

𝑅𝑊𝐶 = ( ) ( )
where FW is the fresh weight measured immediately after sampling; TW is the turgid weight measured after immersing the stem in distilled water for 24 hours (for S-RWC) or after soaking the leaf petiole for 24 hours (or until total weight gain) in distilled water (for L-RWC); and DW is the dry weight of the samples after 24 hours of drying in an oven at 70°c (or until measuring a stable dry weight). All measurements were obtained with a precision scale (METTLER AE 260, DeltaRange ®) and were performed on three healthy leaves or three small stem sections.

Electrolyte leakage

Cell damages induced by drought were determined on three stem samples per plant using the electrolyte leakage test (Zhang andWillison 1987, Sutinen et al. 1992). Briefly, stem samples were cut in 10 slices of 2mm of thickness and put in test tubes containing 15mL of pure water. Test tubes were shaken at 60 shakes/min during 24hours at 5°c to stop enzyme activity. Water conductivity of the effusate (C1) was measured at room temperature using a conductimeter (3310 SET1, Tetracon® 325). Then, all the cells contained in the test tubes were killed by autoclaving the samples at 121°c for 30minutes [START_REF] King | Chilling injury and electrolyte leakage in fruit of different tomato cultivars[END_REF], let cool at room temperature for 60 minutes and the maximal conductivity (C2) measured. The lysis percentage (EL) was then determined using the following equation: 𝐸𝐿 = * 100.

Chlorophyll a fluorescence

Chlorophyll A fluorescence measurements in light conditions (Fv'/Fm') were performed on five healthy leaves per plant using a fluorometer (PAM-210 Chlorophyll Fluorometer).

Measures were done on the adaxial side of one sun-exposed leaf and consisted in applying one saturating pulse of light, emitted with a Red LED (emission peak at 665nm, max intensity 3500µmol.m -2 .s -1 PAR, duration 3µs), and performed approximately in the middle of the leaf while avoiding the mid rib. For XYL'EM, in both species, the PLC was evaluated using three stem samples from each individual. The sample length was at least of 30mm. For both species, the initial conductance (Ki)

was measured at low pressure. In order to determine the maximal conductance (Kmax), Prunus samples were flushed with water at high pressure (2bars) for 20 minutes to remove the embolism.

Pseudotsuga samples, which are composed of tracheids and not vessels, could not be flushed at high pressure. Therefore, in order to remove embolism, Pseudotsuga samples were immersed in water and put under the action of a vacuum pump to create void overnight.

For Micro-CT, for both species, one or two samples (depending on the quantity of plant material) were immersed in liquid paraffin wax to prevent their dehydration during the 21 minutes scan. At the end of the experiment, samples were cut 3mm above the scanned cross section, injected with air (0.1MPa) and re-scanned to visualize all the emptied vessels [Figure 19]. The amount of PLC was computed by determining the amount of cavitated vessels in the samples before and after cutting the sample. This then permitted to determine the PLC for each sample.

Cytology: detecting living cells

Fluorescein diacetate (FDA) (F7378-10G, SIGMA-ALDRICH, Co, St Louis, MO, USA), was used to stain the cytoplasm of living cells and therefore identify qualitatively the amount of living cells and their location while plants were exposed to water stress conditions and after rewatering them. For this, 60µm thick cross sections were obtained with a microtome (Leica RM2165) and stained for 20minutes in a 1% solution of FDA made from a stock solution of 5mg/mL of FDA diluted in acetone [START_REF] Widholm | The use of FDA and phenosafranine for determining viability of cultured plant cells[END_REF]. Cross sections were mounted in a slide and observed using an inverted fluorescence microscope (Axio Observer Z1, ZEISS; YFP filter) within the following hour. Photos of the entire cross section were obtained by assembling automatically tiles (which correspond of different photos of the cross section) and were obtained with Zen 2 software. The number of tiles per image variated between 16 tiles (for small cross sections) to 110 ), the stem water potential (ΨS) (3,4), the leaf water potential (ΨL) (5,[START_REF]Plant material, growth conditions and experimental design[END_REF] in Prunus lusitanica (a) and Pseudotsuga menziesii (b).

(a) For Prunus individuals, the dehydration started on the third day of the experiment (green line) for Prunus 1,2,3 and 4 and ended 8 days later with the beginning of the rehydration phase (blue line). The dehydration of Prunus 6 started on the first day (dashed green line) and ended 8 days later (dashed grey line). ΨS and ΨL consisted of punctual measurements made with the Scholander Pressure Chamber. (b) For Pseudotsuga individuals, the dehydration (DH) started between the 9 and the 16 day of the experiment and ended with the rehydration (RH) between the day 22 and the 41 of the experiment. ΨS was continuously recorded with ICTs psychrometers while ΨL was measured punctually with the Scholander Pressure Chamber. For both species, ΨL measurements were measured until reaching the limit of the pressure chamber (-10MPa).

(5)

(1)

(2)

(6) (4) (3)
tiles for (big cross sections) for both species. Photo processing was done using Zen 2, Image J and Photoshop CS5 softwires.

Statistical analyses

Statistical analyses consisted of paired t-test (after testing for normality and homogeneity of variances) and Wilcoxon test (for non-normal distribution) and were performed using R program to compare the set before and after the drought event and before re-watering and after re-watering.

A one way analysis of variance (ANOVA) (for normal distribution and homogeneity of variance) or a F Test of Welch (without supposing homogeneity of variance) were used to compare individuals within each set for each trait before (Control), after the drought treatment (BRW) and during the rehydration phase (PRW). When the differences were significant, a multiple comparison of means (post hoc Tukey honest significant difference test or Tukey contrast) was carried out. All tests were performed using a level of significance α=0.05.

Results

Water potential and trunk diameter dynamics

Linear Variable Differential Transformer (LVDT) sensors showed a noticeable shrinkage in the stems of Prunus and Pseudotsuga during the time-course of the drought event and a subsequent increase in the stem diameter following rewatering for those individuals that were able to recover from drought. Thus, Prunus 1 and Prunus 6 which reached ΨS values of -10MPa [Figure 20 (3)] showed an increase in stem diameter immediately after being re-hydrated [Figure 20 (1)].

However, Prunus 2,3,4 [Figure 20 (1)], and the others plants ( Prunus 5,7,8 [Annex 1]) which also reached a ΨS of -10MPa did not show any increase in stem diameter after the rehydration, being unable to recover from drought and therefore considered as dead plants. For Pseudotsuga, only Douglas 1 was able to show a recovery in trunk diameter [Figure 20 (2)] after rehydration at ]. For both species, those individuals that were able to recover after drought did not reach the same values for stem diameter than the ones they showed before dehydration despite showing similar ΨS values before being dehydrated and after the rehydration. Leaf water potential (ΨL) [Figure 20 (5,[START_REF]Plant material, growth conditions and experimental design[END_REF]] and stem water potential (ΨS) [Figure 20 (3,4)] decreased progressively along the dehydration phase of the study for both species However, those individuals that recovered from drought only show a recovery in water status at the stem level (i.e. only ΨS recovered and reached similar values than before dehydration). The ΨL did not recover even in recovering plants for both species, remaining at ca. <-10MPa for Prunus and <-8MPa for Pseudotsuga. Those plants that did not show any increment in stem diameter after the recovery irrigation did not show any recovery in plant water status either in ΨS nor ΨL. for those individuals able to recover from drought. Those individuals that were not able to recover, however, showed mean Fv'/Fm' values of 0.130. For both species, differences in Fv'/Fm' were not significant between individuals that were able to recover from drought and those that were not able to recover.

The S-RWC [Figure 21 (b)] followed the same dynamic as the Fv'/Fm' as its percentage significantly dropped from 92.6% in control conditions to 59.3% in Prunus individuals able to recover after the rehydration and 52.3% Prunus individuals that did not recover following rehydration. The differences in S-RWC were not significant when comparing individuals able to recover and those not able to recover. Similar results were observed for Pseudotsuga, showing S-RWC values that decreased from 83.0% for individuals under control conditions to 49.8% for those able to recover after dehydration (e.g. Douglas 1) and 36.9% for those that were not able to recover.

Unlike Prunus individuals, the differences between Pseudotsuga individuals able to recover and those not able to recover were statistically significant, what suggests that this trait could work as a proxy for this species for determining when a plant is able to recover from drought.

Similarly to S-RWC and the Fv'/Fm', L-RWC was significantly impacted in both species by the drought event as it decreased from 94.71% in control conditions to 58.90% and 64.20% in Prunus individuals able to recover and those unable to recover respectively, while it goes from 91.80% (control conditions) to 53.50% (able to recover) or 51.50% (not able to recover) for Pseudotsuga. None of the two species evaluated showed significant differences between recovering and non-recovering individuals before applying the recovery irrigation [Figure 21(c)]. Contrary to the three previous traits, the electrolyte leakage showed a significant increase during the progressive dehydration of the trees for Prunus (control conditions: 30.6%; able to recover : 50.7%

; non able to recover 64.4%) and for Pseudotsuga (control conditions: 50.5%; not able to recover : 78.8%) [Figure 21 (d)]. However, the Pseudotsuga individuals able to recover do not show a higher electrolyte leakage after the drought event (i.e. control: 50.5%; Douglas 1 able to recover: 50.8%) while contrary, Prunus 1 and Prunus 6 that were able to recover showed a higher percentage of electrolyte leakage after the drought event (i.e. BRW: 50.7%%) than in control conditions (i.e. Control: 30.6%).

During the post-rewatering phase (PRW), measurements of Fv'/Fm' and the L-RWC were performed between 27 and 77 days after the rehydration of the plants, with significant differences between the study species [Figure 21(a)]. First of all, in Prunus, the PS II efficiency decreased until reaching 0.263 in Prunus trees that were able to recover from drought according to results from LVDT and water potential. However, for Pseudotsuga a significant increase in the PSII efficiency was observed after the re-hydration of the plants (BRW: 0.186; PRW: 0.284). The L-RWC [Figure 21 (c)] showed the same pattern as the Fv'/Fm', e.g. it continues to decrease in Prunus individuals (BRW: 58.9%; PRW: 38.2%) while it recovered in Pseudotsuga (BRW: 53.5%; PRW: 74.1%). The electrolyte leakage [Figure 21 (d)] remained stable after re-watering the plants for both species (i.e. for Prunus: BRW: 50.7%, PRW: 46.84%; for Pseudotsuga: BRW 50.8%, PRW: 53.1%). The S-RWC [Figure 21(b)] in individuals able to recover increased significantly for both species but only reached its control values for Prunus individuals. Indeed, it increased from 59.3% (BRW) to 90.4% (PRW) for Prunus and from 49.8% (BRW) to 73.5% (PRW) for Pseudotsuga.

Hydraulic failure

The stem PLC values for Prunus and Pseudotsuga when measured via the Xyl'EM technique prior to the drought treatment (under well-watered conditions) was of 6.90% and 18.88%, respectively. PLC prior to the drought, according to the Micro-CT values, was 0.95% and 7.40% for Prunus and Pseudotsuga respectively. The stem PLC increased during the progressive dehydration of the trees, reaching values between 82.00% to 99,90% according to the Xyl'EM values for Prunus (e.g. Prunus 1: 98.60%, Prunus 7: 93.74%) [Table 2 (a)], i.e. above the threshold BRW represents the measurements performed on the individuals the day of the beginning of the re-watering phase while PRW represents measurements performed on the individuals 77 days after re-watering for Prunus 1, 62 days after re-watering for Prunus 6 and 27 days after the beginning of the re-watering phase for Douglas 1.

for recovery and point of death for angiosperms (i.e. P88). For Pseudotsuga [Table 2(b)], and according to the Micro-CT measurements, the PLC values at the end of the dehydration phase of the study variated between 67.90% to 93.85% (e.g. Douglas 1: 67.90%), being therefore much higher than the threshold for recovery and point of death for conifers (i.e. P50). Therefore, for the two species evaluated, some of the trees that reached PLC levels higher than the common threshold for mortality (P50 for conifers and P88 for angiosperms) were able to keep some living cells and to recover from drought once rehydrated [Figure 22]. Also, the levels of PLC tended to diminish when measured 10 and 4 weeks after rehydration for Prunus and Pseudotsuga respectively reaching 77.83% for Prunus 1 (Xyl'EM value) and 57.89% for Douglas 1 (Micro-CT value) [Table 3].

Effect of drought and recovery on living tissues

The use of FDA allowed to establish if living cells were present or not before and after rehydration [Figure 23]. In control conditions, all of the plants were all showing living cells in the bark and in the phloem proving that the technique was working to detect living tissues. However, no living cells could have been detected in the cambium in both species. The application of FDA BRW showed that the amount of living cells in both species decreased and was then affected by the drought. For Prunus, Prunus 4 and Prunus 7 [Table 2(a)] did not show any living cells, all the others showed living cells but the amount was less than in control conditions. However, out of all the plants showing living cells, only Prunus 1 and Prunus 6 [Table 2(a Figure 24: Prunus lusitanica n°1 flushing new leaves after experiencing a 8-days-long drought event and reaching a stem water potential of -10MPa. Before the re-hydration phase of the experiment, measurements showed a loss of conductance at stem level of 98.6% which is higher than the common proxy for angiosperm mortality (88%).

Discussion

1. Is hydraulic failure the main driver for tree mortality?

Results showed that even when trees for both species reached critical levels of embolism (>90% for Prunus and >50% for Pseudotsuga), they still show evidence of living cells and were able to recover from drought when re-watered. These are very relevant and novel results since, until now, the threshold for recovery and point of death for trees were the water potential value inducing 88% and 50% (i.e P88 an P50) of loss in stem conductance for angiosperms and conifers, respectively [START_REF] Brodribb | Hydraulic Failure Defines the Recovery and Point of Death in Water-Stressed Conifers[END_REF], Barigah et al. 2013b[START_REF] Urli | Xylem embolism threshold for catastrophic hydraulic failure in angiosperm trees[END_REF]. Prunus individuals that reached PLC levels of 98.6%, i.e. well above than 88%, were able to recover and even flushed new leaves [Figure 24] after re-watering them to field capacity. Similarly, Pseudotsuga individuals showing PLC levels of 67.92% were also able to recover from drought once re-watered. However, our study was conducted in potted plants that were re-watered at field capacity after the dehydration phase. Under natural conditions, such increments in the amount of water available for the trees rarely occur, what would enhance the survival probability of the plants.

Similar results have been recently reported for loblolly pine (Pinus taeda) by [START_REF] Hammond | Dead or dying? Quantifying the point of no return from hydraulic failure in drought-induced tree mortality[END_REF] which reported a lethal PLC threshold of 80%, i.e. much higher than 50% commonly reported for conifers. However, our study showed that recovery did not occur in Pseudotsuga individuals when PLC reached values above 70%. Therefore, this raises questions on how lethal PLC thresholds vary not only among tree species, but also within species. Our results, therefore, highlight the importance of revising the actual recovery and point of death thresholds suggested for angiosperms and conifers, and of considering the link between the amount of living cells together with the remaining plant hydraulic functioning when evaluating its recovery capacity from drought. This is especially important considering that these threshold values for mortality are crucial when using mechanistic models aimed to estimate the time to death of plants under drought conditions, as the SUREAU model [START_REF] Martin-Stpaul | Plant resistance to drought depends on timely stomatal closure[END_REF] and T-Crit model [START_REF] Blackman | Toward an index of desiccation time to tree mortality under drought[END_REF]. Our results, therefore, show how the level of stem embolism should not be considered as a proxy for tree death solely due to the similar high PLC values reported for both trees that were able to recover and for those that were not.

However because trees able to recover and re-draw water from soil showed similar high PLC values than the ones not able to, this highlights the importance of carrying out accurate PLC measurements both by using the Xyl'EM or the Micro-CT technique. Indeed, the Micro-CT technique could potentially lead to artefactual PLC values higher than 100% in plants able to recover. The reason is that, by using Micro-CT, PLC is determined by comparing the area of the embolized vessels at a given water potential with the total xylem area of the sample, i.e. the total conductive xylem area. To facilitate the determination of the total xylem area, samples are injected with air and re-scanned [START_REF] Cochard | X-ray microtomography (micro-CT): A reference technology for high-resolution quantification of xylem embolism in trees[END_REF]. The second scan can be not exactly at the same position than the first scan so, when the amount of embolized vessels is important, any change in the total amount of vessels just below or above the first scanning point can lead to important overor underestimations of the PLC. In addition, after the cut, it is necessary to immerse the sample in paraffin wax that can go into some vessels and be considered erroneously as fully functional vessels, underestimating therefore the number of embolized vessels. In addition, the estimation of the PLC using the Xyl'EM is based on the ratio between the initial and the maximal conductance of a sample. However, as discussed by [START_REF] Cochard | Methods for measuring plant vulnerability to cavitation: A critical review[END_REF] the presence of already formed but non-functional vessels can lead to an overestimation of the maximal conductance of the sample. In addition, for conifers, pit membranes can remain permanently aspirated against the cell walls of the embolized tracheids leading to an underestimation of the maximal conductance and consequently to unrealistic PLC values [START_REF] Cochard | Methods for measuring plant vulnerability to cavitation: A critical review[END_REF]. Thus, the precision of the values estimated by the Xyl'EM can be discussed and the levels of PLC measured with this apparatus should be interpreted carefully.

Is there a link between water potential and tree mortality?

As established in the bibliography, a clear link between ΨS and mortality should be made.

Water potential is the main driver for PLC and it has been traditionally used to set mortality thresholds, i.e. P50 and P88 [START_REF] Brodribb | Hydraulic Failure Defines the Recovery and Point of Death in Water-Stressed Conifers[END_REF], Barigah et al. 2013b[START_REF] Urli | Xylem embolism threshold for catastrophic hydraulic failure in angiosperm trees[END_REF].

However, values of ca. 100% of PLC were reached at water potentials below -10.0 MPa, as shown by the fitted vulnerability curves to embolism. This, therefore, did not allow us to determine a threshold water potential value for tree mortality since trees with such high PLC values that were able to recover from drought showed water potential values below -10.0, i.e. below the minimum value that the available techniques are able to measure. Indeed, while both the psychrometers and the Scholander provide accurate water potential values for most of the species at mild water stress conditions, any of these two techniques can be used for such high xylem tensions mostly due to technical limitations (psychrometers) or to safety reasons (Scholander chamber). We tried to resolve this technical limitation by trying to measure those low water potentials using a water activity meters (WP4-T, Decagon Devices, Pullman, Washington, USA) that was originally designed to follow soil water potentials up to -100MPa. However, when we compared the water potential values reported by this device and the ´gold-standard´ method, i.e. the Scholander pressure chamber, for both stem and leaf samples in between -0.5MPa and -10MPa, we observed how water potential values were highly underestimated by the WP4-T device. This agrees in fact with previous observations by [START_REF] Martínez | Comparison of two techniques for measuring leaf water potential in vitis vinifera var[END_REF] and invalidated therefore the use of the WP4-T as an alternative to measure the very low ΨL and ΨS values that trees, as in our study, normally reach when evaluating drought-induced tree mortality. Therefore, in order to be able to read accurate water potentials below -10MPa, current techniques need to be improved or new techniques are need to be developed. In fact, after our observations during this study, some suggestions has been made to the manufacturer of the psychrometers (ICT International, Australia) in order to help with the design of new psychrometers able to measure accurately water potential values below -10MPa.

Why do plants die?

3.1. Disruption of the continuum between available water for tissues and soil water For the two species evaluated in this study, and despite the amount of living cells decreasing noticeably during dehydration, trees that were able to recover showed some living cells mostly located in the bark and in the phloem before rehydration. No living cells could be detected in the cambium, in either the control conditions or in recovering plants. This could suggest that either the FDA was not capable of entering the cambial cells probably due to differences in membrane composition or that the staining process was not long enough to allow the transformation from FDA to fluorescein. However, the presence of living cells at the stem level was not always related with the recovery of the plants after rewatering. This was the case for Pseudotsuga for which we did not observe any recovery for plants showing similar amount of living cells and at similar locations than those that were able to recover from drought after rewatering. Also, the Pseudotsuga individual showing living cells before rehydration (e.g. Douglas 6) and that was not able to recover had lost the FDA fluorescence signal 14 days after the beginning of the re-watering phase of the experiment. The explanation could be that under drought conditions, plants can rely on their own water reserves [START_REF] Epila | Capacitive water release and internal leaf water relocation delay drought-induced cavitation in African Maesopsis eminii[END_REF] which could maintain the metabolism of the cell a bit longer.

However, once the water reserves are drained, the tissues would ultimately dry and cells would dehydrate and die. Therefore, and according to these results, we hypothesize that not only the presence of living cells is required for allowing the plant to recover from drought, but also a hydraulic connection between them and the root system that would allow the irrigation of the tissues and, therefore, would trigger the recovery of the plants. Under severe drought conditions, plants would indeed rely on their own water reserves [START_REF] Epila | Capacitive water release and internal leaf water relocation delay drought-induced cavitation in African Maesopsis eminii[END_REF]. Nonetheless, in order to survive a drought event those water reserves need to be refilled, re-establishing a hydraulic continuum from the roots able to transport the water from soil to the living tissues. This hypothesis suggests that even when PLC show value near to 100% for angiosperms or above 50% for conifers, a minimal hydraulic functioning between the soil and the living tissues could be enough to survive from drought if plants have access to water.

In addition, if plants are thus able to survive according to this hypothesis, it would be important to quantify the amount of living cells before the rehydration to answer unresolved questions as, e.g., is a small amount of living cell able to trigger the plant recovery from drought and keep it alive? Also, it is well known that, due to their totipotency capacity, plant cells are able to dedifferentiate and generate a whole organ from any cell/explant [START_REF] Malamy | Organization and cell differentiation in lateral roots of Arabidopsis thaliana[END_REF]Benfey 1997, Laux 2004). Thus, under a drought-stress, is any of the remaining living cells able to trigger this dedifferentiation process or are specific cells the ones necessary for regenerating the plant tissues and recovering from drought? Therefore, long-term drought survival could rely on the ability to be able to grow new xylem tissues as suggested by [START_REF] Hammond | Dead or dying? Quantifying the point of no return from hydraulic failure in drought-induced tree mortality[END_REF].

Disruption of the continuum between cells and plant water reserves

It has been described that under severe drought conditions the water demand of the plant is almost zero as stomata are already closed in order to reduce the evaporative demand of the plant [START_REF] Hochberg | Stomatal Closure, Basal Leaf Embolism, and Shedding Protect the Hydraulic Integrity of Grape Stems[END_REF]. Despite this, the different plants tissues remain irrigated until water potential reaches values low enough for inducing the xylem hydraulic failure and, therefore, the tree death because of the failing of the system to provide enough water to the crucial cells for survival. It was demonstrated that, under severe drought conditions, certain plants rely on their own water reserves and are therefore not reliant on water supply [START_REF] Epila | Capacitive water release and internal leaf water relocation delay drought-induced cavitation in African Maesopsis eminii[END_REF]. Thus, a focus on cell hydration in living tissues could be the main key for plant survival (Martinez-Vilalta et al.

2019

). However, our results in stem relative water content (S-RWC) did not show a clear pathway to understand tree mortality as no significant differences were noticed between plants able to recover and those unable to do so. Therefore, rather than just focusing on the plant water status, a deeper study of water relocation in plants as suggested by [START_REF] Körner | No need for pipes when the well is dry -a comment on hydraulic failure in trees[END_REF] could be the key to understand tree mortality. Indeed, pressing question is to know whether the relocation of water from plant reserves would be enough for the key tissues to survive a drought event.

Fine root failure hypothesis

Contrary to our study which focused on stem hydraulic failure, a recent study on trees drought-driven mortality in Texas suggested that the fine root failure was probably more related to mortality than stem hydraulic failure as the trees which suffers the most important mortality rates (i.e. Juniperus) have lower (more negative) stem P88 values (i.e. more resistance to embolism) than those showing lower mortality rates (i.e. Quercus) [START_REF] Johnson | Co-occurring woody species have diverse hydraulic strategies and mortality rates during an extreme drought[END_REF]. So according to their results, a more detailed study considering the loss in hydraulic functioning in other plants organs as fine roots during drought would be required to evaluate relative role of hydraulic failure at root and stem level on drought-induced tree mortality.

When do plants die? Which trait can be used as proxy for mortality?

Our results show that most of the plants were still showing living cells prior to rehydration however not all of them were able to recover from drought. Therefore, when does a plant cross the point of no return and cannot recover anymore? This question has been widely debated in recent publications [START_REF] Mcdowell | Mechanisms of plants survival and mortality during drought: why do some plants survive while others succumb to drought?[END_REF], Anderegg et al. 2012a), where mortality has been defined as "a complex system failure due to lack of water resources". However, this definition still does not define when a plant crosses from life to death which is a current priority to understand the mechanism causing tree mortality (McDowell et al. 2011a[START_REF] Martinez-Vilalta | Greater focus on water pools may improve our ability to understand and anticipate drought-induced mortality in plants[END_REF].

Unfortunately, our study failed to give a quantitative physiological threshold for droughtinduced tree mortality. Indeed, our results contrast with those reported by [START_REF] Guadagno | Dead or alive? Using membrane failure and chlorophyll fluorescence to predict mortality from drought[END_REF] showing how the loss of variable fluorescence could work as an operational proxy for plant mortality in Brassica rapa. In our study, no significant differences in PS II efficiency were observed between the trees able to recover and those showing no recovery from drought. This difference between Guadagno's and our study could be related with possible differences in main mechanisms related with the drought-mortality in herbaceous and woody species, although more research on this question should be carried out to confirm this hypothesis. However, our results agree with [START_REF] Guadagno | Dead or alive? Using membrane failure and chlorophyll fluorescence to predict mortality from drought[END_REF] in that membrane failure at the cellular scale could be a possible proxy for mortality in conifers since it clearly seems to be the most proximate cause of death.

However the link between the membrane and the hydraulic failures is still unresolved.

As suggested by [START_REF] Martinez-Vilalta | Greater focus on water pools may improve our ability to understand and anticipate drought-induced mortality in plants[END_REF] a focus on plant water status is necessary to understand drought-induced mortality in plants. Therefore, the RWC for both leaf and stem was monitored during this study. However, S-RWC differed between plants that recovered from drought and those that did not in the case of Pseudotsuga. At the leaf level, despite [START_REF] Kursar | Tolerance to low leaf water status of tropical tree seedlings is related to drought performance and distribution[END_REF] showed that tolerance to low leaf water status is correlated with species drought performance [START_REF] Kursar | Tolerance to low leaf water status of tropical tree seedlings is related to drought performance and distribution[END_REF]) [START_REF] Kursar | Tolerance to low leaf water status of tropical tree seedlings is related to drought performance and distribution[END_REF]) [START_REF] Kursar | Tolerance to low leaf water status of tropical tree seedlings is related to drought performance and distribution[END_REF], differences in leaf relative water content before rehydration of the trees in our study were not significant between recovering and non-recovering trees, invalidating this trait as a possible proxy identifying trees able to recover from drought. This discrepancy between [START_REF] Kursar | Tolerance to low leaf water status of tropical tree seedlings is related to drought performance and distribution[END_REF] and our study is probably due to the fact that L-RWC was measured in our study when plants showed levels of water stress much higher than in [START_REF] Kursar | Tolerance to low leaf water status of tropical tree seedlings is related to drought performance and distribution[END_REF]. So, probably, leaves were already hydraulically disconnected from the stems when measuring L-RWC, i.e. just before applying the recovery irrigation, for both species.

This would have favored a faster dehydration of the leaves in comparison with the stems. It is also important to consider that this study was conducted on young trees in pots and that, to confirm if the results obtained can be extrapolated to mature trees growing in the field, a more extended and detailed experiment including both young and mature trees of different species and exposed to different levels of drought would be required in order to improve our knowledge about the key physiological thresholds for drought-induced tree mortality.

Conclusions and perspectives

By combining a living-cell staining process with LVDT sensors and PLC measurements, this study showed that the common thresholds for recovery and point of death considered until now, i.e P50 for conifers and P88 for angiosperms, seem to be not accurate enough for evaluating tree mortality. Our results showed that plants with PLC levels of 98.6% for Prunus (angiosperm) and 67.92% for Pseudotsuga (conifer) were still able to draw water from soil and recover from drought when they were rewatered. As our results were obtained on young trees, i.e. between 2 and 4 years, and for an angiosperm and a conifer species only, a more exhaustive and wider study including several species for each plant group and including individuals within a wider range of ages, i.e. from young to mature trees, would be required in order to confirm if our observations represent a general pattern for trees. So, if P50 and P88 cannot stand for threshold of mortality identification, what physiological trait can be used to this purpose? This question is still unresolved as none of the followed traits during the time-course of this experiment demonstrated a clear threshold for distinguishing, at severe drought conditions, those trees that have succumbed to drought from those that would still be able to recover from it and therefore survive. Therefore, none of them would work as a proxy for tree mortality on their own. The fact that plants that were exposed to severe drought conditions and still show some living tissues at the stem level as well as a minimal hydraulic functioning, however, makes us to hypothesize that, as far as the remaining living tissues of the stem are hydraulically connected to the roots, the recovery of the tree from drought is possible. Still, after the hydraulic disconnection between the stem and the root, some living tissue could be detected at the stem for a given amount of time, what could generate some confusion at the time of determining if a plant is alive, dying or already dead base on these observations only.

Results from our study have generated new interesting questions about the behavior of the living cells during dehydration. Thus, whether isolated living cells are capable of dedifferentiate and renewal the plant tissues during the recovery from drought is still unclear. Similarly, dynamic of cellular death, i.e. what are the first and last tissues to die when trees reach high levels of water stress, is still unclear. After this study, however, we have identify different techniques that could be very helpful for addressing these unsolved questions. Thus, the FDA staining process developed in this study; correlated with the recovering ability of the tree shown by LVDT sensors, would be helpful to identify key tissues for tree survival. Also, if plants rely on their own water reserves to keep the irrigation of the different tissues during drought, it would be interesting to look at the water dynamic in the different plant tissues during a drought event. For this purpose, Nuclear Magnetic Resistance (NMR) imaging could be used like it was done by Barigah et al. (2013a) to identify the water distribution in trees. The use of tracers such as stable isotopes (e.g. deuterium) as suggested by [START_REF] Körner | No need for pipes when the well is dry -a comment on hydraulic failure in trees[END_REF] could also help for identifying if the irrigation of those key tissues or survival are kept during drought until the very end (that is to say, the mortality point).

In addition, if a clear dynamic of water relocation is detected, carrying out a comparison between a wide range of species, with different distribution ranges and different strategies to cope with drought, could be interesting in order to check for possible common evolutionary patterns for the preservation of certain tissues during a drought event.

Figure 1 :Figure 2 :

 12 Figure 1: Annual global anthropogenic carbon dioxide emissions. Half of the cumulative anthropogenic CO2 emissions between 1750-2011 were emitted in the last 40 years mainly due to fossil fuel combustion linked with economic development. (Intergovernmental Panel on Climate Change, 2014)

Figure 3 :

 3 Figure 3: Change in average surface temperature (a) and change in average precipitation (b).The worst-case scenario (RCP8.5) evinced that the temperature elevation could reach between +1.5°c and +11°c in 2100. For precipitations, RCP8.5 scenario show that mid-latitude and subtropical regions will experiment lower means precipitations than actual while in wet regions precipitations will increase.(Intergovernmental Panel on Climate Change, 2014)

Figure 4 :

 4 Figure 4: Water circulation in plants : Soil-Plant-Atmosphere continuum. Xylem sap flow following the cohesion-tension theory. (McElrone et al. 2013)

Figure 5 :Figure 6 :

 56 Figure 5: Consequences of drought on forests and climate change.Anthropic emissions of GHGs are the main reason to global warming causing higher temperatures that will disrupt the water cycle and cause lower precipitations in arid areas. The combination of those two elements is likely to cause an increase in drought frequency, intensity and duration. Drought stress induces higher tree mortality which reduces the number of trees on Earth. Because trees take a long time to regrow and therefore cannot compensate the humans' emissions of CO2, a lower number of trees reduces the potential carbon storage, and, as a result, more carbon dioxide will remain in the atmosphere and thus enhance global warming. (©Marylou MANTOVA)

Figure 7 :Figure 8 :

 78 Figure 7: Cavitation mechanism. As the tension increases at the pit-membrane level, a critical tension is reached and water pass from liquid state to vapor state. Because of xylem tension, the void created extends and generates cavitation. (© 2013 Nature Education Adapted from Tyree & Zimmermann 2002, McElrone et al. 2013)

3. 2 .

 2 Intense drought: hydraulic failure 3.2.1. Hydraulic failure definition and mechanism It is well known now that most of the mortality events occurring under severe drought conditions are induced by the hydraulic failure of the xylem tissue due to the occurrence of cavitation events. (McDowell et al. 2008) [Figure 6]. Thus, during a severe drought event, soil water availability decreases while the evaporative demand and cuticle conductance increase resulting in an increment of the xylem tension that induces the occurrence of cavitation events in the xylem. Cavitation is the change from liquid water to water vapor [Figure 7] (Dixon and Joly 1894, Tyree and Sperry 1989) that results in the formation of gas bubbles (emboli) in the xylem conduits and provokes the hydraulic dysfunction of the xylem reducing the plant water transport capacity.Thus, during intense drought, as VPD and xylem tension increase, the cavitation can spread throughout the entire xylem[START_REF] Choat | Noninvasive Measurement of Vulnerability to Drought-Induced Embolism by X-Ray Microtomography[END_REF]. As the percentage of cavitated vessels increases, the hydraulic conductance of the xylem decreases until the flow of water stops and provokes the desiccation of the plant tissues, the cells death and, as a last resort, the death of the tree. Despite the efforts of plants to reduce water loss by closing stomata before the onset of hydraulic failure [Figure8], plants still suffer from water-stress as the water potential (Ψ) continue decreasing due to the water losses through the cuticle[START_REF] Kerstiens | Cuticular water permeability and its physiological significance[END_REF] and stomatal leakiness[START_REF] Oren | Transpiration in response to variation in microclimate and soil moisture in southeastern deciduous forests[END_REF].

Figure 9 :

 9 Figure 9: Chemical reaction transforming fluorescein diacetate in fluorescein.(https://www.thermofisher.com/order/catalog/product/F1303 modified)

  [Figure 9]. Once excited by wavelength around 490-500nm, the fluorescein molecule restitutes a yellow/green fluorescence [Figure 10], allowing the identification of the living cells [Figure 11].

Figure 13 :

 13 Figure13: Changes in trunk diameter (mm) recorded with LVDT sensors on young peach trees (Prunus persica L.). Surrounded in red is the daily variation of the trunk diameter of 5-year-old peach tree. It is possible to notice a shrink in diameter in light conditions (from 09:00 to 17:00). The growth rate of the peach tree can be determined by looking at the differences between a and b levels in regard to the time variation.[START_REF] Simonneau | Diurnal changes in stem diameter and plant water content in peach trees[END_REF]

Figure 14 :

 14 Figure 14: Summary diagram of the state of the art, ongoing questions and purpose of the master project. (©Marylou MANTOVA)

Figure 15 :

 15 Figure 15: Timeline showing the different phases of the experiment and the time of punctual measurements of the different followed traits associated with the expected variations in stem water potential (ΨS) and stem diameter (ΔLVDT).

Figure 16 :

 16 Figure 16: Vulnerability curves to cavitation for Prunus lusitanica stems (a) and Pseudotsuga menziesii stems (b) a) Vulnerability curve for Prunus lusitanica stems obtained on four different samples using the optical method (Brodribb et al. 2017). The P50 is evaluated at -4.3MPa. b) Vulnerability curve for Pseudotsuga menziesii stems obtained on five different samples using the CAVITRON technique developed by Cochard in 2002. The P50 is evaluated at -3.4MPa. According to these vulnerability curves, Prunus individuals seems to be more resistant to cavitation dans Pseudotsuga individuals making them more resistant to drought.

  During the time course of the experiment and for a continuous monitoring of the water potential, 4 psychrometers (ICT international, Australia) were installed in 4 plants (i.e. one psychrometer per plant) at the stem level and covered with aluminum foil to prevent their direct exposure to the sunlight and minimize the effect of external temperature variations[START_REF] Vandegehuchte | Long-term versus daily stem diameter variation in co-occurring mangrove species: Environmental versus ecophysiological drivers[END_REF] [Figure17]. Psychrometers continuously recorded the stem water potential every 30mn.Regular measurements were also made using a Scholander-type pressure chamber (PMS, Corvallis, Oregon, USA) to monitor the stem xylem water potential (ΨS), the leaf water potential (ΨL), and assess plant water status[START_REF] Mccutchan | Stem-water potential as a sensitive indicator of water stress in prune trees (Prunus domestica L. cv. French)[END_REF]. These additional measurements confirmed the accuracy of the values collected by the psychrometers (for the stem xylem water potential) and also enabled us to monitor a larger number of plants since the amount of ICT's available was limited. Stem water potential was always measured in two fully developed and healthy leaves previously bagged for at least forty minutes prior the measurements to prevent transpiration and promote equilibrium with the plant axis. The stem xylem water potential was then assumed to be the same as the stem bearing the covered leaves[START_REF] Mccutchan | Stem-water potential as a sensitive indicator of water stress in prune trees (Prunus domestica L. cv. French)[END_REF]

Figure 18 :Figure 17 :

 1817 Figure 18: LVDT sensors installed on plants during the time-course of the experiment. a) LVDT sensor installed on Pseudotsuga menziesii trunk; b) LVDT sensor set up on Prunus lusitanica stem. ©Marylou MANTOVA

Figure 19 :

 19 Figure 19: X-ray microtomograph (Micro-CT) scans showing the state of embolism of Prunus lusitanica individuals in a) native scan and b) after the cut and injection with air. The black holes correspond to embolized vessels of xylem. The percentage loss of conductance is determined by computing the amount of cavitated vessels before and after the cut and is here equal to 4.65%.

Figure 20 :

 20 Figure 20: Dynamic of the stem diameter recorded by LVDT (in mV)(1,2), the stem water potential (ΨS)(3,4), the leaf water potential (ΨL)(5,[START_REF]Plant material, growth conditions and experimental design[END_REF] in Prunus lusitanica (a) and Pseudotsuga menziesii (b).(a) For Prunus individuals, the dehydration started on the third day of the experiment (green line) for Prunus 1,2,3 and 4 and ended 8 days later with the beginning of the rehydration phase (blue line). The dehydration of Prunus 6 started on the first day (dashed green line) and ended 8 days later (dashed grey line). ΨS and ΨL consisted of punctual measurements made with the Scholander Pressure Chamber. (b) For Pseudotsuga individuals, the dehydration (DH) started between the 9 and the 16 day of the experiment and ended with the rehydration (RH) between the day 22 and the 41 of the experiment. ΨS was continuously recorded with ICTs psychrometers while ΨL was measured punctually with the Scholander Pressure Chamber. For both species, ΨL measurements were measured until reaching the limit of the pressure chamber (-10MPa).

Figure 21 :

 21 Figure 21: Variation of photosystem II efficiency (Fv'/Fm') (a), stem relative water content (S-RWC) (b), leaf relative water content (L-RWC) (c) and stem electrolyte leakage (EL) (d) in (1) Prunus lusitanica and (2) Pseudotsuga menziesii. Measurements were performed on all individuals in control conditions (Control) and after the drought event (e.g before re-watering the plants: BRW). The post re-watering (PRW) data correspond to the measurements performed on recovering individuals 27 to 76 days after rehydrating the trees. ATR stands for the individuals able to recover from drought while NATR refers to the individuals not able to recover from drought. Different letters indicate statistically significant differences (α=0.05) within individuals for each species.

2 .

 2 Dynamic of photosystem II efficiency, electrolyte leakage and relative water content During the dehydration of the plants, a significant decrease in photosystem II efficiency (Fv'/Fm') and both in leaf (L-RWC) and stem relative water content (S-RWC) were observed for both species [Figure 21]. Thus, Fv'/Fm' mean values for Prunus decreased from 0.763 in control conditions to 0.498 at the end of the dehydration phase for those individuals that were able to recover (as indicated by changes in stem diameter) after irrigation (i.e. Prunus 1 and Prunus 6). However, those individuals that did not show any recovery after being rehydrated showed mean Fv'/Fm' values of 0.322 [Figure 21 (a)]. Similar results were observed for Pseudotsuga [Figure 21 (b)], which showed a PS II efficiency for individuals under control conditions of 0.750 and of 0.190

Table 2 :

 2 Summary of the Prunus lusitanica (a) and Pseudotsuga menziesii (b) drought experiment.Each individual was dehydrated until its water potential surpass the P88 for Prunus and the P50 for Douglas, respectively. The PLC was measured on three stem samples of each plant using one hydraulic technique (Xyl'EM) and one imaging technique (Micro-CT). PSII efficiency (FV'/FM'), stem and leaf relative water content (S-RWC; L-RWC) and electrolyte leakage (EL) were then determined for each individual. Finally, the staining process using

Figure

  Figure 22: Theoretical curve showing variation in stem diameter in trees able to recover (grey) and trees not able to recover from drought (blue). The PLC (loss of conductance) reached at the end of the dehydration phase for each individual was above the common threshold for mortality for both species: P50 for Pseudotsuga and P88 for Prunus.

Figure 23 :

 23 Figure 23: Evolution of the amount of living tissues between control conditions, before re-hydration (BRW), and 27 to 77 days after re-hydration (PRW) in cross section of Prunus and Pseudotsuga stained with fluorescein diacetate (FDA) and observed with a fluorescence microscope.

  )] were able to recover from drought and to maintain the amount of living cells days after the rehydration [Figure 23]. All the other plants that showed living cells before rehydration (Prunus 2, Prunus 3, Prunus 5, Prunus 8) lost the fluorescence signal during the rehydration phase. The same results were obtained for Pseudotsuga. Douglas 1, Douglas 4 [Table 2 (b)] and Douglas 6 [Figure 23] all showed living cells in the bark and phloem BRW. However, only Douglas 1 was able to recover from drought. All the other individuals did not emit fluorescence before rehydration (e.g. Douglas 2 and Douglas 3) [Table 2(b)]. Douglas 1 was able to maintain its amount of living cells during the rehydration phase while all the other ultimately loose the fluorescence signal after re-watering the plants [Figure 23].

Table 1 :

 1

Summary table of the individuals and their associated period of drought event.

Table 3 :

 3 Table summarizing the evolution of the different traits followed during the time-course of the experiment in plants able to recover from drought.

This internship took place at the integrative physics and physiology of tree in fluctuating environment (PIAF) research unit of the French National Institute for Agricultural Research (INRA) station of Clermont-Ferrand.

Supplementary data

Skills

During this internship, I had the opportunity to develop many skills.

First of all, working in an international team helped me to greatly improve my English To finish, my social skills were also greatly improved as I took part of number of activities at, and outside, the research station with my co-workers.


Keywords: Escherichia coli, Yersinia, Enterobacter, Klebsiella, Serratia, Citrobacter, Salmonella, Alcaligenes, Pseudomonas

One of the leader in the production and consumption of eggs in the EU  14.9 billion eggs produced / year  213 eggs consumed /year / person 60 -70 % 30 -40 % Itavi and AND International, 2017 What about egg microbiology? Eggshell surface Contaminated in the environments of hen breeding and egg handling and storage +++ Gram-positive bacteria Streptococcus, Aerococcus, Micrococcus, Staphylococcus, and low occurrence of Bacillus + Gram-negative bacteria

Introduction

How to control microbial spoilage in the sector of egg processing ?

A key challenge for the reduction of food wastes , 2014 ;Miller et al., 2010 ;Baron et al., 2007 ;Protais et al., 2007 ;Protais et al., 2006 ;Wood et Waites, 1988 ;Foegeding et Stanley, 1987 ;Payne et Gooch, 1980 ;Shafi et al., 1970 What are the main features of the bacterial collection coming from whole egg products?

  for egg product stabilization for ensuring egg product safety Sterile except under sporadic cases of contamination by Salmonella (mainly Salmonella Enteritidis) during egg formation Less than 1% of contamination but still 30% of strong evidence of salmonellosis caused by eggs or egg products in the EU • Strong affinity for the avian reproductive tract • Persistence on the eggshell surface • Resistance to egg defence heat-resistant psychrotrophic bacteria in liquid egg products and sensitive food where they are used as ingredients? development of industrial efficient tools to detect food spoilage contaminants or the products of their metabolism Industrial egg-based refrigerated desserts The famous "île flottante" dessert as a research model Custard cream comprising whole egg or egg yolk as ingredient at 55 to 57 °C for 2 to 5 min in France What are the main contaminants of liquid egg white products? STLOpen Days 19-21 March 2019 7 What are the main bacterial contaminants of liquid egg products?

Frequency

  main features of the bacterial collection coming from whole egg products? main features of the bacterial collection coming from whole egg products? Mainly coming from the eggshell surface  contamination at the egg breading step De Reu et al., 2005 and 2006 ; Musgrove et al., 2004 et 2005 ; Mallet et al., 2006 Main contaminants Techer et al., 2015, J. Food Science Persistence after the heat-treatment STLOpen Days 19-21 March 2019 19 What are the main bacterial contaminants of liquid egg white products? -resistant psychrotrophic bacteria in pasteurised liquid egg products Spoilage activities, in the product itself and when used as ingredient, even at low temperature Genetic signature of the spoilage risk due to psychrotrophic B. cereus group bacteria And now? Study of the bacterial spoilage metabolism Development of online tools to early detect food spoilage contaminants or the products of their metabolism THANK YOU FOR YOUR ATTENTION Please visit http://www.rennes.inra.fr/stlo_eng Ici une image sur le thème de votre présentation MERCI STLOpen Days 19-21 March 2019

Table eggs

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		Egg products
		Biscuits, pastries,
		desserts, pasta, chocolate
		and sugar confectionery,
	Source of nutrients	delicatessen, seafood products, wines, etc.
	Source of ingredients
	Foaming
	Emulsifying
	Coloring
	Binding
	Thickening
	Flavoring

Jan et al., 2011. Food Microbiol.