Roberto Luis Salomón

Kathy Steppe
email: kathy.steppe@ugent.

Jean Marc Ourcival
email: jean-marc.ourcival@cefe.cnrs.fr

Selwyn Villers
email: selwyn.villers@ugent.

Jesús Rodriguez-Calcerrada

Roderick Schapman
email: schapmanroderick@hotmail.com

Jean Marc Limousin
email: marc.limousin@cefe.cnrs.fr

Roberto Luis

Hydraulic acclimation in a Mediterranean oak subjected to permanent throughfall exclusion results in increased stem hydraulic capacitance

Keywords: capacitive water, carbon gain optimization, desorption curve, drought-stress, electrical resistivity tomography, frequency domain reflectometry, sapwood area, stem water content, rainfall exclusion, tree acclimation

Stem water storage capacity and hydraulic capacitance (CS) play a crucial role in tree survival under droughtstress. To investigate whether CS adjusts to increasing water deficit, variation in stem water content (StWC) was monitored in vivo for two years and related to periodical measurements of tree water potential in Mediterranean Quercus ilex trees subjected either to permanent throughfall exclusion (TE) or to control conditions. Seasonal reductions in StWC were larger in TE trees relative to control ones, resulting in greater seasonal CS (154 and 80 kg m -3 MPa -1 , respectively), but only during the first phase of the desorption curve, when predawn water potential was above -1.1 MPa. Below this point, CS decreased substantially and did not differ between treatments (<20 kg m -3 MPa -1 ). The allometric relationship between tree diameter and sapwood area, measured via electrical resistivity tomography, was not affected by TE. Our results suggest that (i) CS response to water deficit in the drought-tolerant Q. ilex might be more important to optimize carbon gain during well-hydrated periods than to prevent drought-induced embolism formation during severe drought stress, and (ii) enhanced CS during early summer does not result from proportional increases in sapwood volume, but mostly from increased elastic water.

Introduction

In the early 20 th century, [START_REF] Spalding | Mechanical adjustment of the suaharo (Cereus giganteus) to varying quantities of stored water[END_REF] reported for the first time clear evidence of stem water storage and related diameter changes for the Suaharo desert plant. Later studies described a significant imbalance between leaf transpiration and soil water uptake, and suggested an important role of water storage throughout plant tissues, mainly in the stem, to satisfy the evaporative demand under water limiting conditions (Kramer, 1937, and references therein). This pioneering work demonstrated that tree stems are not inert pipelines along the root-to-leaf continuum. A more recent body of studies has since highlighted the physiological relevance of stem water pools in plant transpiration, hydraulic modelling and survival [START_REF] Waring | Sapwood water storage: its contribution to transpiration and effect upon water conductance through the stems of old-growth Douglas-fir[END_REF][START_REF] Tyree | Water-storage capacity of Thuja, Tsuga and Acer stems measured by dehydration isotherms[END_REF][START_REF] Goldstein | Stem water storage and diurnal patterns of water use in tropical forest canopy trees[END_REF]Zweifel et al. 2001;[START_REF] Steppe | A mathematical model linking tree sap flow dynamics to daily stem diameter fluctuations and radial stem growth[END_REF][START_REF] Meinzer | Xylem hydraulic safety margins in woody plants: coordination of stomatal control of xylem tension with hydraulic capacitance[END_REF][START_REF] Scholz | Hydraulic Capacitance: Biophysics and Functional Significance of Internal Water Sources in Relation to Tree Size[END_REF][START_REF] Salomón | Stem hydraulic capacitance decreases with drought stress: implications for modelling tree hydraulics in the Mediterranean oak Quercus ilex[END_REF][START_REF] Körner | No need for pipes when the well is dry-a comment on hydraulic failure in trees[END_REF][START_REF] Martinez-Vilalta | Greater focus on water pools may improve our ability to understand and anticipate drought-induced mortality in plants[END_REF]. These studies have helped to more comprehensively understand drought-driven embolism formation and plant hydraulic functioning.

When a plant transpires, a gradient in water potential (Ψ) along the soil-plant-atmosphere continuum is generated. This vertical Ψ gradient results in a radial Ψ gradient between stem xylem conduits and surrounding tissues that drives a dynamic radial water flow within tree stems according to transpiration needs [START_REF] Steppe | Diel growth dynamics in tree stems: linking anatomy and ecophysiology[END_REF]. As a result, stem water pools are depleted when the evaporative demand exceeds tree water uptake by the root system. Inversely, stem water pools are replenished when root water uptake exceeds tree transpiration until stem radial Ψ gradients reach hydraulic equilibrium. Under drought stress conditions, water tension in xylem conduits increases and the xylem functionality is endangered by the risk of embolism, which may hinder tree transpiration and carbon gain. Capacitive water release from tree stems buffers increases in xylem tension thereby delaying or avoiding embolism formation in situations of water limitation [START_REF] Meinzer | Coordination of leaf and stem water transport properties in tropical forest trees[END_REF][START_REF] Meinzer | Xylem hydraulic safety margins in woody plants: coordination of stomatal control of xylem tension with hydraulic capacitance[END_REF][START_REF] Scholz | Hydraulic Capacitance: Biophysics and Functional Significance of Internal Water Sources in Relation to Tree Size[END_REF][START_REF] Vergeynst | Cavitation: A blessing in disguise? New method to establish vulnerability curves and assess hydraulic capacitance of woody tissues[END_REF]. Water release from stem tissues to the transpiration stream across a gradient of xylem Ψ is described by the stem desorption curve, theoretically divided into three phases [START_REF] Tyree | Water-storage capacity of Thuja, Tsuga and Acer stems measured by dehydration isotherms[END_REF][START_REF] Tyree | The hydraulic architecture of trees and other woody plants[END_REF][START_REF] Pratt | Conflicting demands on angiosperm xylem: Tradeoffs among storage, transport and biomechanics[END_REF][START_REF] Steppe | The potential of the tree water potential[END_REF]). The first phase of this curve occurs at relatively high xylem Ψ, when the plant is well hydrated.

Here, a relatively large amount of capillary water is released for a small change in xylem Ψ in a nearly linear manner. Capillary water is mostly located in intercellular spaces and open conduits (i.e. conduits with open ends), tracheids and fibres. The desorption curve flattens along the second phase as water is released at a slower rate for a comparatively large reduction in stem Ψ. Along this phase, water is primarily released from elastic living cells, integrating parenchyma, cambium and peridermal tissues. Finally, the desorption curve steepens during the third phase, when water is mostly released from embolized vessels or tracheids before total stem dehydration. The amount of water released at this point largely depends on the amount and lumen size of embolized conduits. Recent studies performed in vivo using X-ray microtomography question, however, whether this sequential release of capacitive water sources along the desorption curve, which has been derived from excised branches, can be extrapolated to living plants [START_REF] Knipfer | Storage compartments for capillary water rarely refill in an intact woody plant[END_REF][START_REF] Knipfer | Spatiotemporal coupling of vessel cavitation and discharge of stored xylem water in a tree sapling[END_REF].

Stem hydraulic capacitance (CS; kg m -3 MPa -1 ) is defined by the amount of stem water released (ΔW; kg) for a given change in xylem water potential (ΔΨ; MPa) per unit of tissue volume (V; m 3 ) [START_REF] Tyree | The hydraulic architecture of trees and other woody plants[END_REF] :

𝐶 𝑆 = 𝛥𝑊 𝛥𝛹 1 𝑉

Eqn. 1

Stem capacitance largely varies according to the stem water status [START_REF] Vergeynst | Cavitation: A blessing in disguise? New method to establish vulnerability curves and assess hydraulic capacitance of woody tissues[END_REF][START_REF] Salomón | Stem hydraulic capacitance decreases with drought stress: implications for modelling tree hydraulics in the Mediterranean oak Quercus ilex[END_REF]). However, it is commonly estimated by the initial slope of the desorption curve [START_REF] Meinzer | Whole-tree water transport scales with sapwood capacitance in tropical forest canopy trees[END_REF][START_REF] Barnard | Climate-related trends in sapwood biophysical properties in two conifers: avoidance of hydraulic dysfunction through coordinated adjustments in xylem efficiency, safety and capacitance[END_REF][START_REF] Mcculloh | The dynamic pipeline: hydraulic capacitance and xylem hydraulic safety in four tall conifer species[END_REF][START_REF] Vergeynst | Cavitation: A blessing in disguise? New method to establish vulnerability curves and assess hydraulic capacitance of woody tissues[END_REF], when the stem is well hydrated. Stem storage capacity is largely determined by stem size [START_REF] Meinzer | Dynamics of transpiration, sap flow and use of stored water in tropical forest canopy trees[END_REF][START_REF] Scholz | Hydraulic Capacitance: Biophysics and Functional Significance of Internal Water Sources in Relation to Tree Size[END_REF], and more specifically, by sapwood volume [START_REF] Goldstein | Stem water storage and diurnal patterns of water use in tropical forest canopy trees[END_REF]: the larger the tree, the more room for water storage.

Moreover, beyond this intuitive relationship, larger trees seem to rely to a greater extent on stored water to fulfil transpiration needs [START_REF] Phillips | Reliance on stored water increases with tree size in three species in the Pacific Northwest[END_REF][START_REF] Scholz | Hydraulic Capacitance: Biophysics and Functional Significance of Internal Water Sources in Relation to Tree Size[END_REF] and thus have greater CS [START_REF] Domec | Cavitation and water storage capacity in bole xylem segments of mature and young Douglas-fir trees[END_REF][START_REF] Mcculloh | The dynamic pipeline: hydraulic capacitance and xylem hydraulic safety in four tall conifer species[END_REF], although the underlying causes of a direct relation between CS and sapwood volume remain unexplored. This suggests, nevertheless, that CS might be a relatively plastic trait related to biomass allocation patterns: as trees grow and allocate most of their biomass to the stem [START_REF] Poorter | Biomass allocation to leaves, stems and roots: meta-analysis of interspecific variation and environmental control[END_REF], the amount of conducting and storing biomass increases to the detriment of transpiring tissues (i.e., leaves).

A trade-off between CS and xylem resistance to embolism has been reported, with "drought-avoidant" species characterized by low wood density and embolism-vulnerable xylem having large CS, which dampens the reductions in xylem Ψ, and "drought-tolerant" species characterized by high wood density and xylem conduits able to maintain functionality at low Ψ having low CS [START_REF] Meinzer | Coordination of leaf and stem water transport properties in tropical forest trees[END_REF][START_REF] Meinzer | Xylem hydraulic safety margins in woody plants: coordination of stomatal control of xylem tension with hydraulic capacitance[END_REF][START_REF] Mcculloh | The dynamic pipeline: hydraulic capacitance and xylem hydraulic safety in four tall conifer species[END_REF]. Accordingly, evergreen sclerophyll species across an aridity gradient exhibited lower wood density, greater CS and less negative xylem Ψ in high-rainfall sites [START_REF] Richards | Sapwood capacitance is greater in evergreen sclerophyll species growing in high compared to low-rainfall environments[END_REF]. This pattern, however, does not seem to hold at the intraspecific level. Comparisons in ponderosa pine and Douglas-fir showed that, for both species, trees growing in drier locations had greater CS and sapwood thickness, and were more vulnerable to embolism formation [START_REF] Barnard | Climate-related trends in sapwood biophysical properties in two conifers: avoidance of hydraulic dysfunction through coordinated adjustments in xylem efficiency, safety and capacitance[END_REF]. These authors suggested that enhanced CS under drier conditions may partially explain the lack of acclimation of structural traits related to the resistance of xylem to embolism, which has been demonstrated to be limited in intraspecific comparisons across aridity gradients [START_REF] Martínez-Vilalta | Hydraulic adjustment of Scots pine across Europe[END_REF]Wortemann et al. 2011;[START_REF] Lamy | Limited genetic variability and phenotypic plasticity detected for cavitation resistance in a Mediterranean pine[END_REF][START_REF] Schuldt | How adaptable is the hydraulic system of European beech in the face of climate change-related precipitation reduction?[END_REF][START_REF] Rosas | Adjustments and coordination of hydraulic, leaf and stem traits along a water availability gradient[END_REF], as well as in manipulative experiments that alter soil water availability [START_REF] Limousin | Change in hydraulic traits of Mediterranean Quercus ilex subjected to long-term throughfall exclusion[END_REF][START_REF] Hudson | Impacts of long-term precipitation manipulation on hydraulic architecture and xylem anatomy of piñon and juniper in Southwest USA[END_REF]. On the other hand, structural adjustment of biomass allocation is a key strategy to optimize water use [START_REF] Magnani | Adjustment of tree structure in response to the environment under hydraulic constraints[END_REF][START_REF] Choat | Triggers of tree mortality under drought[END_REF], and reductions of the leaf-to-sapwood area ratio are commonly reported as a mechanism to maintain homeostatic minimum stem Ψ, and hence hydraulic functionality, while supplying water to the canopy Funder drought stress [START_REF] White | The influence of drought on the relationship between leaf and conducting sapwood area in[END_REF][START_REF] Mencuccini | The ecological significance of long-distance water transport: short-term regulation, long-term acclimation and the hydraulic costs of stature across plant life forms[END_REF][START_REF] Martínez-Vilalta | Hydraulic adjustment of Scots pine across Europe[END_REF][START_REF] Martin-Stpaul | The temporal response to drought in a Mediterranean evergreen tree: comparing a regional precipitation gradient and a throughfall exclusion experiment[END_REF][START_REF] Rosas | Adjustments and coordination of hydraulic, leaf and stem traits along a water availability gradient[END_REF].

The need for better considering the role of stem water pools and CS in drought-driven tree mortality has recently been highlighted [START_REF] Körner | No need for pipes when the well is dry-a comment on hydraulic failure in trees[END_REF][START_REF] Martinez-Vilalta | Greater focus on water pools may improve our ability to understand and anticipate drought-induced mortality in plants[END_REF]. However, we still lack direct assessments on the effect of drought stress on CS and related metrics. Permanent rainfall manipulations in the field provide an excellent opportunity to investigate long-term plant acclimation to increasing water limitation, as this approach allows straightforward comparison of the surveyed trait in the same species and at the same site, thus minimizing confounding co-factors that may vary over space and time. To advance our knowledge on stem water pools and hydraulic acclimation to drought stress, cutting-edge technologies were applied to measure in vivo stem water content (StWC), CS, sap flow and sapwood area (SA) in Quercus ilex trees subjected to control conditions and long-term, partial throughfall exclusion (TE) in a Mediterranean stand. Variation in StWC was continuously monitored for two years using frequency domain reflectometry (FDR), and SA was discretely measured applying electrical resistivity tomography. Based on a previous modelling exercise at the study site, in which the reliance on stored water to daily transpiration increased with drought stress [START_REF] Salomón | Stem hydraulic capacitance decreases with drought stress: implications for modelling tree hydraulics in the Mediterranean oak Quercus ilex[END_REF], and given that CS is beneficial to buffer xylem Ψ reductions [START_REF] Meinzer | Xylem hydraulic safety margins in woody plants: coordination of stomatal control of xylem tension with hydraulic capacitance[END_REF], we hypothesized that TE would enhance CS, particularly under conditions of severe drought stress. If true, we expected that potential increases in CS would be related to allometric adjustments reflected in the relationship between stem diameter and SA.

Materials and methods

Site study and experiment setup

The study was conducted in the Puéchabon State Forest (Occitanie, France), in an experimental plot dominated by Q. ilex (43°44029″ N, 3°35045″ E, 270 m.a.s.l.). The site was historically coppiced until 1942.

Stand density is 4700 stems ha -1 , average dominant height is 5.5 m and most stems (> 70%) have a stem diameter at breast height (DBH) between 4 and 10 cm. Climate is Mediterranean with an annual mean temperature of 13.5 ºC and 953 mm of annual precipitation. Summer is hot and dry with less than 20% of the annual precipitation falling during this season. Air temperature, precipitation and relative humidity are measured in a weather station located in the experimental site (see e.g. [START_REF] Rambal | How drought severity constrains gross primary production (GPP) and its partitioning among carbon pools in a Quercus ilex coppice?[END_REF] for further details on the site). A permanent partial throughfall exclusion (TE) experiment started in spring 2003 in three plots of 10 × 10 m. PVC rain gutters were suspended below the canopy at ~ 1.5 m above the soil in order to cover one third of the plot area and drive throughfall outside the plots. This experimental set-up was shown to effectively reduce the total net precipitation by 28 % [START_REF] Limousin | Modelling rainfall interception in a mediterranean Quercus ilex ecosystem: Lesson from a throughfall exclusion experiment[END_REF]). In the control plots the same PVC gutters were suspended upside down to avoid microclimatic differences between treatments (see [START_REF] Rodríguez-Calcerrada | Leaf CO2 efflux is attenuated by acclimation of respiration to heat and drought in a Mediterranean tree[END_REF] for a photograph and further details on the TE setup). Four trees per treatment located within one of the replicate plots were selected in December 2016 to measure StWC, predawn and midday leaf Ψ, CS as the reduction in StWC for a given drop in Ψ (see below) and stem sap flow. Stem DBH of selected trees did not differ between treatments (P = 0.71), with mean values of 11.40 ± 0.85 cm. Ten trees in each treatment were selected to measure SA across a wide range of stem DBH in spring 2019.

Stem water content

Stem water content (StWC) was monitored by frequency domain reflectometry (FDR) with GS3 moisture probes (Decagon Instruments, Pullman, Wash., USA) [START_REF] Hao | Investigating xylem embolism formation, refilling and water storage in tree trunks using frequency domain reflectometry[END_REF][START_REF] Carrasco | Water storage dynamics in the main stem of subtropical tree species differing in wood density, growth rate and life history traits[END_REF][START_REF] Matheny | Observations of stem water storage in trees of opposing hydraulic strategies[END_REF]. According to the allometric relationship between stem diameter and SA previously observed at the experimental site (Eqn. 1 in [START_REF] Limousin | Longterm transpiration change with rainfall decline in a Mediterranean Quercus ilex forest[END_REF], the estimated sapwood depth in monitored trees (3.25 ± 0.23 cm) was smaller than the probe needle length (5.5 cm). Probe needles were therefore shortened to 3 cm with a rotary tool to avoid needle penetration into heartwood [START_REF] Matheny | Observations of stem water storage in trees of opposing hydraulic strategies[END_REF]. Probes were individually calibrated before installation in order to ensure the accuracy of StWC estimates from raw readings of dielectric permittivity (ε). For this, Q. ilex trees were felled outside the experimental plots and cut into 15-cm-long stem segments of similar diameter to the selected trees. Bark was sanded in non-crooked sections before segments were immersed into deionized water overnight. Volume of stem segments was estimated the next day by Archimedes' principle to the nearest 0.1 mL, and probes were installed afterwards. Three parallel holes were radially drilled into the stem using a customized drill guide where bark was previously sanded. To ensure a tight contact between stem and needle, the drill bit diameter was equal to that of the probe needle. A dead-blow hammer was used to carefully push the needles into the holes, and non-caustic silicone was applied to seal the stem-probe interface. After probe installation, stem segments were weighted to the nearest 0.1 g near water saturation point and as stems dehydrated at a room temperature of ~ 20 ºC, while ε was recorded every minute with a data logger (Campbell CR3000, Campbell Scientific Ltd, Shepshed, UK). Stem segments were disconnected from the logger to be weighted, twice per day during the first week of dehydration and then once per day during the second week. After two weeks, probes and silicon were detached and weighted to correct weight readings of the stem segment. Dry weight was determined after four additional weeks in a dry oven at 60 ºC until constant weight, and StWC (kg m -3 ) was determined according to:

𝑆𝑡𝑊𝐶 = 𝐹𝑟𝑒𝑠ℎ 𝑤𝑒𝑖𝑔ℎ𝑡-𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑉𝑜𝑙𝑢𝑚𝑒 Eqn. 2
The linear relationship between StWC and the mean √𝜀 recorded during the hour before and after the fresh weight reading was established according to manufacturer indications:

𝑆𝑡𝑊𝐶 = 𝑎 √𝜀 + 𝑏 Eqn. 3
where a and b are the slope and intercept of this linear relationship, respectively. A preliminary linear model showed that the intercept of this relationship significantly varied with each probe (P < 0.001, n = 9).

Considering probe as a random (intercept) factor, the slope of this relationship was significant (P < 0.001), whereas the interaction √𝜀 × probe was not (P > 0.1) denoting equal slopes among probes (Figure S1). The Because ε can be affected by temperature [START_REF] Chanzy | Correcting the temperature influence on soil capacitance sensors using diurnal temperature and water content cycles[END_REF], two additional probes were installed in dead stems to evaluate the potential effect of the temperature sensitivity of ε (α = Δε /ΔT; ºC -1 ) on estimates of ΔStWC. For this, α was estimated on a daily basis as the slope between ε and dead stem temperature (TSTEM; ºC) recorded by the probe, and the relationship between α and mean daily ε was evaluated across the surveyed period. A direct sub-daily relationship between ε and TSTEM in dead stems was observed during summer (ε < 4-5) resulting in roughly constant and positive temperature sensitivity of ε (α ≈ 0.04 ºC -1 , Figure S2). As dead stems rehydrated during wetter months, the sub-daily relationship between ε and TSTEM became non-significant, and even inversed at the highest range of ε (> 8-9) resulting in negative α (down to -0.03 ºC -1 ). This variable pattern of α in relation to ε throughout the year and the lack of overlap between ε ranges measured in dead (ε < 10) and living (ε > 10) trees prevented any temperature-correction of ε to refine ΔStWC estimates.

Gradients in water potential and stem water content

To calculate gradients in Ψ on seasonal and sub-daily bases, seven and six Ψ measurement campaigns were performed encompassing the dry season of 2017 (DOYs 171, 185, 234, 251, 268, 284 and 298) and 2018 (DOYs 171, 187, 133, 256, 276 and 297), respectively. In each campaign, leaf Ψ was measured at predawn and midday (ΨPD and ΨMD, respectively) in trees equipped with GS3 probes, except on DOY 268 of 2017 and DOY 256 of 2018, when uniquely ΨPD was measured. Leaf Ψ was measured with a pressure chamber (PMS1000, PMS Instruments, Corvallis, OR, USA) on freshly excised leafy shoots sampled at similar tree height and solar exposition. Two leafy shoots per tree were sampled, and a third one was added when the first two Ψ readings differed by more than 0.2 MPa. In cases of missing Ψ data for a particular tree at a given date (9 cases for a total of 104; 8 trees × 13 dates), treatment mean values were applied. Seasonal ΔΨ was estimated as the reduction in ΨPD relative to the spring maxima (-0.45 MPa; [START_REF] Limousin | Morphological and phenological shoot plasticity in a Mediterranean evergreen oak facing long-term increased drought[END_REF].

Ideally, stem Ψ readings would be necessary to estimate CS. Nevertheless, assuming hydraulic equilibrium between leaf and stem Ψ at predawn, the potential bias in seasonal CS estimates is negligible. Sub-daily ΔΨ was calculated as the difference between ΨPD and ΨMD (i.e., the sap flow driving force at midday; [START_REF] Limousin | Longterm transpiration change with rainfall decline in a Mediterranean Quercus ilex forest[END_REF]. Hence, sub-daily CS estimates are likely underestimated as sub-daily shoot ΔΨ can be greater than that of the stem, at least during well-hydrated conditions when leaf transpiration is substantial. To evaluate the potential error in sub-daily CS incurred when using shoot ΔΨ instead of stem ΔΨ, we alternatively used indirect estimates of stem ΔΨ to estimate sub-daily CS, as similarly approached in a modelling exercise at this study site [START_REF] Salomón | Stem hydraulic capacitance decreases with drought stress: implications for modelling tree hydraulics in the Mediterranean oak Quercus ilex[END_REF]. Briefly, leaf and stem Ψ were simultaneously measured at predawn and midday across a drought-stress gradient in a set of Q. ilex seedlings [START_REF] Rodríguez-Calcerrada | Drought-induced shoot dieback starts with massive root xylem embolism and variable depletion of nonstructural carbohydrates in seedlings of two tree species[END_REF]. Stem Ψ was measured by means of covering leaves with aluminium foil for 1h, until hydraulic equilibrium was reached. The ratio between stem and leaf ΔΨ along a gradient in ΨPD observed in Q. ilex seedlings was adjusted with a sigmoidal fit and applied here to alternatively estimate sub-daily CS.

To estimate seasonal and sub-daily CS following Eq. 1, seasonal and sub-daily ΔStWC were estimated for those days in which Ψ measurement campaigns were performed. Seasonal ΔStWC was estimated as the cumulative reduction in StWC relative to the spring maxima registered before summer drought (DOY 156 in 2017, andDOY 151 in 2018). Sub-daily ΔStWC was estimated as the difference between daily minimum StWC registered during high-transpiration hours (from 10:00 to 19:00 h) minus daily maximum StWC during low-transpiration hours (from 3:00 to 8:00 h). Additionally, the relationship between seasonal and sub-daily ΔStWC (not restricted to dates of Ψ measurement campaigns) was evaluated across the summer drought periods of 2017 and 2018. To ensure that sub-daily ΔStWC was driven by transpiration, and was not a consequence of water refilling following rain events, days with positive ΔStWC were excluded from analyses. On average, 9% of the days during the dry seasons in 2017 and 2018 were excluded, which is lower than the number of days in which rain was registered (15%).

Contribution of stored water to transpiration

Daily transpiration was estimated as the daily cumulative sap flow (kg day -1 ). Sap flow was estimated as the product of sap flux density (kg cm -2 h -1 ) and sapwood area (SA, cm 2 ). Sap flux density was measured with 20-mm long thermal dissipation probes designed according to [START_REF] Granier | Evaluation of transpiration in a Douglas-fir stand by means of sap flow measurements[END_REF] and installed at 1.2 m height.

Probes were shielded with aluminium cover to avoid direct solar radiation and rainfall. Sap flux density was recorded every 30 minutes with a data logger (model CR1000, Campbell Scientific)see [START_REF] Limousin | Longterm transpiration change with rainfall decline in a Mediterranean Quercus ilex forest[END_REF] for further details. Sapwood area was estimated from stem diameter according to the allometric relationship obtained for the study site [START_REF] Limousin | Longterm transpiration change with rainfall decline in a Mediterranean Quercus ilex forest[END_REF]; see discussion). Daily release of stored water (kg day -1 ) was estimated as the product of sub-daily ΔStWC in absolute terms (kg m -3 ) by sapwood volume (m 3 ). Sapwood volume was estimated assuming tree cylindrical shape as the product of SA by stem height measured in each tree equipped with GS3 probes. Finally, the contribution of stored water to transpiration was estimated as the ratio between daily values of stored water release and transpiration.

Sapwood area via electrical resistivity tomography

Electrical resistivity tomography (ERT) was used to quantify SA in a non-destructive manner following the approach described in [START_REF] Benson | Estimating conductive sapwood area in diffuse and ring porous trees with electronic resistance tomography[END_REF]. Briefly, twelve nails were inserted around each measured stem at approximately 1 m above the soil until the outer sapwood layer was reached. The geometry of the stem and the location of each nail was recorded using a digital calliper and a measuring tape. Nails were connected with electrodes to a 12-channel resistivity system (PiCUS TreeTronic, Argus Electronic Gmbh, Rostock, Germany), and the electrical resistivity (ER) among nails was recorded by the PiCUS software following voltage application and accounting for the geometry of the stem cross section. The resulting tomogram consists of a triangular-based lattice that informs about the spatial variability in ER throughout the stem cross section. The sapwood-heartwood boundary is commonly characterized by a sharp change in water content reflected by a corresponding change in ER, and can therefore be identified as the point of steepest slope of ER along the radial profile of a tomograph (see Figure S3 for an illustration) [START_REF] Guyot | Using electrical resistivity tomography to differentiate sapwood from heartwood: Application to conifers[END_REF][START_REF] Wang | Quantifying sapwood width for three Australian native species using electrical resistivity tomography[END_REF]). To estimate SA, it is necessary to delineate the boundary between heartwood and sapwood in two dimensions at a fine spatial resolution, which is achieved by means of rotating the surveyed radial profile at a given angle [START_REF] Benson | Estimating conductive sapwood area in diffuse and ring porous trees with electronic resistance tomography[END_REF]. For this, optimal bandwidth and the order of the polynomial function (n) used for fitting the gridded data generated by ERT need to be selected a priori.

Here, highest R 2 were obtained with a bandwidth of one cm and n = 7 for all surveyed trees. The R script provided as supplementary material in [START_REF] Benson | Estimating conductive sapwood area in diffuse and ring porous trees with electronic resistance tomography[END_REF] was used for SA determination. Total crosssection area derived from tape measurements (including bark) was consistently higher than that obtained via ERT (16.4 ± 8.6 %), likely because the tip of the electrodes were located just beneath the cambium layer so the bark area was unaccounted for in the tomograms. Bark thickness was therefore set to zero in the R script to estimate SA from ERT. Irregularities around the perimeter of the tomography leading to equivocal detection of the sapwood-heartwood boundary were excluded from the ER radial profile.

ERT readings were performed during spring 2019 (DOYs 105-109) in ten trees per treatment across a range of DBH between 6 and 16 cm. To minimize any potential effect related to sub-daily variation in temperature [START_REF] Schapman | How does sapwood area measured with ERT covary with stem growth in Fagus sylvatica L.?[END_REF]) and compare SA between treatments, ERT readings were performed around midday and alternatively between C and TE trees. Additionally, the accuracy of this approach was tested by examining the relationship between SA estimated via ERT and visual observation of wet sapwood depth in four freshly cut trees with similar range of DBH located outside the experimental plot (hereafter ´validation´ trees).

Validation trees were cut immediately after ERT readings to visually demarcate sapwood and heartwood, and SA was determined using the image-processing software ImageJ (version 1.52p, FIJI).

Data analyses

R software (version 3.6.1) was used for statistical analyses. Cubic smoothing splines were fitted to daily ΔStWC time series to minimize noise in ε readings after rain and at TSTEM below 0º C. Seasonal desorption curves exhibited a biphasic shape with different slopes at the beginning and at the end of the dry season (phases I and II hereafter). Comparisons of seasonal CS between treatments, years and between phases I and II (CS_I and CS_II hereafter) were performed by adjusting linear segmented mixed models with the segmented.lme function [START_REF] Muggeo | Segmented mixed models with random changepoints: A maximum likelihood approach with application to treatment for depression study[END_REF]. Seasonal ΔStWC was determined considering treatment, year, ΨPD and their interaction as fixed factors, whereas tree was considered as a random slope factor.

Treatment and inter-annual differences in the breakpoint along the desorption curve (ΨPD separating phases I and II) and the difference-in-slope parameters (difference in slope of the desorption curve before and after the breakpoint) were also tested. Random variation in the breakpoint and the difference-in-slope parameters was allowed using a diagonal random effects covariance matrix. Stepwise backward selection was applied for model selection. In this model, the significance of the interaction between ΨPD and treatment or year denotes different slope of the desorption curve, and hence treatment or inter-annual differences in CS, respectively.

Sub-daily CS was estimated as the ratio between sub-daily ΔStWC and ΔΨ (ΨPD -ΨMD) according to Eqn.

1. To test the effect of TE and ΨPD on sub-daily CS, linear mixed models were adjusted considering treatment, ΨPD and their interaction as fixed factors and tree as a random factor using the lme function. The relationship between sub-daily ΔStWC and ΔΨ was further examined by a non-linear mixed model using the nlme function, as an exponential fit reduced model residual error and AIC relative to the linear fit. The relationship between ΨPD and ΔΨ was analysed likewise. To display non-linear fits, 95% confidence intervals were estimated by first-order Taylor expansion and Monte Carlo simulation (predictNLS function in propagate library). On a daily basis, treatment differences in ΨPD, transpiration, water storage release and the contribution of stored water to transpiration were tested with ANOVA. Because of small sample size (n = 4), differences with strong (P < 0.05) and moderate (P < 0.10) statistical significance were reported. The allometric relationship between SA and stem diameter was compared between treatments by adjusting loglog linear models and applying stepwise forward selection.

Results

The summer of 2017 was exceptionally dry, with 41.2 mm of rain from early June (DOY 156) to mid-October (DOY 290), when first heavy rains took place (Figure 1A). Summer drought in 2018 was less intense with 97.6 mm of rain from DOY 156 to 279. During these summer drought periods, mean daily air temperature ranged between 13.6 and 30.4 ºC in 2017, and between 14.9 and 28.7 ºC in 2018 (Figure 1B).

Mean daily TSTEM did not differ between treatments throughout the surveyed period, nor during 2017 or 2018 summers (P > 0.78). The more intense summer drought in 2017 relative to 2018 was reflected by the lower yearly minimum ΨPD in 2017 (P < 0.001). Significant differences in ΨPD between treatments were observed in both years (P < 0.01; Figure 1C). Minimum ΨPD in 2017 was -4.8 ± 0.1 and -5.9 ± 0.3 MPa in C and TE trees, respectively, whereas minimum ΨPD in 2018 was -4.2 ± 0.1 and -4.6 ± 0.1 MPa in C and TE trees, respectively.

Seasonal and sub-daily variability in stem water content and hydraulic capacitance

Stem water content consistently decreased during the dry season in 2017 and 2018, reaching lowest values immediately before autumn first heavy rains (Figure 1D). After 2017 autumn rains, StWC rapidly reached pre-drought levels in C trees, whereas stem refilling was slower in TE trees and additional rain during winter 2018 was necessary to reach pre-drought values. In 2018, stem water refilling following autumn rains was similar between treatments. The larger initial seasonal decline in StWC with decreasing ΨPD allowed to separate phases I and II of the seasonal desorption curve by their different slopes (P < 0.001, Table 1, Figure 2) and hence capacitances (CS_I and CS_II, respectively). The breakpoint of the seasonal desorption curve was observed at a ΨPD of -0.97 MPa and -1.10 MPa in 2017 and 2018, respectively, and did not differ between treatments (Table 1, Figure 2). Above this ΨPD, the interaction ΨPD × treatment was significant (P < 0.01, Table 1), denoting higher CS_I in TE trees (154 kg m -3 MPa -1 ) compared to C ones (80 kg m -3 MPa - 1 ). On the contrary, the slope of the desorption curve below the breakpoint substantially decreased (Figure 2), and the interaction ΨPD × treatment became not significant, denoting low and similar CS_II between treatments (< 20 kg m -3 MPa -1 ). Unexpectedly, a larger reduction in seasonal StWC was observed in 2018 than in 2017 (Figure 1D), resulting in a significantly higher CS_II in 2018 relative to 2017 (18 and 7 kg m -3

MPa -1 , respectively; Figure 2). The interaction ΨPD × treatment × year was not significant above or below the breakpoint point, denoting consistency in the inter-annual treatment comparisons of both CS_I and CS_II.

Sub-daily CS estimated using shoot Ψ measurements to calculate the midday sap flow driving force (ΔΨ = ΨPD -ΨMD) was 19.73 ± 5.92 kg m -3 MPa -1 . When indirect estimates of stem ΔΨ were applied (see Figure S4a), sub-daily CS increased to 33.38 ± 6.70 kg m -3 MPa -1 . Independently of the ΔΨ applied for calculation, sub-daily CS did not differ between treatments nor across a ΨPD gradient (P > 0.1). Accordingly, parameters of the non-linear relationship between sub-daily ΔStWC and shoot ΔΨ did not differ between treatments (Figure 3). Sub-daily ΔStWC increased (in absolute terms) with ΔΨ, which was in turn determined by ΨPD (inset Figure 3), and exhibited highest values when ΔΨ was above 2 MPa, before drought stress exacerbated.

Figure S4b shows these relationships when indirect estimates of stem ΔΨ were used for calculation.

Seasonal and sub-daily ΔStWC were not linearly related (Figure 4A). Sub-daily ΔStWC (in absolute terms)

did not peak at the start of the dry season, but after mild dehydration, when the evaporative demand was likely higher. Highest sub-daily ΔStWC, ranging between 25-35 and 18-25 kg m -3 , were reached in early summer of 2017 and 2018, respectively (Figure 4A-B). Following seasonal dehydration, sub-daily ΔStWC progressively decreased down to 5 kg m -3 for both years (Figure 4A, C-D). Overall, sub-daily ΔStWC was not significantly affected by TE. Mean values of sub-daily ΔStWC were moderately higher in TE trees uniquely in sparse days along phase I of the seasonal desorption curve (e.g. Figure 4B, P < 0.1). tended to be higher in C than TE trees when ΨPD was high, although this difference was not significant (Figure 6A). Transpiration decreased with decreasing ΨPD (P < 0.001), with the interaction ΨPD × treatment being significant (P < 0.05), reflecting that TE trees were able to maintain similar transpiration rates at more negative ΨPD. The release of stored water to the transpiration stream decreased with ΨPD (P < 0.001; Figure 6B). It tended to be higher in TE trees, although this difference was not significant when considering the whole surveyed period; differences were uniquely observed in early summer of 2018 (Figure 5D). The daily contribution of stored water to transpiration was not related to ΨPD (Figure 6C). Again, although this ratio tended to be higher in TE trees, differences were not significant considering the whole surveyed period and were uniquely and sparsely observed during the summer of 2018 (Figure 5F).

Daily transpiration and release of stored water

Sapwood area

The range of electrical resistivity for each surveyed stem is displayed in Figure 7. Overall, individual ER minima and maxima largely varied independently of the treatment. Minimum ER values were mostly below 100 Ωm, and maximum ER rarely exceeded 1600 Ωm. The slope and intercept of the log-log linear regression between stem diameter (D) and SA did not differ between treatments (P > 0.05; Figure 8). Pooling trees from C and TE treatments, the coefficients a and b of the allometric relationship between SA (cm 2 ) and D (cm):

𝑆𝐴 = 𝑎 𝐷 𝑏 Eqn. 5
were 0.682 and 1.911, respectively. These coefficients did not differ significantly from those previously obtained at this site, in which SA area was visually determined [START_REF] Limousin | Longterm transpiration change with rainfall decline in a Mediterranean Quercus ilex forest[END_REF]. The coefficient of determination between SA estimated visually and via ERT in validation trees was 0.96, with ERT estimates being higher than those visually determined (Figure S5).

Discussion

Methodological considerations to quantify stem water content

Methodological constrains to accurately monitor StWC in vivo have limited our knowledge about spatial and temporal dynamics in CS and its response to stress elicitors, although it might be of primary relevance within the context of drought-driven tree mortality [START_REF] Körner | No need for pipes when the well is dry-a comment on hydraulic failure in trees[END_REF][START_REF] Martinez-Vilalta | Greater focus on water pools may improve our ability to understand and anticipate drought-induced mortality in plants[END_REF]. Frequency domain reflectometry is becoming increasingly applied to the detriment of time domain reflectometry due to its comparable potential to provide continuous and long-term data on StWC in a non-destructive manner for a relatively lower cost (e.g. [START_REF] Hao | Investigating xylem embolism formation, refilling and water storage in tree trunks using frequency domain reflectometry[END_REF][START_REF] Carrasco | Water storage dynamics in the main stem of subtropical tree species differing in wood density, growth rate and life history traits[END_REF][START_REF] Matheny | Observations of stem water storage in trees of opposing hydraulic strategies[END_REF][START_REF] Saito | Monitoring of stem water content of native and invasive trees in arid environments using GS3 soil moisture sensors[END_REF][START_REF] Beedlow | Seasonal patterns of bole water content in old growth Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco)[END_REF]. The need for accurate FDR probe calibration in the wood medium is, however, important to highlight. Some earlier works have seen an agreement between laboratory and factory calibration before applying parameters given by the manufacturers to estimate StWC from ε readings (e.g. [START_REF] Hao | Investigating xylem embolism formation, refilling and water storage in tree trunks using frequency domain reflectometry[END_REF][START_REF] Carrasco | Water storage dynamics in the main stem of subtropical tree species differing in wood density, growth rate and life history traits[END_REF]. Nevertheless, these probes are originally designed and calibrated to measure soil water content and the relationship between √𝜀 and water content could largely differ according to the medium (Wullschleger et al. 1996;[START_REF] Matheny | Observations of stem water storage in trees of opposing hydraulic strategies[END_REF][START_REF] Saito | Monitoring of stem water content of native and invasive trees in arid environments using GS3 soil moisture sensors[END_REF] Similarly, TSTEM variations may also affect ε readings as it occurs with soil temperature variations [START_REF] Chanzy | Correcting the temperature influence on soil capacitance sensors using diurnal temperature and water content cycles[END_REF]. Our attempt to account for TSTEM effect on StWC estimates by means of monitoring dead trees to avoid transpiration-driven fluctuations in ε did not result in a consistent relationship between α and ε (Figure S2) that could be extrapolated to living trees. The lack of overlap between ε ranges measured in dead and living trees hindered the determination of a reliable α that could be applied in living trees for temperature correction of ε across the surveyed period, during which variation in the proportion of free and bound water possibly alters the temperature sensitivity of ε. Specific calibration tests applying temperature changes under controlled StWC [START_REF] Saito | Monitoring of stem water content of native and invasive trees in arid environments using GS3 soil moisture sensors[END_REF] could help to reduce this potential bias in future studies.

Further measurement artifacts related to the wounding of stem tissues surrounding the probe needles may also affect ε readings. A pioneering study applying time domain reflectometry reported apparent StWC increases with time since probe installation (Wullschleger et al. 1996), which could hinder inter-annual comparisons in ΔStWC. It is possible that the comparatively larger ΔStWC (and CS_II) registered in 2018 than in 2017 was partially explained by a measurement artefact. However, inter-annual variation in ΔStWC could be also be explained by different meteorological conditions. Smaller ΔStWC in 2017 was caused by the inter-annual difference in spring StWC maxima, which was lower in 2017 than in 2018, with the summer StWC minima being similar during both years (Figure 1D). This observation could also suggest that stem water reservoirs were not fully replenished in 2017 before the onset of the seasonal desorption curve.

Incomplete stem refilling in 2017 could be possibly attributed to relatively drier winter and spring months (Figure 1A). At this time, cumulative rain previous to the spring StWC maxima in 2017 was 39, 35, 45, 53 and 51% of that registered in 2018 during the 1-, 2-, 3-, 4-and 5-month period preceding spring StWC maxima, respectively.

Seasonal and sub-daily variation in stem water content and capacitance

Stem water pools were largely depleted during summer drought, as similarly observed in several species subjected to water deficit [START_REF] Hernández-Santana | TDR measurement of stem and soil water content in two Mediterranean oak species[END_REF][START_REF] Hao | Investigating xylem embolism formation, refilling and water storage in tree trunks using frequency domain reflectometry[END_REF][START_REF] Matheny | Observations of stem water storage in trees of opposing hydraulic strategies[END_REF][START_REF] Beedlow | Seasonal patterns of bole water content in old growth Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco)[END_REF]. The seasonal desorption curve exhibited a steep decrease under well-hydrated conditions, which flattened as drought-stress exacerbated. Therefore, CS decreased along the dry season, with the breakpoint of the desorption curve being observed at ΨPD between -0.97 and -1.10 MPa (Figure 2). Capillary water is mostly released at water potentials above -0.2 MPa [START_REF] Tyree | Water-storage capacity of Thuja, Tsuga and Acer stems measured by dehydration isotherms[END_REF]) and largely depleted below -0.5 MPa [START_REF] Tyree | The hydraulic architecture of trees and other woody plants[END_REF][START_REF] Pratt | Conflicting demands on angiosperm xylem: Tradeoffs among storage, transport and biomechanics[END_REF], which is in accordance with the negative pressure required to drain capillaries as calculated from Laplace´s law [START_REF] Knipfer | Storage compartments for capillary water rarely refill in an intact woody plant[END_REF].

Therefore, capillary water might be physiologically irrelevant during phase I of the desorption curve (ΨPD between -0.45 and -1.10 MPa) and CS_I could be primarily attributed to elastic living cells. The subsequent reduction in CS during phase II might result from the limited contribution of largely depleted elastic pools together with a small capacitive water release from vessel embolism given the remarkable embolism resistance of Q. ilex, with P12 values down to -4.93 MPa [START_REF] Lobo | Assessing inter-and intraspecific variability of xylem vulnerability to embolism in oaks[END_REF]. Remarkably, the breakpoint of the desorption curve closely matched the threshold for stem growth interruption imposed by water deficit at the study site (ΨPD = -1.1 MPa; [START_REF] Lempereur | Growth duration is a better predictor of stem increment than carbon supply in a Mediterranean oak forest: implications for assessing forest productivity under climate change[END_REF] and was not modified by TE. Stem growth being a turgor-driven process, it requires sufficient turgor pressure in the cambial cells for cell division and enlargement [START_REF] Hsiao | Sensitivity of with of roots versus leaves to water stress: Biophysical analysis and relation to water[END_REF][START_REF] Steppe | Diel growth dynamics in tree stems: linking anatomy and ecophysiology[END_REF]. Stem living cells during phase I were probably still sufficiently hydrated to build-up the turgor pressure above the yield threshold, while the large depletion of water pools during phase II of this curve constrained cambial turgor below this yield threshold. The coincidence of these two ΨPD thresholds for stem growth and stem hydraulic capacitance suggests that stem elastic water pools are responsible for both the stem capacitive water release along phase I and the turgor pressure that drives cell wall yielding during growth.

Stem capacitance is commonly estimated as the initial slope of the desorption curve. Initial capacitance in Q. ilex was below 160 kg m -3 MPa -1 , which is consistent with previous reports in evergreen sclerophyll species [START_REF] Richards | Sapwood capacitance is greater in evergreen sclerophyll species growing in high compared to low-rainfall environments[END_REF], and is lower than the capacitance commonly observed in temperate and tropical trees [START_REF] Scholz | Hydraulic Capacitance: Biophysics and Functional Significance of Internal Water Sources in Relation to Tree Size[END_REF]. The shape of the seasonal desorption curve obtained here via FDR was similar to that previously modelled for control trees (cf. Figure 5 in [START_REF] Salomón | Stem hydraulic capacitance decreases with drought stress: implications for modelling tree hydraulics in the Mediterranean oak Quercus ilex[END_REF], using a model that only accounted for the capacitive water release from outer tissues (cambium, phloem and bark).

Although FDR cannot discriminate between sapwood and outer tissues as water sources, it would mostly reflect sapwood capacitance as most of the probe needle length (~ 85%) was in contact with sapwood.

Modelled CS_I of outer tissues in control trees (62 kg m -3 MPa -1 ; [START_REF] Salomón | Stem hydraulic capacitance decreases with drought stress: implications for modelling tree hydraulics in the Mediterranean oak Quercus ilex[END_REF] was lower than measured CS_I here (80 kg m -3 MPa -1 ), suggesting a greater capacitance of sapwood tissues on a volume basis, as similarly observed in Neotropical savanna trees [START_REF] Scholz | Biophysical properties and functional significance of stem water storage tissues in Neotropical savanna trees[END_REF].

As initially hypothesized, permanent throughfall exclusion increased seasonal CS. However, and contrary to our expectations, differences were observed during phase I of the desorption curve, when trees were still well-hydrated, instead of during phase II, when reliance on stored water might play a more important role in maintaining xylem hydraulic functionality. These results suggest that capacitive water release to limit Ψ reductions represents a secondary strategy to face drought in Q. ilex, as this drought-tolerant species is highly resistant to drought-driven embolism, with P50 values of -7.13 MPa [START_REF] Lobo | Assessing inter-and intraspecific variability of xylem vulnerability to embolism in oaks[END_REF]. Contrarily to drought-avoidant species, the significance of CS in drought-tolerant species such as Q. ilex might be more important to maximizing carbon gain during well-hydrated conditions than to dampening Ψ reductions under drought stress. According to this idea, the use of stem water pools increased carbon gain by 18% in Douglas-fir trees [START_REF] Phillips | Reliance on stored water increases with tree size in three species in the Pacific Northwest[END_REF] and by up to 12% in tropical forests [START_REF] Bartlett | Predicting shifts in the functional composition of tropical forests under increased drought and CO2 from trade-offs among plant hydraulic traits[END_REF], which can be achieved by enlarging periods of transpiration at sub-daily and seasonal timescales [START_REF] Goldstein | Stem water storage and diurnal patterns of water use in tropical forest canopy trees[END_REF][START_REF] Beedlow | Seasonal patterns of bole water content in old growth Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco)[END_REF][START_REF] Bartlett | Predicting shifts in the functional composition of tropical forests under increased drought and CO2 from trade-offs among plant hydraulic traits[END_REF]. Moreover, TE trees exhibited a more gradual decline in transpiration with decreasing ΨPD (Figure 6A). This pattern is consistent with the reduced sensitivity of transpiration to short-term fluctuations in atmospheric vapour pressure deficit (VPD) and soil moisture (SM)

following long-term reductions in soil water availability [START_REF] Grossiord | Manipulative experiments demonstrate how long-term soil moisture changes alter controls of plant water use[END_REF]. Lower sensitivity of transpiration to VPD and SM allows TE trees to maintain transpiration across a wider gradient of ΨPD but comes at the cost of reduced annual tree transpiration. Transpiration of our sample of trees was 8% and 16% lower in TE relative to C trees in 2017 and 2018, respectively, but a significant reduction of transpiration of approximately 23% has been observed over the long-term course of the experiment and related to a reduction of the trees' leaf area [START_REF] Limousin | Longterm transpiration change with rainfall decline in a Mediterranean Quercus ilex forest[END_REF][START_REF] Gavinet | Rainfall exclusion and thinning can alter the relationships between forest functioning and drought[END_REF]. Reduced sensitivity of transpiration to VPD can also limit water loss under conditions of very high temperature and evaporative demand, which are not necessarily optimal in terms of photosynthetic carbon gain (e.g. [START_REF] Drake | Trees tolerate an extreme heatwave via sustained transpirational cooling and increased leaf thermal tolerance[END_REF]. Therefore, although TE limits tree transpiration and carbon gain in absolute terms following leaf area reductions [START_REF] Limousin | Longterm transpiration change with rainfall decline in a Mediterranean Quercus ilex forest[END_REF], greater CS_I together with reduced sensitivity of transpiration to VPD and SM could better couple transpiration dynamics with optimal photosynthetic conditions [START_REF] Phillips | Reliance on stored water increases with tree size in three species in the Pacific Northwest[END_REF], and thereby improve whole-tree water use efficiency.

Mean sub-daily CS estimates increased from 20 to 33 kg m -3 MPa -1 when direct shoot ΔΨ measurements and indirect estimates of stem ΔΨ were used for calculation, respectively, thereby highlighting the need of direct stem Ψ measurements in future research to more accurately estimate sub-daily CS. Regardless of the ΔΨ used for calculation, sub-daily CS was similar between treatments and across the surveyed ΨPD gradient.

Similar sub-daily CS concomitant with increased seasonal CS_I in TE trees suggests that, although the difference between sub-daily StWC maximum and minimum remained homeostatic between treatments, daily refilling along phase I of the seasonal desorption curve was more limited in TE trees (Figure 4B), eventually resulting in a greater cumulative reduction in StWC at a longer (seasonal) temporal scale.

Furthermore, although the contribution of stored water to daily transpiration tended to be higher in TE, especially during the summer 2018 (Figure 5E-F), treatment differences were not significant for most of the surveyed period, as similarly observed for ponderosa pines across an aridity gradient [START_REF] Maherali | Influence of climate-driven shifts in biomass allocation on water transport and storage in ponderosa pine[END_REF]. It is important to note, however, that the small sample size in this study (n = 4) limits the statistical power of tests applied to detect any treatment effect. Daily stored water release and transpiration decreased proportionally with decreasing ΨPD, resulting in a roughly constant contribution of stored water to transpiration of around 10% (Figure 6), a value within the lower range of observations made for other species (from 10 to 50%; reviewed by [START_REF] Scholz | Hydraulic Capacitance: Biophysics and Functional Significance of Internal Water Sources in Relation to Tree Size[END_REF]. A sustained contribution of sapwood water pools to daily transpiration contrasts with the modelled result that the contribution to transpiration of stored water from outer tissues increased with decreasing ΨPD [START_REF] Salomón | Stem hydraulic capacitance decreases with drought stress: implications for modelling tree hydraulics in the Mediterranean oak Quercus ilex[END_REF]. This apparently contradictory observation could be explained by the spatial location of the different water reservoirs along the stem radial profile. Sapwood water pools are intimately connected to conducting xylem vessels, possibly acting as immediate water sources to fulfil transpiration needs in a proportional manner. Contrastingly, outer tissues located over the cambium layer are indirectly connected to xylem vessels via radial ray parenchyma, possibly acting as delayed reservoirs with lower initial capacitance but able to remain hydrated for longer periods, hence increasing its contribution to transpiration as drought stress progresses.

Hydraulic capacitance in relation to hydraulic allometry

Stem water storage capacity, reliance on stored water to fulfil transpiration needs and CS are relatively plastic traits related to sapwood volume and tree allometry [START_REF] Scholz | Hydraulic Capacitance: Biophysics and Functional Significance of Internal Water Sources in Relation to Tree Size[END_REF]. Allometric adjustments to chronic water limitation, assessed along aridity gradients, favour biomass allocation to conducting and storing sapwood to the detriment of transpiring leaves [START_REF] Maherali | Influence of climate-driven shifts in biomass allocation on water transport and storage in ponderosa pine[END_REF][START_REF] Martínez-Vilalta | Hydraulic adjustment of Scots pine across Europe[END_REF][START_REF] Martin-Stpaul | The temporal response to drought in a Mediterranean evergreen tree: comparing a regional precipitation gradient and a throughfall exclusion experiment[END_REF][START_REF] Rosas | Adjustments and coordination of hydraulic, leaf and stem traits along a water availability gradient[END_REF]. Accordingly, intra-specific comparisons in two conifer species have shown greater CS in drier sites, consistent with larger sapwood thickness, number of tree rings in sapwood, percentage of tree rings in sapwood, and a (non-significant) tendency of higher sapwood thickness relative to stem diameter [START_REF] Barnard | Climate-related trends in sapwood biophysical properties in two conifers: avoidance of hydraulic dysfunction through coordinated adjustments in xylem efficiency, safety and capacitance[END_REF]. In view of these observations, we hypothesized that potential changes in CS could be attributed to a structural acclimation of the sapwood depth in response to long-term water limitation. To test this hypothesis, we performed in vivo measurements of SA via ERT in ten trees from each treatment. The allometric relationship between SA and stem diameter did not differ between treatments (Figure 8) refuting our hypothesis. This comparison, however, should be taken with caution due to unaccounted sources of variation in ER (e.g., electrolyte content, wood density; [START_REF] Bär | Electrical resistivity tomography: patterns in Betula pendula, Fagus sylvatica, Picea abies and Pinus sylvestris[END_REF], illustrated here by large differences in the ER range registered among stems (Figure 7). Unexplained variability in ER may shift the point of steepest change in ER along the stem radial profile, commonly resulting in overestimation of SA via ERT [START_REF] Wang | Quantifying sapwood width for three Australian native species using electrical resistivity tomography[END_REF][START_REF] Benson | Estimating conductive sapwood area in diffuse and ring porous trees with electronic resistance tomography[END_REF], as similarly observed here in the four validation trees cut outside the experimental plots. Nevertheless, this bias did not significantly affect the relationship between stem diameter and SA relative to that visually obtained considering a larger sample size (n = 18; [START_REF] Limousin | Longterm transpiration change with rainfall decline in a Mediterranean Quercus ilex forest[END_REF], confirming the validity of allometric extrapolations applied at the site.

Differences in CS were observed during phase I of the seasonal desorption curve, when elastic water pools might be the largest source of capacitive water release. Regardless of similar SA between treatments, elastic water pools could be larger in TE trees, as suggested by the lower wood density previously observed in branches of TE trees [START_REF] Limousin | Change in hydraulic traits of Mediterranean Quercus ilex subjected to long-term throughfall exclusion[END_REF]. Wood density is directly related to Young´s modulus of elasticity [START_REF] Niklas | Worldwide correlations of mechanical properties and green wood density[END_REF]. Therefore, less dense wood with lower Young´s modulus undergoes greater elastic deformation for a given pressure, hence enabling greater release and refill of water for a given change in xylem Ψ. This is the reason why wood density is inversely related to CS [START_REF] Meinzer | Xylem hydraulic safety margins in woody plants: coordination of stomatal control of xylem tension with hydraulic capacitance[END_REF][START_REF] Mcculloh | The dynamic pipeline: hydraulic capacitance and xylem hydraulic safety in four tall conifer species[END_REF]. We further speculate that reductions in the leaf-to-sapwood area ratio (LA/SA) could be related to the observed increase in CS_I. Previous studies have shown that leaf biomass production in the TE plot was significantly and consistently reduced by 23 % [START_REF] Gavinet | Rainfall exclusion and thinning can alter the relationships between forest functioning and drought[END_REF] and that the leaf area index was also lower because increased leaf life span or decreased leaf mass per area did not compensate for the lower leaf production [START_REF] Limousin | Morphological and phenological shoot plasticity in a Mediterranean evergreen oak facing long-term increased drought[END_REF]. Significant reductions in LA/SA were thus observed at the branch level (Limousin et al. 2010a;[START_REF] Limousin | Morphological and phenological shoot plasticity in a Mediterranean evergreen oak facing long-term increased drought[END_REF] and confirmed here at the whole tree level considering homeostatic SA measured via ERT and LA reductions observed in previous studies at the site.

The mechanism linking LA/SA ratios with CS remains, however, unclear and dedicated experiments should be performed to address this hypothetical relation.

Further research in wood anatomy and tree water content regulation under drought stress [START_REF] Knipfer | Spatiotemporal coupling of vessel cavitation and discharge of stored xylem water in a tree sapling[END_REF][START_REF] Martinez-Vilalta | Greater focus on water pools may improve our ability to understand and anticipate drought-induced mortality in plants[END_REF]) could help to more accurately discriminate how stem capacitive water sources are preferentially used during drought stress, and to better understand the underlying link between CS and the regulation of water potential and carbon and water exchange. More long-term data on stem water pools, CS and allometric adjustments applying non-destructive techniques across different species would be necessary to evaluate the generality of the pattern observed here and advance knowledge on hydraulic acclimation strategies across plant functional types. Research in drought-prone Mediterranean regions could be particularly informative as the largest body of research in stem water pools has so far focused on more mesic (temperate and tropical) species, whose response to severe drought events could substantially differ from that of drought-tolerant ones.

Table 1. Fixed effects of the segmented mixed linear model for the seasonal relationship between predawn water potential (ΨPD) and the reduction in stem water content relative to the spring maxima (ΔStWC) in trees subjected to throughfall exclusion (TE) and control conditions across two consecutive years (2017 and 2018). Mean values per treatment (n = 4) and the smooth curve adjusted by local polynomial regression with its

  coefficient of determination of the individual fits between StWC and √𝜀 ranged from 0.94 to 0.99. Given the linear relationship between √𝜀 and StWC at any time step (Eqn. 3), the temporal (sub-daily and seasonal) variation in StWC (ΔStWC) can be estimated by means of the time derivative of √𝜀: , it is possible to determine ΔStWC rather than absolute values from ε readings, thereby avoiding the use of random (unknown) intercept values. After calibration, probes were installed in the field following the protocol described above. Probes were installed in selected trees at approximately 0.5 m above the soil and on the north face of the stem to avoid direct solar radiation. Readings were performed every 30 minutes and recorded with a data logger (model CR1000, Campbell Scientific Ltd, Shepshed, UK). More than two years of data are shown here, from sensor installation in December 2016 (DOY 341) until DOY 365 in 2018.

Figure 5

 5 Figure 5 illustrates dynamics in transpiration, stored water release and the contribution of stored water to

Figure 1 .

 1 Figure 1. Daily values of precipitation (A), mean daily temperature (B), leaf water potential (Ψ) at predawn

Figure 2 .

 2 Figure 2. Seasonal relationship between predawn water potential (ΨPD) and the cumulative reduction in

Figure 3 .

 3 Figure 3. Relationship between midday sap flow driving force (difference between predawn and midday

Figure 4 .

 4 Figure 4. Relationship between seasonal (cumulative) and sub-daily variation in stem water content

  Figure 1 834

  . Here, a constant slope of the relationship between √𝜀 and gravimetric StWC allowed reliable evaluation of ΔStWC at different timescales, but random variation of the intercept prevented the estimation of absolute StWC values. Custom probe shortening might have affected the parameters defining the √𝜀-StWC relationship

[START_REF] Matheny | Observations of stem water storage in trees of opposing hydraulic strategies[END_REF]

. Further calibration tests across different species and needle lengths would help to evaluate the suitability of fixed parameters given by the manufacturers to estimate StWC under different experimental conditions. Moreover, as calibration tests are, for practical reasons, generally performed during medium dehydration and not rehydration, any potential hysteretic relation between √ε and StWC could affect the StWC readings in studies with multiple dehydration-rewetting cycles.

Acknowledgements

We are grateful to Karim Piquemal for assistance during calibration, installation and maintenance of GS3 probes, to Vito Muggeo for his valuable support on the statistical analyses of mixed segmented models, and to Jonas von der Crone for his support and guidance in the analyses of ERT data. We thank two anonymous reviewers for their thoughtful comments on an earlier version of this manuscript. The Puéchabon experimental site belongs to the SOERE F-ORE-T, which is supported annually by Ecofor, Allenvi and the French national research infrastructure ANAEE-F, and the OSU-OREME of Montpellier. This project has received funding from the FWO and the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no 665501 granted to RLS.

Conflict of Interest Statement

The authors declare no conflict of interest. Sub-daily ΔStWC was moderately (P < 0.1) higher in TE trees at the seasonal spring maximum (B), whereas differences became non-significant at the breakpoint of the seasonal desorption curve (C), and at the end of the dry season immediately before first autumn rain (D). Horizontal and vertical axes in each tomograph illustrate the dimension of the stem cross-section in m. The range of electrical resistivity (ER, Ωm) is depicted by the colour bar. In some cases, irregularities around the perimeter of the cross section resulted in high ER. These irregularities were excluded from the ER radial profile to locate the heartwood-sapwood boundary. Note that images directly generated by the PICUS software do not illustrate the actual range of ER values, but maximize the colour contrast towards the centre of the tomography by adjusting the range of the colour map.

Genotypic variability and phenotypic plasticity of cavitation resistance in