Zwicke Marine

Picon-Cochard Catherine
email: catherine.cochard@clermont.inra.fr

Morvan-Bertrand Annette

Prud'homme Marie-Pascale

Volaire Florence

What functional strategies drive drought survival and recovery of perennial species from upland grassland?

Keywords: dehydration avoidance, dehydration tolerance, drought survival, forbs, fructans, grasses, membrane stability, resilience index, root system, sucrose



Backgrounds and aims. Extreme climatic events such as severe droughts are expected to increase with climate change and limit grassland perennity. We investigated the plant strategies involved in drought survival and recovery of temperate herbaceous species.



Methods. Monocultures of six native perennial species from upland grasslands and one Mediterranean drought resistant cultivar were compared under semi-controlled and nonlimiting rooting depth conditions. Above-and below-ground traits were measured under irrigation in spring and during drought in summer (50 days of withholding water) to characterise resource use and drought resistance strategies. They were then rehydrated to assess survival (15 days) and recovery (1 year).

 Key results. Dehydration avoidance through water uptake was associated with species that had deep roots (> 1.2 m) and high root mass (> 4 kg m -3 ). Cell membrane stability ensuring dehydration tolerance of roots and meristems was positively and negatively correlated to fructan and sucrose contents, respectively. Species that survived and recovered best combined high resource acquisition in spring (leaf elongation rate > 9 mm d -1 and rooting depth > 1.2 m) and both high dehydration avoidance and tolerance strategies.



Conclusion.

The variability of plant strategies observed in these species emphasizes the role of functional diversity in sustaining the drought resilience of perennial grasslands.

INTRODUCTION

In Europe, grasslands cover 22% of the EU-25 land area (FAOSTAT data, http://faostat.fao.org) and provide various ecosystemic services such as forage production, carbon storage, soil protection and biodiversity preservation (Millenium Ecosystem Assessment, 2005). Climate change will likely impact this ecosystem through shifts in grassland composition and production potential [START_REF] Grime | The response of two contrasting limestone grasslands to simulated climate change[END_REF][START_REF] Gilgen | Response of temperate grasslands at different altitudes to simulated summer drought differed but scaled with annual precipitation[END_REF][START_REF] Craine | Functional consequences of climate changeinduced plant species loss in a tallgrass prairie[END_REF]Zwicke et al., 2013). In temperate areas, more extreme rainfall regimes are expected to increase the duration and severity of drought [START_REF] Knapp | Consequences of more extreme precipitation regimes for terrestrial ecosystems[END_REF][START_REF] Seneviratne | Changes in climate extremes and their impacts on the natural physical environment. Managing the risks of extreme events and disasters to advance climate change adaptation[END_REF]. In this context, more knowledge is required to understand the mechanisms of resistance, survival under severe drought and recovery of herbaceous species after drought [START_REF] Chaves | Understanding plant responses to droughtfrom genes to the whole plant[END_REF][START_REF] Volaire | Designing resilient and sustainable grasslands for a drier future: adaptive strategies, functional traits and biotic interactions[END_REF].

There is evidence for a persistence-productivity trade-off, species with a high maximum growth rate having a higher mortality under restricted resources [START_REF] Sibly | A life-cycle theory of responses to stress[END_REF][START_REF] Wright | Functional traits and the growthmortality trade-off in tropical trees[END_REF]. In the case of drought tolerance, evidence for this trade-off has been described for woody plants [START_REF] Reich | The world-wide 'fast-slow' plant economics spectrum: a traits manifesto[END_REF], but the relationships between plant growth rate under optimal conditions and subsequent performance under severe drought has been scantly explored in herbaceous species [START_REF] Pérez-Ramos | Tradeoffs between functional strategies for resource-use and drought-survival in Mediterranean rangeland species[END_REF]. Perennial herbaceous species are interesting plant models, since they can resist and survive severe drought through adaptive strategies contributing to dehydration avoidance and dehydration tolerance [START_REF] Ludlow | Strategies of response to water stress[END_REF][START_REF] Blum | Crop responses to drought and the interpretation of adaptation[END_REF]. Under moderate drought, dehydration avoidance ensures the maintenance of plant tissue hydration and osmotic potential by maximizing water uptake and minimizing water losses [START_REF] Ludlow | Strategies of response to water stress[END_REF][START_REF] Volaire | Summer drought survival strategies and sustainability of perennial temperate forage grasses in Mediterranean areas[END_REF]. Under severe drought, once complete leaf senescence is reached, dehydration tolerance ensures plant survival by the maintaining cell integrity in meristematic tissues through cell membrane stabilization, accumulation of water soluble carbohydrates (WSC) and dehydrins (Volaire et al., 1998a, b;[START_REF] Verslues | Methods and concepts in quantifying resistance to drought, salt and freezing, abiotic stresses that affect plant water status[END_REF].

Grass species of temperate areas accumulate large amounts of reserve carbohydrates mainly as fructans in leaf meristems [START_REF] Chatterton | Carbohydrate partitioning in 185 accessions of Gramineae grown under warm and cool temperatures[END_REF][START_REF] Pollock | Fructan metabolism in grasses and cereals[END_REF]. It has been shown that this accumulation during drought improves plant survival after drought (Volaire et al., 1998a;[START_REF] Clark | Tissue-specific changes in remobilisation of fructan in the xerophytic tussock species Festuca novaezelandiae in response to a water deficit[END_REF]. Fructans in particular were shown to stabilize membranes in vitro by interaction with lipids under stress [START_REF] Vereyken | Fructans insert between the headgroups of phospholipids[END_REF][START_REF] Vereyken | The effect of fructan on membrane lipid organization and dynamics in the dry state[END_REF][START_REF] Hincha | Specific effects of fructo-and gluco-oligosaccharides in the preservation of liposomes during drying[END_REF][START_REF] Hincha | Fructans from oat and rye: composition and effects on membrane stability during drying[END_REF] and because they may act as antioxidants [START_REF] Peshev | Towards understanding vacuolar antioxidant mechanisms: a role for fructans?[END_REF]. These compounds play an indirect role in drought survival when they are hydrolysed to fuel growth after rehydration (Volaire et al., 1998b;[START_REF] Thomas | Partitioning of sugars in Lolium perenne (perennial ryegrass) during drought and on rewatering[END_REF][START_REF] Amiard | Fructans, but not the sucrosyl-galactosides, raffinose and loliose, are affected by drought stress in perennial ryegrass[END_REF].

Under progressive soil drying, higher biomass allocation to roots [START_REF] Poorter | Biomass allocation to leaves, stems and roots: meta-analyses of interspecific variation and environmental control[END_REF]) and extensive root system have been commonly observed in drought resistant species [START_REF] Comas | Root traits contributing to plant productivity under drought[END_REF], since deep roots can take up water from moister soil layers. Under severe soil drought conditions root mortality increases due to tissue dehydration [START_REF] Eissenstat | The ecology of root lifespan[END_REF][START_REF] Facette | Responses of maize roots to dryinglimits of viability[END_REF]. However, it has been shown in ryegrass that some roots can survive very dry conditions (soil water content < -10 MPa), especially root apices, and that production of lateral roots from existing roots is observed after soil rewetting [START_REF] Jupp | Morphological and anatomical effects of severe drought on the roots of Lolium perenne L[END_REF]. Thus maintaining live roots in severe soil drying conditions should enhance plant recovery [START_REF] Weaver | Length of life of roots of ten species of perennial range and pasture grasses[END_REF][START_REF] Huang | Drought-resistance mechanisms of seven warmseason turfgrasses under surface soil drying: II. Root aspects[END_REF]. However, despite of their crucial role in water uptake under drought, below-ground plant traits remain poorly quantified compared with leaf traits [START_REF] Reich | The world-wide 'fast-slow' plant economics spectrum: a traits manifesto[END_REF]. The combination of these plant responses depends on species and genotypes, and can be investigated by assessing leaf and root traits [START_REF] Pérez-Ramos | Tradeoffs between functional strategies for resource-use and drought-survival in Mediterranean rangeland species[END_REF] associated with water status and WSC metabolism [START_REF] Volaire | Plant traits and functional types to characterise drought survival of plurispecific perennial herbaceous swards in Mediterranean areas[END_REF]. The present study aimed (i) to characterize the adaptive responses whereby temperate grassland species resist, survive and recover from a severe drought and (ii) to explore the relationships between plant resource use and drought resistance strategies. To this end, we compared six dominant perennial herbaceous species (five grasses and one forb) originating from upland temperate grasslands [START_REF] Louault | Plant traits and functional types in responses to reduced disturbance in semi-natural grassland[END_REF], according to their resistance to and recovery from summer extreme (Zwicke et al., 2013), with a Mediterranean grass cultivar used as control for its high drought survival. These species were grown in semi-controlled and non-limiting rooting depth conditions to analyse leaf and root traits. Optimum resource-use strategies were identified under irrigation in spring, dehydration avoidance strategy was assessed under moderate drought (20 days of withholding water) and dehydration tolerance was estimated under severe drought (50 days). Plant survival and resilience indices (according to [START_REF] Van Ruijven | Diversity enhances community recovery, but not resistance, after drought[END_REF] were respectively measured two weeks and one year after rehydration. We tested the following hypotheses: (i) the drought survival of temperate species mainly depends on dehydration avoidance through water acquisition strategies, (ii) accumulation of WSC, especially fructans and sucrose in surviving organs including not only leaf meristems, but also roots and root apices, is associated with dehydration tolerance, and (iii) there is a trade-off between high resource-use under non-limiting conditions and high ability to survive and recover from severe drought.

MATERIALS AND METHODS

Experimental set-up and conditions

The experiment was conducted outdoors at Clermont-Ferrand (45°47'N, 03°05'E, 350 m a.s.l.), under a semi-continental climate (mean annual temperature, 12.4 °C; mean annual precipitation, 579 mm). An automatic weather station recorded air temperature, global radiation, wind speed and vapour pressure deficit, allowing the calculation of potential evapotranspiration (PET) (Table S1).

In summer 2010, granitic brown soil (12% clay, 17% loam, 59% sand, 13% organic matter), extracted from an upland grassland (45°43'N, 03°01'E, 850 m a.s.l.), was sieved at 7 mm and left to dry in the air. Plastic sleeves were placed inside 105 deep tubes (PVC, 150 cm deep, 10 cm diameter) for easy sampling of the whole root system. The tubes were filled with the soil mixed with slow-release fertiliser (3.5 kg m -3 , NPK 14-7-14, Multicote 12, Haifa, Israel). In September 2010, seeds of native species: Dactylis glomerata (Dg), Festuca arundinacea (Fa), Poa pratensis (Pp), Poa trivialis (Pt), Trisetum flavescens (Tf) and Taraxacum officinale (To) from an upland grassland (45°43'N, 3°01'E, 880 m a.s.l., [START_REF] Pontes | Seasonal productivity and nutritive value of temperate grasses found in semi-natural pastures in Europe: responses to cutting frequency and N supply[END_REF] and seeds of a Mediterranean cultivar of D. glomerata 'Medly' (RAGT, France) (Md), were sown at a density of 2500 seeds m -2 in each tube. This Mediterranean cultivar was used as a control for high drought survival [START_REF] Volaire | Drought survival in Dactylis glomerata and Festuca arundinacea under similar rooting conditions in tubes[END_REF]. All the tubes were placed outside in soil trenches during the winter and kept well-watered with rainfall and additional watering.

In spring 2011 a drip irrigation system was set up (day of year, DOY 78) to maintain soil water content (SWC) near field capacity. In addition, three volumetric SWC (m 3 m -3 ) probes (ECHO-5, Decagon, USA) were inserted horizontally at depths 10, 40 and 100 cm on three tubes per species, and connected to a datalogger (EM50, Decagon, USA). From May to November (DOY 132 to 326), SWC was measured every 15 min (Fig. 1). Three tubes per species were also installed on a weighing scale (60 x 60 cm, Arpege Master K, type N PAC + SAT MB, France) to record plant water-use (WU, g kg -1 ) by gravimetry. To limit soil warming due to light radiation, the tubes were insulated with polystyrene (50 mm thick, Styrodur ®, BASF, France).

The drought treatment (50 days of withholding water) was induced from DOY 182 by stopping irrigation and intercepting all precipitations with a transparent polycarbonate shelter (12.5 x 10.8 m, 6.2 m high, 90 % transmitted PAR, Batiroc, France). This shelter was automatically controlled by a rain sensor. The drought treatment consisted first of 20 days of gradual soil drying until cessation of leaf growth (moderate drought, DOY 182-201), and then 30 days at SWC < 0.1 m 3 m -3 (severe drought, DOY 202-231) (Fig. 1). This treatment was applied on 12 tubes per species. Three tubes per species were maintained under full irrigation for the control treatment. From DOY 232, the tubes were rehydrated and maintained under full irrigation until the growing season ended (DOY 306). All the tubes then received local precipitation until the end of the experiment in June 2012 (DOY 174). Finally 21 and 84 tubes were used respectively for control and drought treatments.

During the experiment, plants were cut to a height of 5 cm, six times in 2011 (DOY 97,116,136,165,230,271) and then twice until spring 2012 (DOY 78,151).

Leaf and root traits

Leaf length (mm) of 36 mature tillers per species was measured every 2-3 days from DOY 146 to 193 (moderate drought conditions) to calculate leaf elongation rate (LER, mm d -1 , [START_REF] Carrère | Tissue turnover within grass-clover mixed swards grazed by sheep. Methodology for calculating growth, senescence and intake fluxes[END_REF]. During the drought treatment , leaf senescence (LS, 0-100 %) was visually assessed every 2-5 days.

Three tubes per species were harvested at four sampling dates (SD), corresponding to the end of the watering period (DOY 180, SD1), the end of moderate drought after the cessation of leaf growth (DOY 200,SD2), the end of severe drought (DOY 231, SD3) and to the control treatment (DOY 242, SD4) in which plants were not subjected to drought (controls).

For each SD, nine mature leaves per species were collected and rehydrated to measure leaf relative water content (LRWC, %) according to Volaire et al. (1998b). Specific leaf area (SLA, m 2 kg -1 ) and leaf dry matter content (LDMC, g g -1 ) were measured at SD1. Leaf meristems (enclosed leaf bases) were sampled after removal of mature leaves [START_REF] Lattanzi | Defoliation effects on carbon and nitrogen substrate import and tissue-bound efflux in leaf growth zones of grasses[END_REF], and fresh and dry (oven-dried at 60°C for 48h) weighed to determine their water content (MWC, %). For P. trivialis and T. flavescens, stolons (storage organs) were also collected for subsequent WSC measurements. The whole intact root system was extracted from each tube and carefully washed with tap water. Maximum rooting depth was measured (Dmax, cm), then deepest root apical zone (15 cm length, called root apices) and other roots were collected according to five soil layers (0-15, 15-30, 30-40, 40-90, and 90-150 cm, hereafter called roots). For P. pratensis and T. officinale, rhizomes and tap root (storage organs) were separated from fine roots. Root dry matter content (RDMC, g g -1 ) and total root mass (RM, kg m -3 ) were calculated. The 95% rooting depth (D 95 , cm), i.e. the depth including 95% of the root biomass, was calculated according to [START_REF] Schenk | Rooting depths, lateral root spreads and belowground/above-ground allometries of plants in water-limited ecosystems[END_REF].

Cell membrane stability (CMS)

At the end of severe drought (SD3, SD4), three sub-samples of leaf meristems, roots from each soil layer, root apices and storage organs were harvested to measure CMS (%), according to [START_REF] Volaire | Growth, carbohydrate reserves and drought survival strategies of contrasting Dactylis glomerata populations in a Mediterranean environment[END_REF] and [START_REF] Charrier | The timing of leaf fall affects cold acclimation by interactions with air temperature through water and carbohydrate contents[END_REF]. The CMS of each root layer was averaged (CMSr).

Water soluble carbohydrate (WSC) analysis

At each SD, fresh sub-samples of leaf meristems, roots per soil layer, root apices and storage organs (rhizomes, stolons, and tap roots) were quickly fresh-weighed, dropped into liquid nitrogen and stored at -80 °C before freeze-drying (-100 °C for 48 h). Fine powder samples (30-50 mg) were extracted in 80 % ethanol, and purified in mini-columns (Mobicols from MoBITec, Göttingen, Germany) with ion-exchange resins (see Amiard et al., 2003 for details). Water soluble carbohydrates (mg g -1 ) were analysed by high performance liquid chromatography (HPLC) on a cation exchange column (Sugar-PAK, 300 X 6.5 mm, Millipore Waters, Milford, MA, USA) eluted at 0.5 mL min -1 and 85 °C with 0.1 mM Ca-EDTA in water and quantified using a refractive index detector (2410 Differential Refractometer, Millipore Waters). External standards used to quantify carbohydrates were glucose, fructose, sucrose and Cichorium intybus inulin (Sigma-Aldrich, MO, USA). The HPLC system enabled us to separately quantify fructans with degrees of polymerization 3 and 4 (low degree of polymerization, LDP) and over 5 (high degree of polymerization, HDP). The mean WSC content of the whole root system was calculated from the WSC content (mg g -1 DW) and the dry mass of each root layer. In addition, amount of WSCs (mg per plant) in roots (QWSCr) was calculated by multiplying dry mass of roots (g per plant) with WSC content (mg g -1 DW) measured after severe drought.

Plant survival, recovery and resilience indices

Two weeks after rehydration (DOY 246) and in the following spring 2012 (DOY 158), percentage of green tissue in aerial tissues was visually assessed on three tubes per species to determine plant survival (SURV) and one-year recovery (RECOV).

In spring 2011 (DOY 97,116,136,165) and spring 2012 (DOY 78, 151) above-ground biomass was oven-dried (60 °C for 48 h) and weighed to determine pre-and post-drought forage production. In addition, three tubes per species were harvested in spring 2011 (SD1) and in spring 2012 (DOY 172) to assess pre-and post-drought standing root mass (kg m -3 ).

Resilience indices of spring forage production (RI) and spring standing root mass (RRI) were calculated as the ratio of post-drought (2012) to pre-drought (2011) biomass and standing root mass, respectively [START_REF] Van Ruijven | Diversity enhances community recovery, but not resistance, after drought[END_REF].

Statistical analyses

Statistical analyses were carried out with R software (R Core Team, 2012). Data were transformed when necessary (arcsine-square root-transformed, square root) before analysis to conform to the assumptions of normality (Shapiro-Wilk test) and homogeneity of variances (Fligner Killeen test). For each SD, single factor (species, treatment) variance analysis (ANOVA) and the post hoc Tukey test were performed with the 'multcomp' package [START_REF] Hothorn | The multcomp Package[END_REF]. Soil water content was estimated at the end of leaf growth (SWC el ) and for 50% of leaf senescence (SWC s ) from linear and sigmoid regressions between LER with SWC and LS with SWC, respectively. In addition, a Student test was used to compare means of two variables (green tissue percentage, resilience indices) for each species.

Pearson's coefficients of correlation between plant traits were calculated with the 'Hmisc' package [START_REF] Harrel | The Hmisc Package[END_REF]. Plant functional strategies for resource acquisition, dehydration avoidance and dehydration tolerance were analysed with matrices crossing traits and species replicates, using three principal component analyses (PCA) with the 'ade4' package [START_REF] Dray | The ade4 package: Implementing the duality diagram for ecologists[END_REF]. The first PCA tested seven traits measured before drought.

The second PCA tested six traits measured during the moderate drought. The third PCA tested 10 traits measured during moderate or severe drought. For each PCA, SURV, RECOV, RI and RRI were added as supplementary variables. Multiple regressions with backward selection then identified the traits most closely correlated with plant performance.

RESULTS

Soil volumetric water content

Before drought, SWC in shallow soil layers (depths 10 cm and 40 cm) fluctuated according to irrigation or rainfall events and plant transpiration, and averaging 0.139 m 3 m -3 across species.

At depth 100 cm, SWC varied less and averaged 0.250 m 3 m -3 (Fig. 1). One week after the last irrigation, SWC in shallow soil layers rapidly decreased and stabilized at 0.045 m 3 m -3 . At 100 cm depth and during the moderate drought, SWC declined to either 0.102 m 3 m -3 (F. arundinacea, T. officinale, D. glomerata 'Medly') or 0.279 m 3 m -3 (T. flavescens, P. pratensis and P. trivialis), and remained stable during the severe drought. After rehydration , SWC in shallow and deep layers reached 0.174 and 0.295 m 3 m -3 , respectively.

Plant traits before drought

All traits differed significantly between species (Table 1). Specific leaf area (SLA) ranged between 49.5 (P. trivialis) and 22.8 (P. pratensis) m 2 kg -1 . D. glomerata, D. glomerata 'Medly' and T. officinale had the highest elongation rate (LER > 9 mm d -1 ), while T. flavescens, F. arundinacea, P. trivialis and P. pratensis had the lowest (LER < 7 mm d -1 ).

Total root mass (RM 1 , P < 0.001) discriminated T. officinale and grasses due to the high tap root mass (7.5 kg m -3 ). Within grasses, F. arundinacea, D. glomerata and D. glomerata 'Medly' had the highest RM 1 and P. trivialis the lowest. Higher values of maximum rooting depth (Dmax 1 ) and 95% rooting depth (D95 1 ) were observed for T. officinale, F. arundinacea, D. glomerata and D. glomerata 'Medly', whereas P. trivialis had the shortest root system (P < 0.001, Fig. 2). Finally, water used (WU 1 ) was significantly higher for T. officinale and F. arundinacea (> 120 g kg -1 , P < 0.001) than for species with shallower root systems, such as T. flavescens, P. pratensis and P. trivialis. Root dry matter content (RDMC 1 ) ranged between 0.101 (To fine roots) to 0.228 g g -1 (P. pratensis), D. glomerata showing significantly lower values (0.189 g g -1 ) than D. glomerata 'Medly' (0.211 g g -1 , P < 0.01).

For all species and all analysed tissues, fructans with a high degree of polymerization (DP ≥ 5, HDP fructans) were the dominant sugar compounds (73.2 ± 1.6 %) of total water soluble carbohydrates (WSC), followed by sucrose (17.8 ± 1.2 %), fructans with a low degree of polymerization (DP = 3-4, LDP fructans, 7.1 ± 0.4 %) and monosaccharides (glucose and fructose, 4.4 ± 0.6 %). The present study focuses on the dominant carbohydrates (HDP fructans and sucrose). Results for the other sugars (LDP fructans and monosaccharides) are presented as supplemental data [Fig. S1,S2]. Before drought, HDP fructan contents in leaf meristems and root apices were not significantly different between species (Fig. 3A,3E). By contrast, species were discriminated by HDP fructans and sucrose contents in roots (P < 0.001) to form three groups: (i) T. officinale and T. flavescens, (ii) P. pratensis, (iii) D. glomerata, D. glomerata 'Medly', F. arundinacea and P. trivialis (Fig. 3C,3D). Three species (T. officinale, P. pratensis, and P. trivialis) accumulated fructans and sucrose in storage organs (Fig. 3G,3H).

Plant responses to moderate drought

For all the species, LER rapidly declined and stopped 10-12 days after the last irrigation when SWC el reached on average 0.090 m 3 m -3 and LS 2 = 23%, respectively (Table 1). Leaf relative water content (LRWC 2 ), meristem water content (MWC 2 ) and WU 2 decreased (P < 0.001), RDMC 2 and LS 2 increased (P < 0.001) in comparison with irrigated treatment, whereas RM 2 , Dmax 2 , and D95 2 did not change significantly. However all trait responses to moderate drought differed between species (Table 2). The forb species (T. officinale) showed higher values of LRWC 2 and MWC 2 and lower values of RDMC 2 than the grass species. Within grasses, LRWC 2 and MWC 2 values were higher than 33% and 46%, respectively, for D. glomerata, F. arundinacea, D. glomerata 'Medly' and lower for the remaining species (Table 1). Leaf senescence (LS 2 ) was the highest for P. trivialis, RDMC 2 of T. flavescens was significantly lower than that of D. glomerata 'Medly' and F. arundinacea. For RM 2 , Dmax 2 and D95 2 , between-species differences followed the same pattern as observed before drought (Fig. 2). Water used (WU 2 ) of T. officinale was about twice higher that of T. flavescens, P. pratensis and P. trivialis (Table 1).

Across species HDP fructan contents did not change in response to the moderate drought in leaf meristems and in root apices (Fig. 3A,3E, Table 2), whereas sucrose contents increased in all organs, especially in leaf meristems (+177 %, P < 0.001) and root apices (+72 %, P < 0.01) (Fig. 3B, 3F, Table 2). The pattern of HDP fructans in roots and the pattern of sucrose in leaf meristems, roots and root apices differed between species. For T. officinale, HDP fructan and sucrose contents in roots and taproot were maintained whereas significant increases in HDP fructans (+61%) and sucrose (+73%) contents were observed in roots of D. glomerata 'Medly' (Fig. 3C, 3D, P < 0.001). Sucrose content in leaf meristems remained stable for D. glomerata 'Medly' and D. glomerata, but strongly increased in T. flavescens, P. pratensis, F. arundinacea and P. trivialis (+200 % on average, Fig. 3B, P < 0.01). An accumulation of sucrose was also observed in roots of D. glomerata and F. arundinacea (+92 % and +56 %, respectively, P < 0.01), and in root apices of D. glomerata (+362 %, P < 0.05).

Conversely for T. flavescens and P. trivialis, HDP fructan content in roots decreased (-52 % on average, P < 0.01), whereas sucrose content in roots and root apices remained stable (Fig. 3B, 3C, 3D). Also, sucrose content increased in stolons of P. trivialis (+219 %, P < 0.001) but did not change significantly in rhizomes of P. pratensis (Fig. 3H). Content of fructans with a low degree of polymerization (3-4, LDP fructans) decreased in root apices (-71 %, Fig. S1F).

At the end of moderate drought, HDP fructan content in leaf meristems was more than twice as high in D. glomerata 'Medly' as in the upland species, whereas sucrose content was much higher in T. flavescens, P. pratensis and F. arundinacea than in T. officinale, D. glomerata 'Medly' and D. glomerata (Fig. 3A,3B). In roots, HDP fructan content of T. officinale, P. pratensis, T. flavescens and D. glomerata 'Medly' was higher than that of D. glomerata, F. arundinacea, P. trivialis (Fig. 3C). Roots of P. pratensis showed the highest sucrose content, 130 % higher than in roots of P. trivialis (Fig. 3D). T. flavescens had eight times higher HDP fructan content in root apices than D. glomerata, F. arundinacea, and P. trivialis (Fig. 3E). Regarding the storage organs, taproot of T. officinale had the highest HDP fructan content, whereas sucrose content was higher in the rhizomes of P. pratensis (Fig. 3G,3H).

Plant responses to severe drought

For all the species, leaf senescence (LS 3 ) increased, reaching on average 93 % (P < 0.001, Table 1). Leaf relative water content (LRWC 3 ) of the remaining living leaves and MWC 3 declined on average to 18 % and 31 %, respectively (P < 0.001), and WU 3 was half that under the moderate drought (P < 0.001). Root dry matter content (RDMC 3 ) increased significantly (P < 0.001) whereas RM 3 and Dmax 3 were unchanged between SD 2 and SD 3 . Contents of HDP fructan and sucrose in leaf meristems were not affected by the severe drought (Table 2).

However, in roots, HDP fructan content significantly decreased by 49 % on average for T. officinale, D. glomerata 'Medly' and P. pratensis (P < 0.01), and sucrose content also declined for T. officinale, D. glomerata 'Medly', D. glomerata, F. arundinacea and P. pratensis (P < 0.01, Fig. 3C,3D). Production of stolons appeared in T. flavescens during the severe drought, and this organ accumulated HDP fructan and sucrose contents similar to those of P. trivialis (Fig 3G ,3H).

Cell membrane stability (CMS) significantly declined in response to the drought (P < 0.01), except in leaf meristems of D. glomerata 'Medly' and T. flavescens, in root apices of D. glomerata 'Medly' and P. trivialis, and in stolons of T. flavescens (Fig. 4). Considering both treatments and all the species together, CMS of leaf meristems, roots and storage organs was positively correlated to the HDP fructan content of the corresponding tissues (r² = 0.16, P < 0.01; r² = 0.31, P < 0.001; r² = 0.10, P < 0.01, respectively), measured after severe drought in stressed and control plants ( Fig. 5A,5C,5E). By contrast, CMS was negatively correlated to sucrose content (r² = 0.24, P < 0.01 in leaf meristems and r² = 0.24, P < 0.01 in storage organs). At species level, correlation between CMS and HDP fructans could not be found in leaf meristems but in roots CMS and HDP fructan content were positively linked (r² > 0.39; P < 0.001) for each species except P. trivialis (Fig. 5C). Cell membrane stability was negatively correlated to sucrose content in leaf meristems of F. arundinacea and T. officinale, in roots of D. glomerata, D. glomerata 'Medly', P. pratensis and T. flavescens, and in the storage organs of the four species considered (Fig. 5B, 5D, 5F). Cell membrane stability was not correlated with carbohydrate contents in root apices (data not shown).

Plant survival and one-year recovery after drought

All the species were able to regrow two weeks after rehydration, but the percentage of green tissue in aerial tissue (SURV) differed significantly between species (P < 0.001, Fig. 6A,6B).

Thus, three groups of species were identified: (i) high survival for T. officinale and D. glomerata 'Medly' (green tissue > 90 %), (ii) intermediate survival for D. glomerata, T. flavescens and P. pratensis (50-70 %), and (iii) low survival for F. arundinacea and P. trivialis (< 20 %). However, greenness measured one-year after drought application (RECOV) was not significantly different between species, but high standard error values were noted for T. flavescens, F. arundinacea and P. trivialis (Fig. 6B).

Resilience index of spring forage production (RI) was significantly higher than that for D. glomerata 'Medly' (P < 0.01), and large between-species differences were observed (P < 0.001, Fig. 6C). D. glomerata 'Medly', P. pratensis and F. arundinacea showed the highest RI and T. officinale, T. flavescens and P. trivialis the lowest. Resilience index of spring standing root mass (RRI) was close to one, except for D. glomerata 'Medly' and D. glomerata (P < 0.05), which showed the highest values.

Plant strategies during drought and after rehydration

Pearson's correlation coefficients (Table 3) showed positive and significant correlations between plant survival (SURV) and some of the traits measured before drought (LER, RM 1 , Dmax 1 ), after moderate drought (HDPr 2 , LRWC2, RM 2 , SWCel, WU 2 ) and during severe drought (MWC 3 , CMSm and SOr3). SURV was negatively correlated with LS 3 , RDMC 2 , and SOm 2 . The resilience index of spring forage production (RI), was positively correlated with traits measured before drought (Dmax 1 , D95 1 and RDMC 1 ), and also during severe drought (CMSr, QWSCr and SOa 3 ). The resilience index of spring forage production was also negatively correlated to SLA and SWCs. However, only positive correlation with SWCel was observed for RECOV and RRI, which were correlated with each other and not with SURV.

The first two axes of the PCA performed with traits related to resource acquisition before drought (Fig. 7A), dehydration avoidance (Fig. 7B) and dehydration tolerance during drought treatment (Fig. 7C) accounted for 75.9%, 91.1% and 54.7% of the total variance observed among species, respectively. For each PCA, SURV was closely correlated to the first axis: 0.65, -0.74 and -0.73, respectively. The resilience index of spring forage production was correlated to the second axis of the PCA related to resource acquisition (0.74) and dehydration avoidance (0.66). However, RECOV and RRI were not explained by these syndromes of traits. The multiple regressions performed with these traits showed that the contribution to SURV differed according to the strategies with the following decreasing ranking: dehydration tolerance strategy (r² = 0.76, P < 0.001), dehydration avoidance (r² = 0.54, P < 0.001) and resource acquisition before drought (r² = 0.44, P < 0.001). Conversely, RI was mostly associated with the resource acquisition strategy (r²= 0.68, P < 0.001).

The first axis of PCAs, related to resource acquisition (Fig. 7A), and drought avoidance (Fig. 7B) showed a main contrast between the species with small root systems (P. trivialis, P. pratensis and T. flavescens) and those with deep ones (D. glomerata, F. arundinacea, D. glomerata 'Medly' and T. officinale) (Fig. 7A, 7B, P < 0.001). Species were also segregated by the first axis of the PCA related to dehydration avoidance with a main contrast between the forb (T. officinale) and the grass species (Fig. 7B, P < 0.001), while the first axis of the PCA related to tolerance strategy mostly opposed the upland species from the Mediterranean cultivar of D. glomerata (Fig. 7C, P < 0.001).

When considering only the six species from the upland grassland, the multiple regressions show a modified contribution of traits of both dehydration avoidance (r² = 0.65, P < 0.01), and dehydration tolerance after backward selection (HDPr, SOm, MWC 3 and CMSm, r² = 0.67, P < 0.001) on drought survival.

DISCUSSION

Temperate and Mediterranean species display different strategies for dehydration avoidance

Against our first hypothesis, the results show a similar contribution of dehydration avoidance and tolerance to drought survival in species from temperate upland grassland. Dehydration avoidance is achieved through a combination of responses favouring maximized water uptake and minimized water loss under drought [START_REF] Ludlow | Strategies of response to water stress[END_REF]. The changes in relative water content in tissues (MWC and LRWC, Table 1) are indicators of the cellular adjustments (accumulation of solutes and/or cell wall hardening) made by the plant to achieve a low water potential while avoiding water loss [START_REF] Verslues | Methods and concepts in quantifying resistance to drought, salt and freezing, abiotic stresses that affect plant water status[END_REF]. In this study, LRWC at moderate drought was correlated to RM, WU and plant survival (Fig. 7B, Table 3), which confirms that maximizing water uptake during drought is an important mechanism in maintaining the hydration of lamina [START_REF] Passioura | Water collection by roots[END_REF][START_REF] Chaves | Water stress, CO 2 and climate change[END_REF][START_REF] Volaire | Summer drought survival strategies and sustainability of perennial temperate forage grasses in Mediterranean areas[END_REF][START_REF] Lelièvre | Water-use efficiency and drought survival in Mediterranean perennial forage grasses[END_REF]. As previously shown for other species [START_REF] Jackson | Root water uptake and transport: using physiological predictions in global predictions[END_REF][START_REF] Pinheiro | Drought tolerance is associated with rooting depth and stomatal control of water-use in clones of Coffea canephora[END_REF][START_REF] Volaire | Plant traits and functional types to characterise drought survival of plurispecific perennial herbaceous swards in Mediterranean areas[END_REF], water uptake is mainly associated with initial root traits (Table 3). The species with higher RM and D max , such as T. officinale, D. glomerata 'Medly', D. glomerata and F. arundinacea, had a higher water-use before and during moderate drought (Table 1,Table 3, Fig. 7B). In arid and semi-arid environments, relatively small proportions of roots in deeper, moister soil layers may suffice to sustain water absorption of plants [START_REF] Ehleringer | Water uptake by plants: perspectives from stable isotope composition[END_REF]. The maintenance of water uptake by roots during a drought period is mainly driven by hydraulic continuity between soil, roots and leaves, which all depend on plant transpiration, hydraulic properties of roots [START_REF] Passioura | Water transport in and to roots[END_REF] and also on root morphology and anatomy [START_REF] North | Changes in hydraulic conductivity and anatomy caused by drying and rewetting roots of Agave deserti (Agavaceae)[END_REF][START_REF] Rieger | Root system hydraulic conductivity in species with contrasting root anatomy[END_REF][START_REF] Huang | Linking hydraulic conductivity to anatomy in plants that vary in specific root length[END_REF][START_REF] Steudle | Water uptake by roots: effects of water deficit[END_REF]Hernández et al., 2010). Between-species differences observed on leaf and root traits such as LRWC, MWC and RDMC (Table 1) suggest differences in water acquisition strategy, and therefore in the ability to maintain water uptake, explained by rooting depth (Fig. 7A, 7B; Table 3) and differences in root diameter and tissue density [START_REF] Picon-Cochard | Effect of species, root branching order and season on the root traits of 13 perennial grass species[END_REF]. Overall, root lifespan maintenance during drought could be related to root hydration maintenance by the hydraulic lift mechanism [START_REF] Bauerle | Importance of internal hydraulic redistribution for prolonging the lifespan of roots in dry soil[END_REF], cell osmotic increase in cortex cells [START_REF] Sharps | Solute regulation and growth by roots and shoots of waterstressed maize plants[END_REF], and suberization of the endoderm protecting xylem cells to dehydration, which could explain at least part of the increase in RDMC and decrease in plant water content (Table 1). The species able to maintain a root production during moderate drought, such as T. officinale and D. glomerata 'Medly' (Table 1, Fig. 2) had higher drought survival rates (Fig. 6B). Root growth maintenance under moderate water stress was also identified as an important mechanism to avoid dehydration by maximizing water uptake through an increase in root absorption area [START_REF] Passioura | Water collection by roots[END_REF][START_REF] Passioura | Water transport in and to roots[END_REF][START_REF] Vartanian | Some aspects of structural and functional modifications induced by drought in root systems[END_REF][START_REF] Pérez-Ramos | Tradeoffs between functional strategies for resource-use and drought-survival in Mediterranean rangeland species[END_REF].

Other mechanisms, such as modifications in water-use efficiency and regulation of stomatal conductance are involved in dehydration avoidance [START_REF] Jones | Mechanisms of drought resistance[END_REF][START_REF] Chaves | Water stress, CO 2 and climate change[END_REF][START_REF] Golluscio | Water-use efficiency of twenty-five co-existing Patagonian species growing under different soil water availability[END_REF]. In addition, when stress intensifies, increased leaf senescence contributes to the minimization of water loss by reducing leaf area and evaporation [START_REF] Ludlow | Strategies of response to water stress[END_REF][START_REF] Gepstein | Leaf senescence -not just a 'wear and tear' phenomenon[END_REF]. In this study, leaf senescence, which was inversely correlated to plant survival, was not adaptive but an indicator of drought vulnerability (Fig. 7C, Table 3). Conversely, gradual foliage senescence was positively correlated with plant survival in a range of Mediterranean grasses (Volaire et al., 1998b;[START_REF] Volaire | Drought survival in Dactylis glomerata and Festuca arundinacea under similar rooting conditions in tubes[END_REF][START_REF] Pérez-Ramos | Tradeoffs between functional strategies for resource-use and drought-survival in Mediterranean rangeland species[END_REF]. This suggests differences in plant strategies for dehydration avoidance between temperate and Mediterranean herbaceous plants, which can exhibit summer dormancy with induced foliage senescence, as a specific strategy to survive extreme drought [START_REF] Volaire | Summer dormancy in perennial temperate grasses[END_REF].

Water soluble carbohydrate metabolism is involved in dehydration avoidance and in dehydration tolerance

This study shows that plant survival is associated with high leaf meristem hydration and cell membrane stability under severe drought (Table 3, Fig. 4), which confirms that the ability of plant to protect leaf meristems is a key mechanism of dehydration resistance (including both avoidance and tolerance strategies) to ensure plant survival (Volaire et al., 1998b;[START_REF] Volaire | Drought survival in Dactylis glomerata and Festuca arundinacea under similar rooting conditions in tubes[END_REF]. Higher dehydration avoidance and dehydration tolerance were both previously attributed to WSC, through their involvement in osmotic adjustment and cell membrane stabilization [START_REF] Thomas | Accumulation and consumption of solutes in swards of Lolium perenne during drought and after rewatering[END_REF]Volaire et al., 1998b;[START_REF] Livingston | Fructan and its relationship to abiotic stress tolerance in plants[END_REF].

Interestingly, among non-specific storage organs, it was in the leaf meristems of all the species that total WSC content was highest at the end of severe drought, but it was not discriminating between species [Fig. S1A]. However, each carbohydrate was modulated differently by drought according to the species considered (Fig. 3, Fig. S1, Fig. S2). This indicates that carbohydrate composition rather than carbohydrate content is involved in drought resistance mechanisms as already suggested [START_REF] Ingram | The molecular basis of dehydration tolerance in plants[END_REF].

When the photosynthetic machinery is impaired by prolonged drought [START_REF] Chaves | Water stress, CO 2 and climate change[END_REF], carbohydrate contents are modified, which may act as metabolic signals to promote leaf senescence and reserve mobilization [START_REF] Zeppel | Mechanistic causes of tree drought mortality: recent results, unresolved questions and future research needs[END_REF][START_REF] Mcdowell | Mechanisms linking drought, hydraulics, carbon metabolism, and vegetation mortality[END_REF][START_REF] Sala | Carbon dynamics in trees: feast or famine?[END_REF]. In the sensitive species, T. flavescens, P. pratensis, F. arundinacea and P. trivialis (Fig. 6A,6B), sucrose was accumulated in leaf meristems during the moderate drought (Fig. 3B), and HDP fructan content declined significantly in the most sensitive ones, F. arundinacea and P. trivialis (Fig. 3A). Accumulation of sucrose might result from sucrose synthesis enhancement and/or from fructan degradation. Although increasing sucrose synthesis could be stimulated by drought [START_REF] Ingram | The molecular basis of dehydration tolerance in plants[END_REF], more studies on enzyme activities are needed to elucidate the role of fructan hydrolysis in sucrose accumulation under drought. Hydrolysis of the polymers and concomitant synthesis of sucrose could be the mechanism whereby plant tissues survive water deficits [START_REF] Spollen | Response of fructan to water-deficit in growing leaves of Tall Fescue[END_REF][START_REF] Xue | Use of expression analysis to dissect alterations in carbohydrate metabolism in wheat leaves during drought stress[END_REF][START_REF] Saeedipour | Comparison of the drought stress responses of tolerant and sensitive wheat cultivars during grain filling: impact of invertase activity on carbon metabolism during kernel development[END_REF]. As sucrose is known to play an important role in osmotic adjustment during drought [START_REF] Jones | Mechanisms of drought resistance[END_REF][START_REF] Thomas | Accumulation and consumption of solutes in swards of Lolium perenne during drought and after rewatering[END_REF]Volaire et al., 1998a,b), these results suggest that sucrose is involved in the dehydration avoidance strategy for the most sensitive populations. Depending on species and duration of drought, fructans are either accumulated [START_REF] Volaire | Production, persistence, and water-soluble carbohydrate accumulation in 21 contrasting populations of Dactylis glomerata L. subjected to severe drought in the south of France[END_REF]Volaire et al., 1998b;[START_REF] De Roover | Drought induces fructan synthesis and 1-SST (sucrose:sucrose fructosyltransferase) in roots and leaves of chicory seedlings (Cichorium intybus L.)[END_REF], modified in chain length [START_REF] Thomas | Accumulation and consumption of solutes in swards of Lolium perenne during drought and after rewatering[END_REF]Volaire et al., 1998b;[START_REF] Thomas | Partitioning of sugars in Lolium perenne (perennial ryegrass) during drought and on rewatering[END_REF], or reduced [START_REF] Thomas | Accumulation and consumption of solutes in swards of Lolium perenne during drought and after rewatering[END_REF][START_REF] Spollen | Response of fructan to water-deficit in growing leaves of Tall Fescue[END_REF][START_REF] Clark | Tissue-specific changes in remobilisation of fructan in the xerophytic tussock species Festuca novaezelandiae in response to a water deficit[END_REF]. In this study, maintenance of HDP fructan content in leaf meristems during drought (Fig. 3A) could contribute to higher survival in the more tolerant species, T. officinale, D. glomerata 'Medly' and D. glomerata (Fig. 6), as already reported for Mediterranean cultivar [START_REF] Volaire | Effects of drought on water relations, mineral utpake, watersoluble carbohydrate accumulation and survival of two contrasting populations of cocksfoot (Dactylis glomerata L.)[END_REF]Volaire et al., 1998b;[START_REF] Volaire | Plant traits and functional types to characterise drought survival of plurispecific perennial herbaceous swards in Mediterranean areas[END_REF]. Despite a decline in CMS, this comparative study clearly reveals a positive correlation between HDP fructan content and CMS in roots for all the species, except for the most sensitive one, P. trivialis (Fig. 5C). This can be attributed to the role of fructans in cell membrane protection [START_REF] Valluru | Plant fructans in stress environments: emerging concepts and future prospects[END_REF][START_REF] Livingston | Fructan and its relationship to abiotic stress tolerance in plants[END_REF]. While several studies have demonstrated this role through in vitro approaches [START_REF] Vereyken | Fructans insert between the headgroups of phospholipids[END_REF][START_REF] Hincha | Specific effects of fructo-and gluco-oligosaccharides in the preservation of liposomes during drying[END_REF][START_REF] Hincha | Fructans from oat and rye: composition and effects on membrane stability during drying[END_REF], only one study on D. glomerata supported it in planta (Volaire et al., 1998b). Present data corroborate the protective role of fructans in D. glomerata and support it in a broader panel of temperate species, including forbs and grasses.

In leaves of resurrection plants which do not accumulate fructans, sucrose is the main carbohydrate contributing to drought tolerance by membrane and protein stabilization [START_REF] Ingram | The molecular basis of dehydration tolerance in plants[END_REF][START_REF] Scott | Resurrection plants and the secrets of eternal leaf[END_REF]. In fructan-accumulating plants, fructans might play this role.

In addition, we found that CMS of roots was not significantly correlated to plant survival (Table 3). This suggests that dehydration tolerance of the root system was not enough to ensure plant survival, underlining the role of leaf meristems in re-growth capacity after rehydration in species from upland grasslands and especially in the Mediterranean genotype.

The combination of resource acquisition, dehydration avoidance and tolerance strategies enhance drought survival and recovery of temperate species

As in [START_REF] Ludlow | Strategies of response to water stress[END_REF], present results show that species were able to combine strategies for resource acquisition and strategies for drought resistance (Fig. 7, Table 3). The highest drought survival and recovery (RI index) was observed for the fast-growing species with higher LER and deeper roots, such as T. officinale, D. glomerata 'Medly' and D. glomerata (Table 1, Fig. 6, Fig. 7, Table 3). A higher LER or SLA can confer a competitive advantage for maintaining photosynthesis and root growth, although the resulting greater leaf area may be less efficient in avoiding dehydration [START_REF] Pérez-Ramos | Tradeoffs between functional strategies for resource-use and drought-survival in Mediterranean rangeland species[END_REF]. As LER and WU were positively correlated (Fig. 7A, Table 3), our results confirm that differences among species in dehydration avoidance strategy are closely associated with strategy to acquire water depending on root mass and depth [START_REF] Chaves | How plants cope with water stress in the field? Photosynthesis and growth[END_REF].

However, a dehydration avoidance strategy mainly based on maximizing water acquisition was not efficient enough to ensure plant survival and fast recovery in the case of F. arundinacea. This species had a deep root system and a high root mass, but was unable to maintain LRWC and MWC, and therefore had a low survival rate (Table 1, Fig. 6B). This could be explained by higher leaf transpiration and lower water-use efficiency in comparison with T. officinale, D. glomerata 'Medly' and D. glomerata [START_REF] Brock | Drought tolerance in the Alpin Dandelion, Taraxacum ceratophorum (Asteraceae), its exotic congenert T. officinale, and interspecific hybrids under natural and experimental conditions[END_REF][START_REF] Milbau | Ecophysiological and morphological parameters related to survival in grass species exposed to an extreme climatic event[END_REF]. However, at a longer time scale, F. arundinacea was able to recover to the level of D. glomerata 'Medly', D. glomerata and T. officinale. In addition, species that were least efficient in avoiding dehydration, but which had storage organs, such as T. flavescens and P. pratensis, had higher drought survival than F. arundinacea (Fig. 5B), probably due to the amount of carbohydrate reserves in their storage organs (Fig. 3G, 3H, Fig. S1G). These results therefore confirm that storage organs can contribute to short-term plant re-growth [START_REF] Klimešová | Bud banks and their role in vegetative regeneration -A literature review and proposal for simple classification and assessment[END_REF][START_REF] Carter | Drought-mediated stem and below-ground bud dynamics in restored grasslands[END_REF]. However, at a longer time scale, P. pratensis recovered better than T. flavescens and was able to extend its root system (RRI > 1, Fig. 6C). Higher ion solutes, WSC contents in roots, and root development after drought treatment [START_REF] Jiang | Effects of drought or heat stress alone and in combination on Kentucky bluegrass[END_REF] may explain the better recovery capacity of P. pratensis than of T. flavescens and P. trivialis (Fig. 6C).

All the species showed dehydration avoidance and tolerance mechanisms involving sucrose accumulation and/or fructan maintenance in plant organs. Also, differences between carbohydrate dynamics in response to drought were as high among the grasses as between the grasses and the forb (T. officinale) (Fig. 3, Fig. S1, Fig. S2). The Mediterranean cultivar of D. glomerata survived better than the ecotype of cocksfoot of upland origin, probably because of the higher initial fructan level in its leaf meristems, confirming previous results for this cultivar [START_REF] Volaire | Growth, carbohydrate reserves and drought survival strategies of contrasting Dactylis glomerata populations in a Mediterranean environment[END_REF][START_REF] Volaire | Drought survival in Dactylis glomerata and Festuca arundinacea under similar rooting conditions in tubes[END_REF]. However, the recovery of D. glomerata and its Mediterranean cultivar one year after drought was similar, emphasizing the capacity of resilience of the native species after a severe drought.

This study on forage grassland species showed diversity in plant strategies to survive and recover after severe drought, as observed by [START_REF] Craine | Global diversity of drought tolerance and grassland climate-change resilience[END_REF] or Pérez [START_REF] Pérez-Ramos | Tradeoffs between functional strategies for resource-use and drought-survival in Mediterranean rangeland species[END_REF].

Against our third hypothesis, no trade-off was found between high resource acquisition under non-limiting conditions and drought survival within the plant material tested originating from upland areas subjected to infrequent severe drought. This performance results mainly from dehydration avoidance and tolerance strategies associated with a strong allocation of carbon to the root system ensured by an efficient carbon acquisition at the whole plant level. It is also noteworthy that plant survival observed two weeks after rehydration was not a good indicator of one-year recovery. This emphasizes the need to assess recovery after drought at a longer time scale and to take into consideration storage organs as key organs for resilience capacity of species. Our results also suggest that most of the native forage species studied are able to survive and recover from extreme drought but with various time lags. This wide range of drought resistance strategies in native forage upland species raises the question of high resilience of diverse plant communities for maintenance of grassland services after extreme events. Dmax: maximum rooting depth (cm); D95: 95% rooting depth (cm); LER: leaf elongation rate (mm d -1 ); LDMC: leaf dry matter content (g g -1 ); LRWC: leaf relative water content (%); LS: leaf senescence (%); MWC: leaf meristem water content (%); RM: total root mass (kg m -3 ); RDMC: root dry matter content (g g -1 ); SWCel: soil water content at the end of leaf growth (m 3 m -3 ); SLA: specific leaf area (m 2 kg -1 ); SWCs: soil water content at 50% of leaf senescence (m 3 m -3 ); WU: water used (g kg -1 ). Lower script (1, 2, 3) of traits when present corresponds to sampling dates (SD). Dg: Dactylis glomerata; Fa: Festuca arundinacea; Md: Dactylis glomerata 'Medly'; Pp: Poa pratensis; Pt: Poa trivialis; Tf: Trisetum flavescens; To: Taraxacum officinale. : maximum rooting depth (cm); D95: 95% rooting depth (cm); HDP: fructan DP ≥ 5 content in leaf meristems (m), roots (r) and root apices (a) (mg g -1 DM); LER: leaf elongation rate (mm d -1 ); LDMC: leaf dry matter content (g g -1 ); LRWC: leaf relative water content (%); LS: leaf senescence (%); MWC: leaf meristem water content (%); QWSCr: amount of WSC in roots (mg); RI: resilience index of spring forage production; RECOV: one-year recovery of aerial green tissue (%); RRI: resilience index of standing root mass; RM: total root mass (kg m -3 ); RDMC: root dry matter content (g g -1 ); SO: sucrose content in leaf meristems (m), roots (r), and root apices (a) (mg g -1 DM); SURV: plant survival rate two weeks after rehydration (%); SWCel: soil water content at the end of leaf growth (m 3 m -3 ); SLA: specific leaf area (m 2 kg -1 ); SWCs: soil water content at 50% of leaf senescence (m 3 m -3 ); WU: water used (g kg -1 ). Lower script (1, 2, 3) of traits when present corresponds to sampling dates (SD). Different letters correspond to between-species differences for canopy green tissue measured two weeks after rehydration. HDP Fructans content (mg g -1 DM)

Zwicke M, Alessio

Sucrose content (mg g -1 DM)

Figure legends Figure 1 .

 legends1 Figure legends Figure 1. Time course of soil volumetric water content (SWC, m 3 m -3 ) for Taraxacum officinale (A), Dactylis glomerata 'Medly' (B), Dactylis glomerata (C), Trisetum flavescens (D), Poa pratensis (E), Festuca arundinacea (F), Poa trivialis (G), irrigated and during drought treatment, at 10 cm (solid lines), 40 cm (dashed lines) and 100 cm (dotted lines) below soil surface. Vertical lines represent last watering (black solid line), end of leaf growth (grey solid line) and rehydration (black dashed line), delimiting periods of moderate drought (DOY 182-201) and severe drought (DOY 202-231), respectively. Arrows mark the four sampling dates: SD1 (DOY 180); SD2 (DOY 200); SD3 (DOY 235); SD4 (DOY 242).

Figure 2 .

 2 Figure 2. Root mass (kg m -3 ) profile in the soil of Taraxacum officinale (A), Dactylis glomerata 'Medly' (B), Dactylis glomerata (C), Trisetum flavescens (D), Poa pratensis (E), Festuca arundinacea (F), Poa trivialis (G) measured before drought treatment (black circles), during moderate drought (white circles) and severe drought (white squares), and in the control plants at the end of drought (black squares). For Taraxacum officinale, insert corresponds to tap root mass profile. Mean values are shown ± se (n = 3).

Figure 3 .

 3 Figure 3. Fructans with degree of polymerization ≥ 5 (HDP) (A, C, E, G) and sucrose contents (B, D, F, H) in leaf meristems (A, B), in roots (C, D), in root apices (E, F) of Taraxacum officinale (filled orange triangles), Dactylis glomerata 'Medly' (open blue circles), Dactylis glomerata (filled blue circles), Trisetum flavescens (open red triangles), Poa pratensis (filled green squares), Festuca arundinacea (filled red triangles), Poa trivialis (open green squares) and in storage organs (G, H) of Taraxacum officinale (taproot), Trisetum flavescens (stolon), Poa pratensis (rhizome) and Poa trivialis (stolon) during the experimental drought (DOY 182 -231) and in the control plants (DOY 242). Mean values are shown ± se (n = 3). Symbols indicate significant differences between species after one-way ANOVA: + P ≤ 0.1; * P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001.

Figure 4 .

 4 Figure 4. Cell membrane stability measured in leaf meristems (A), roots (R), and root apices (C) of Taraxacum officinale (To), Dactylis glomerata 'Medly' (Md), Dactylis glomerata (Dg), Trisetum flavescens (Tf), Poa pratensis (Pp), Festuca arundinacea (Fa), Poa trivialis (Pt), and in storage organs (D) of To (taproot), Tf (stolon), Pp (rhizome) and Pt (stolon) measured at the end of severe drought in control (black) and drought (white) plants. Mean

Figure 5 .

 5 Figure 5. Correlations between cell membrane stability and fructans with degree of polymerization ≥ 5 (high degree of polymerization, HDP) (A, C, E), and sucrose contents (B, D, F) measured in leaf meristems (A, B), in roots (C, D) of Taraxacum officinale (filled orange triangles), Dactylis glomerata 'Medly' (open blue circles), Dactylis glomerata (filled blue circles), Trisetum flavescens (open red triangles), Poa pratensis (filled green squares), Festuca arundinacea (filled red triangles), Poa trivialis (open green squares) and in storage organs (e, f) of Taraxacum officinale (taproot), Trisetum flavescens (stolon), Poa pratensis (rhizome) and Poa trivialis (stolon) measured at the end of the severe drought in control and drought plants. Lines represent the significant linear regressions (P < 0.05) for all species (black line) and for each species (To: orange line; Md: dashed blue line; Dg: solid blue line; Tf: dashed red line; Pp: solid green line; Fa: solid red line; Pt: dashed green line).

Figure 6 .

 6 Figure 6. Pictures of Taraxacum officinale (To), Dactylis glomerata 'Medly' (Md), Dactylis glomerata (Dg), Trisetum flavescens (Tf), Poa pratensis (Pp), Festuca arundinacea (Fa), Poa trivialis (Pt) two weeks after rehydration (A) and one year after rehydration on drought plants (B), canopy greenness of the tissue (%) two weeks after rehydration (white bars) and one year (hashed bars) after rehydration (C), and resilience index of spring forage production (D) and standing root mass (D) of the seven species. Mean values ± se are shown (n = 3). Symbols indicate significant effect of periods on canopy green tissue, spring forage production and standing root mass for each species (+: P < 0.1; *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001).

Figure 7 .

 7 Figure 7. Analyses of principal component combining traits related to strategies of resource acquisition (A), dehydration avoidance (B), and dehydration tolerance (C), measured on Taraxacum officinale (To), Dactylis glomerata 'Medly' (Md), Dactylis glomerata (Dg), Trisetum flavescens (Tf), Poa pratensis (Pp), Festuca arundinacea (Fa), Poa trivialis (Pt) under full irrigation (SD1), during moderate drought (SD2) and severe drought (SD3), and after rehydration. CMSa, CMSm, CMSr: cell membrane stability in root apices, leaf meristems and roots; Dmax: maximum rooting depth; HDPm, HDPr: fructans content with degree of polymerization ≥ 5 in leaf meristems and roots; LER: leaf elongation rate; LDMC:

Figure S1 .Figure

 S1 Figure S1. Total water-soluble carbohydrate (WSC) (A, C, E, G) and fructans with degrees of 1 polymerization and 4 (low degree of polymerization, LDP) contents (B, D, F, H) in leaf 2 meristems (A, B), roots (C, D), root apices (E, F) of Taraxacum officinale (filled orange 3 triangles), Dactylis glomerata 'Medly' (open blue circles), Dactylis glomerata (filled blue 4 circles), Trisetum flavescens (open red triangles), Poa pratensis (filled green squares), 5 Festuca arundinacea (filled red triangles), Poa trivialis (open green squares) and in storage 6 organs (G, H) of Taraxacum officinale (taproot), Trisetum flavescens (stolon), Poa pratensis 7 (rhizome) and Poa trivialis (stolon) during the experimental drought (DOY 182 -231) and in 8 the control plants (DOY 242). Mean values are shown ± se (n = 3). Symbols indicate 9 significant differences between species after one-way ANOVA: + P ≤ 0.1; * P ≤ 0.05; **: P ≤ 10 0.01; ***: P ≤ 0.001. 11 12 13

  Figure 4

  Figure 6

GA, Thiery L, et al. 2013.

  Lasting effects of climate disturbance on perennial grassland above-ground biomass production under two cutting frequencies.

	APPENDIX
	A1: List of abbreviations
	Global Change Biology 19: 3435-3448. Abbreviation Variable measured	Units
	CMS	Cell membrane stability	%
	D 95	95% rooting depth	cm
	D max	Maximum rooting depth	cm
	HDP	fructans content with degrees of polymerization	mg g -1 DW
		over 5 (high degree of polymerization)
	LDMC	Leaf dry matter content	g g -1
	LDP	fructans content with degrees of polymerization 3	mg g -1 DW
		and 4 (low degree of polymerization)
	LER	Leaf elongation rate	mm d -1
	LRWC	Leaf relative water content	%
	LS	Leaf senescence	%
	MWC	Leaf meristem water content	%
	PET	Potential evapotranspiration	mm d -1
	QWSCr	amount of WSCs in roots	mg per plant
	RDMC	Root dry matter content	g g -1
	RECOV	Plant one-year recovery	%
	RI	Resilience index of spring forage production	-
	RM	total root mass	kg m -3
	RRI	Resilience index of spring standing root mass	-
	SD	Sampling date	-
	SLA	Specific leaf area	m 2 kg -1
	SO	Sucrose content	mg g -1 DW
	SURV	Plant survival rate	%
	SWC	Soil water content	m 3 m -3
	SWC el	Soil water content at the end of leaf elongation	m 3 m -3
	SWCs	Soil water content for 50% leaf senescence	m 3 m -3
	WSC	Water soluble carbohydrate	mg g -1 DW
	WU	Plant water used	g kg -1

Table 1 .

 1 Plant traits measured under irrigation before drought, at the end of moderate and severe drought. Mean values are shown (n = 3). Letters show significant differences between the seven species, except for tap root of To, according to one-way ANOVA and Tukey tests (P < 0.05).

	Periods	Traits	To taproot	To	Md	Dg	Tf	Pp	Fa	Pt
	Before drought	LDMC		168 c	259 ab	242 ab	91 d	311 a	223 bc	267 ab
		SLA		29.2 bc	24.9 bc	27.3 bc	33.5 b	22.8 c	24.5 c	49.5 a
		LER		9.4 abc	10.3 ab	11.8 a	6.7 c	4.6 c	6.6 bc	3.3 c
		LRWC 1		96 ab	98 a	97 ab	96 ab	95 ab	96 ab	88 b
		MWC 1		80 a	77 ab	80 a	75 ab	74 ab	80 a	69 b
		RM 1	7.5	1.7 c	4.9 a	4.6 ab	3.3 b	3.6 bc	5.0 a	2.6 c
		RDMC 1	0.257	0.101 d	0.211 ab	0.189 c	0.195 bc	0.228 a	0.206 bc	0.204 bc
		Dmax 1		146 a	134 a	122 ab	70 cd	100 bc	146 a	57 d
		D95 1	47	98 a	82 ab	71 bc	62 bc	60 bc	106 a	48 c
		WU 1		125 a	106 ab	111 ab	78 b	75 b	133 a	82 b
	Moderate drought LRWC 2		69 a	53 ab	39 ab	30 b	29 b	33 b	27 b
		MWC 2		63.0 a	47.8 bc	46.4 bc	40.5 cd	38.0 d	49.6 b	38.3 d
		LS 2		20.8 a	6.7 a	19.2 a	20.8 a	12.5 a	23.3 a	54.2 b
		RM 2	11.5	2.1 d	5.9 a	4.7 ab	3.7 cd	4.0 bc	4.9 ab	3.0 cd
		RDMC 2	0.340	0.154 c	0.305 a	0.278 ab	0.243 b	0.286 ab	0.306 a	0.302 ab
		Dmax 2		145 a	129 ab	134 a	78 c	95 bc	147 a	67 c
		D95 2	53	91 a	74 bc	67 c	58 c	67 c	98 ab	49 c
		WU 2		103	75	76	46	47	70	37
		SWC el		0.122	0.100	0.110	0.071	0.065	0.099	0.062
	Severe drought	LRWC 3		16	28	19	11	16	-	-
		MWC 3		43.8 a	46.2 a	35.5 ab	28.5 ab	26.3 ab	22.2 ab	14.6 b
		LS 3		90 bc	81 c	96 ab	97 ab	90 bc	100 a	100 a
		RM 3	13.0	1.8 c	5.3 a	4.9 a	3.6 b	3.7 b	5.0 a	2.8 bc
		RDMC 3	0.431	0.237 b	0.343 a	0.339 a	0.266 ab	0.291 ab	0.326 a	0.371 a
		Dmax 3		149 a	133 a	136 a	85 b	91 b	150 a	61 b
		D95 3	57	113 a	78 bc	67 cd	61 cd	53 d	93 ab	56 cd
		WU 3		70	28	36	20	40	20	19
		SWC s		0.085	0.052	0.092	0.029	0.018	0.053	0.061

Table 3 .

 3 Pearson's correlation coefficients between all pairs of plant traits measured under irrigation (DOY 78-181), moderate drought (DOY 182-201) and severe drought (DOY 202-231) treatments. Significant correlations are shown in bold (P < 0.05) and underlined characters (P < 0.001).

		SURV	RECOV	RI	RRI
	LER	0.68	0.35	0.26	0.32
	SLA	-0.38	-0.38	-0.76	-0.38
	RM 1	0.62	0.31	0.05	-0.14
	Dmax 1	0.53	0.41	0.48	0.18
	D95 1	0.00	0.01	0.47	0.01
	RDMC 1	-0.42	-0.10	0.48	0.08
	LDMC	-0.19	0.29	0.35	0.26
	WU 1	0.32	0.23	0.29	0.05
	LRWC 2	0.67	0.39	0.10	0.31
	RDMC 2	-0.51	-0.27	0.34	0.14
	RM 2	0.65	0.35	-0.08	-0.02
	WU 2	0.72	0.43	0.23	0.20
	HDPm 2	0.28	0.07	0.21	0.10
	SOm 2	-0.50	-0.22	0.23	-0.14
	HDPr 2	0.60	0.26	0.10	0.02
	SOr 2	0.26	0.06	0.32	0.15
	HDPa 2	0.09	-0.02	-0.13	-0.05
	SOa 2	0.30	0.22	0.03	0.01
	SWCel	0.45	0.46	0.14	0.49
	LS3	-0.70	-0.24	-0.32	-0.25
	SWCs	-0.42	-0.22	-0.72	-0.25
	MWC3	0.76	0.34	0.31	0.21
	CMSm	0.51	0.09	0.37	0.09
	CMSr	0.06	-0.09	0.50	0.09
	CMSa	0.36	0.17	0.39	0.38
	HDPm3	0.07	0.39	0.40	0.42
	SOm3	-0.35	0.08	0.05	0.21
	HDPr3	0.29	0.13	0.32	0.20
	SOr3	0.56	0.25	0.25	0.08
	HDPa3	0.04	-0.05	0.47	0.21
	SOa3	-0.10	-0.11	0.65	0.04
	QWSCr	0.30	0.14	0.61	0.36
	SURV	1	0.28	0.12	0.24
	RECOV		1	0.40	0.66
	RI			1	0.55
	RRI				1
	Dmax

ACKNOWLEDGMENTS

We thank Boris Adam, Caroline Bernard, Robert Falcimagne, Patrick Pichon, Alexandre Salcedo, Christophe Serre, Lionel Thiery and Patrice Chaleil for their help with the experimental set-up, data collection and site management.

FUNDING This work was supported by the INRA project Climagie (ACCAF Metaprogram: Adaptation to Climate Change of Agriculture and Forest), Auvergne Région and European funding for Regional Development (« L'Europe s'engage en Auvergne ») through a doctoral fellowship awarded to Marine Zwicke, and a Marie Louise FURNESTIN-FAURE Scholarship 2011 (French association of graduated French women).