Frederic C Do
email: frederic.do@ird.fr

Alain Rocheteau

Amadou L Diagne

Venceslas Goudiaby

André Granier

Jean-Paul Lhomme

Stable annual pattern of water use by Acacia tortilis in Sahelian Africa

Keywords: deep roots, dry savanna, heat dissipation method, hydraulic conductance, rainfall, transpiration, xylem sap flow

Water use by mature trees of Acacia tortilis (Forsk.) Hayne ssp. raddiana (Savi) Brenan var. raddiana growing in the northern Sahel was continuously recorded over 4 years. Water use was estimated from xylem sap flow measured by transient heat dissipation. Concurrently, cambial growth, canopy phenology, leaf water potential, climatic conditions and soil water availability (SWA) were monitored. In addition to the variation attributable to interannual variation in rainfall, SWA was increased by irrigation during one wet season. The wet season lasted from July to September, and annual rainfall ranged between 146 and 367 mm. The annual amount and pattern of tree water use were stable from year-to-year despite interannual and seasonal variations in SWA in the upper soil layers. Acacia tortilis transpired readily throughout the year, except for one month during the dry season when defoliation was at a maximum. Maximum water use of about 23 l (dm sapwood area) -2 day -1 was recorded at the end of the wet season. While trees retained foliage in the dry season, the decline in water use was modest at around 30%. Variation in predawn leaf water potential indicated that the trees were subject to soil water constraint.

The rapid depletion of water in the uppermost soil layers after the wet season implies that there was extensive use of water from deep soil layers. The deep soil profile revealed (1) the existence of living roots at 25 m and (2) that the availability of soil water was low (-1.6 MPa) down to the water table at a depth of 31 m. However, transpiration was recorded at a predawn leaf water potential of -2.0 MPa, indicating that the trees used water from both intermediary soil layers and the water table. During the full canopy stage, mean values of whole-tree hydraulic conductance were similar in the wet and dry seasons. We propose that the stability of water use at the seasonal and annual scales resulted from a combination of features, including an extensive rooting habit related to deep water availability and an effective regulation of canopy conductance. Despite a limited effect on tree water use, irrigation during the wet season sharply increased predawn leaf water potential and cambial growth of trunks and branches.

Introduction

The northern Sahel represents one of the driest savannas in the world [START_REF] Houérou | The grazing land ecosystems of the African Sahel[END_REF]. Potential evaporation is in excess of 2000 mm year -1 . The wet season lasts only three months, and rainfall averages between 200 and 400 mm year -1 , with high interannual variability (coefficient of variation of 37%). Groundwater is usually deeper than 30 m. Acacia tortilis (Forsk.) Hayne ssp. raddiana (Savi) Brenan var. raddiana, a dominant tree species in the area [START_REF] Breman | Woody plants in agro-ecosystems of semi-arid regions, with emphasis on the Sahelian countries[END_REF]Kessler 1995, Le Floc'h and[START_REF] Floc'h | Acacia raddiana, un arbre des zones arides à usages multiples[END_REF], represents an efficient model of drought adaptation, but little is known about its water use and the influence of rainfall on its seasonal and annual patterns of water use. The control of water use by trees in water-limited biomes has been much debated [START_REF] Eamus | Dry season conditions determine wet season water use in the wet-dry tropical savannas of northern Australia[END_REF] and has important consequences for strategies of drought adaptation and optimal balance between carbon gain and water loss [START_REF] Eamus | Ecophysiology of trees of seasonally dry tropics: comparisons among phenologies[END_REF]. Moreover, a detailed knowledge of seasonal tree water use and factors affecting its regulation is fundamental to understanding the impacts of climate change on hydrological and carbon balances at the ecosystem level.

Rainfall would be expected to have a major influence on tree water use in a water-limited biome, such that water use would be highest in the wet season, declining with soil water availability (SWA) during the dry season despite an increase in evaporative demand, thereby limiting canopy desiccation. There are few long-term studies of whole-tree water use in the seasonally dry tropics [START_REF] Eamus | Ecophysiology of trees of seasonally dry tropics: comparisons among phenologies[END_REF]; however, several studies have shown that, contrary to expectation, tree water use in such a climate does not differ greatly [START_REF] Cook | Water balance of a tropical woodland ecosystem, Northern Australia: a combination of micro-meteorological, soil physical and groundwater chemical approaches[END_REF][START_REF] Roupsard | Reverse phenology and dry-season water uptake by Faidherbia albida (Del.) A. Chev. in an agroforestry parkland of Sudanese west Africa[END_REF], or at all [START_REF] Meinzer | Atmospheric and hydraulic limitations on transpiration in Brazilian cerrado woody species[END_REF][START_REF] Eamus | Dry season conditions determine wet season water use in the wet-dry tropical savannas of northern Australia[END_REF][START_REF] Hutley | Monsoonal influences on evapotranspiration of savanna vegetation of northern Australia[END_REF], between the wet and dry seasons. Season-to-season uniformity in water use can be at-tributed to constancy in the balance between evaporative demand of the atmosphere and canopy conductance as determined by factors such as SWA, hydraulic conductivity of xylem, leaf area and stomatal conductance. In particular, leaf phenology, which roughly reflects seasonal changes in leaf area, is a principal driver of seasonal changes in transpiration.

Acacia tortilis ssp. raddiana is a semi-evergreen or drought-deciduous species [START_REF] Breman | Woody plants in agro-ecosystems of semi-arid regions, with emphasis on the Sahelian countries[END_REF]Kessler 1995, Le Floc'h and[START_REF] Floc'h | Acacia raddiana, un arbre des zones arides à usages multiples[END_REF]. In the northern Sahel, the full canopy stage of this species lasts between 6 and 8 months, including the 3-month rainy season and part of the dry season [START_REF] Do | Environmental influence on canopy phenology in the dry tropics[END_REF]. The ability to maintain leaf area and transpiration during the dry season may be explained by high water availability in the upper soil layers, as in the case of riparian vegetation, or a deep rooting habit that gives access to water from the subsoil or the water table, or both [START_REF] Cook | Water balance of a tropical woodland ecosystem, Northern Australia: a combination of micro-meteorological, soil physical and groundwater chemical approaches[END_REF][START_REF] Roupsard | Reverse phenology and dry-season water uptake by Faidherbia albida (Del.) A. Chev. in an agroforestry parkland of Sudanese west Africa[END_REF]. That transpiration does not increase during the dry season in response to increasing evaporative demand, despite the availability of soil water, suggests strong stomatal regulation as reported in other dry habitats (Meinzer et al. 1999, Eamus and[START_REF] Eamus | Ecophysiology of trees of seasonally dry tropics: comparisons among phenologies[END_REF]. Where deep water supply is limited, [START_REF] Eamus | Dry season conditions determine wet season water use in the wet-dry tropical savannas of northern Australia[END_REF] and [START_REF] Hutley | Monsoonal influences on evapotranspiration of savanna vegetation of northern Australia[END_REF] hypothesize that stable dry season water use results from limited xylem conductivity associated with the ability to tolerate low water potentials without runaway xylem embolism. If this interpreation is correct, it implies that dry season conditions determine wet season water use by defining the tree's hydraulic architecture.

These studies did not consider variability within and among years, because they were conducted over short measurement periods at the end of the wet and dry seasons within a single year. Moreover, they were conducted under environmental conditions that are less dry than the northern Sahel, and where, in particular, SWA is higher during the dry season.

To investigate water use of A. tortilis growing in the northern Sahel, we measured sap flow in mature trees continuously over a 4-year period, with concurrent measurements of leaf water potential, SWA and climatic conditions. In addition to the natural interannual variation of rainfall, water availability in the upper soil was maximized during one rainy season by irrigation. Our objectives were, first, to quantify seasonal and annual patterns of water use by A. tortilis, and second, to analyze changes in water use between the wet and dry seasons and assess the effect of increased water availability during the wet season. Cambial growth was monitored concurrently. Our hypotheses were that during the full canopy stage, tree water use declines throughout the dry season, the decrease being greatest during the period of defoliation and that higher water availability during the wet season enhances seasonality and increases both water use and growth.

Materials and methods

Site

The study was carried out in northern Senegal, near the village of Souilene (16°20′ N, 15°25′ W, 5-10 m a.s.l.) located 20 km south of Dagana in the grazing land ecosystem of the sandy Ferlo. Annual rainfall averages 280 mm, mean annual temper-ature is 28.7 °C, and mean maximum and minimum temperatures are 41 and 22 °C for the hottest month (May), respectively and 31 and 14 °C for the coldest month (January), respectively. The sparse woody community is dominated by three species: A. tortilis and Balanites aegyptiaca (L.) Del, comprising the tree layer (90 ha -1 ), and Boscia senegalensis (Pers.) Lam in the shrub layer (50 ha -1 ). The soil is deep and sandy, with mean contents of sand, clay, organic matter, nitrogen and total phosphorus (P 2 O 5 ) of 90%, 6%, 0.18%, 0.15‰ and 0.3‰ in the first meter of soil, respectively.

The studied population of 39 mature individuals was located in a 1-ha area that has been protected since 1989. Trunk diameter 30 cm above the soil varied from 5 to 45 cm, with 70% of the population having diameters between 15 and 30 cm. Tree height ranged from 5 to 10 m. Three trees of average diameter were chosen in a grove for the long-term study of water use. Mean tree height was 7 m, crown projected area was 30 m 2 , and mean trunk diameter was 17.4 cm at a height of 1.3 m.

Sap flow measurement

Tree water use was estimated from xylem sap flow measured with a transient thermal dissipation (TTD) system (Do and Rocheteau 2002a), which is a modification of the continuous thermal dissipation method of [START_REF] Granier | Une nouvelle méthode pour la mesure des flux de sève dans le tronc des arbres[END_REF]. The modification avoids the influence of passive temperature gradients in the experimental trees (Do and Rocheteau 2002b). The TTD system is based on the Granier probe design, heating power and empirical response formulation, but with a cyclic schedule of heating and cooling and a specific calibration (Do and Rocheteau 2002a). A cycle of 45 min heating and 15 min cooling was used. Zero flux signal was determined each night assuming that sap flow was negligible at the end of the night (see details below). Total sap flow (F) was calculated as: F = (SFD)A sw , where SFD (l dm -2 h -1 ) is sap flow density per sapwood area and A sw (dm 2 ) is cross-sectional area of conducting wood at the level of the heating probe. The area of conducting wood was determined from observations of whole sections of freshly cut trees and cores. Heartwood was distinguished from sapwood by its distinct color, which was redder and duller than the sapwood. To account for cambial growth, sapwood area was calculated from the external trunk diameter based on the formula:

A R R sw w 2 h 2 = - π( )
where R w is wood radius (excluding bark) and R h is heartwood radius. The radii were related to the external diameter (D ext ; cm) by following relationships based on 14 samplings of external diameters ranging from 9 to 36 cm: R w = 0.49D ext -0.39 (r 2 > 0.99); and R h = 0.48D ext -5.02 (r 2 = 0.93).

Probes were inserted into the trunks 1.3 m above the soil, at which height estimated mean sapwood area was 1.73 dm 2 , representing an approximate conductive radius of 4.7 cm after bark removal, which was 0.5 cm thick. Probes 2 cm in length were inserted into a hole 3 cm deep where they were fully within the conductive sapwood. To account for circumfer-ential variation in each tree, three probes were inserted at different azimuths (North, South-East and South-West). After probe insertion, the exposed parts of the needles were coated with silicone. The trunk area containing the probes was shielded from direct sunlight. Probes were connected to a data logger. Hourly SFD were cumulated over 24 h to calculate daily SFD measurements or daily total flow. Daily total flow is an estimate of daily tree water use or transpiration, ignoring changes in tree water storage.

Estimates of tree water use from sap flow measurements are subject to various uncertainties [START_REF] Köstner | Sapflow measurements in forest stands: methods and uncertainties[END_REF][START_REF] Lu | Granier's thermal dissipation probe (TDP) method for measuring sap flow in trees: theory and practice[END_REF], one being in the estimation of the signal at zero flux. The classical method considers that the maximum value occurs each night to correspond with zero flux, and that seasonal variation in the maximum signal at night results mainly from changes in xylem water content. To test this hypothesis, we compared three methods of estimating the zero flux signal based on maximum signals recorded at the end of the night: (1) maximum value recorded each night; (2) maximum values calculated from a spline curve fitted over 10 days; (3) maximum values calculated from a spline curve fitted over 30 days. The estimates of daily maximum sap flow density based on the three methods were compared with the gradients between minimum and predawn leaf water potentials recorded on the same day, which are also related to transpiration. The first method gave the best agreement (r 2 = 0.36, n = 46) compared with the other methods (r 2 = 0.28 and 0.27, respectively), so we used Method 1, i.e., the classical method.

Other uncertainties concerning estimates of tree water use from sap flow measurements include the number of probes needed to account for circumferential variation in sap flow and the aging of probes during our long-term study. After 2 years, new sets of three probes were installed in each tree between the old probes at approximate azimuths East, South and West. There were no significant differences between the estimates of mean tree water use obtained with the new and old sets of probes. Within a set, probe dynamics were highly synchronous, although absolute values differed markedly and coefficients of variation ranged between 30 and 40%. Circumferential (intra-individual) variations were stable at around 30% and systematically higher than inter-individual variations. The size of the measuring tip of the probe (2 cm), compared with the estimated 4.7 cm radius of conducting xylem, allowed integration of radial variation in sap flow density.

Cambial growth

Stem diameter variation was continuously recorded to ± 0.010 mm on one main branch of each experimental tree every hour with linear displacement transducers (Enertec Sclumberger, Paris, France). Probe supports were made of IN-VAR. Measurement systems were shielded from direct sunlight, and probes were glued onto the bark surface. Linear transducers were cleaned and checked monthly. At the same time, measurements of each branch diameter were made at opposite axes with an electronic calliper (Sumitoyo, Tokyo, Japan).

Climatic measurements

Air temperature, relative humidity, incoming short wave radiation and rainfall were measured with automatic instruments in a field 50 m from any grove edge. A reference potential evapotranspiration (PET) was calculated according to the Penman formula (1948).

Soil water availability

A soil water profile to the water table was kindly provided by Dr. Abdoulaye Faye (Geology Department, Dakar University), which was obtained by measurements on soil samples taken with an auger every 0.5 m. In the uppermost soil layers (0-4 m), soil water content was monitored with a neutron probe (Solo 25, Nardeux S.A., Les Ulis, France). The bases of the three experimental trees in the grove approximately mark the corners of an isosceles triangle with sides of 5 m, and neutron probe access soil tubes were located in the middle of each side of the triangle. The soil wetting front rarely exceeded 1 m in depth. Soil water availability was calculated for the 0-100 cm deep layer, based on the difference between soil water content at field capacity and the lowest soil water content. Within the lowest measured soil zone, water content corresponded to a soil water potential of less than -1.6 MPa. During the wet season of 1998, irrigation was applied twice weekly during August and September to maintain soil water content in the 0-100 cm soil layer (80 mm) at field capacity, as determined by neutron probe measurements.

Phenological stages

For each tree, leaf phenology was assessed in six categories [START_REF] Grouzis | A method for the phenological study of browse populations in the Sahel: the influence of some ecological factors[END_REF]): 0, leafless; 1, leaf buds opening; 2, start of leaf expansion on 10-50% of branches; 3, leaves fully expanded on more than 50% of branches; 4, start of leaf senescence as indicated by color change on less than 50% of branches; 5, all leaves senescent or fallen on more than 10% of branches. These phenological stages were converted to an index of canopy fullness: 0 for Stage 0; 1 for Stages 1 and 5; 2 for Stages 2 and 4; and 3 for Stage 3. The total leaf area of three of the experimental trees was estimated in the first week of February 2000 at the end of the full canopy stage. Total leaf biomass of each tree was measured, and specific leaf area (SLA) was determined to estimate total leaf area.

Leaf water potential

Predawn leaf water potential (Ψ pd ) was measured with a Scholander type pressure chamber (PMS 1000, PMS Instrument Company, Corvallis, OR) between 0600 and 0645 h, and minimum leaf water potential (Ψ min ) was measured between 1400 and 1445 h on two randomly selected leafy twigs on each of the three experimental trees (n = 6). For all the days when both leaf water potentials and daily maximum SFD were available, root-to-leaf sapwood-specific hydraulic conductance (K h ; l dm -2 h -1 ) was estimated as: K h = SFD max /(Ψ pd -Ψ min ), where SFD max is daily maximum sap flow density. Predawn leaf water potential was taken as an approximation of root water potential, which was assumed to be constant throughout the day. This formula ignores capacitance and gravitational effects.

Data recording and analysis

From March 1996 to January 2000, phenological observations and measurements of soil water content and leaf water potential were carried out monthly in the dry season and twice monthly in the wet season. Characteristic values of daily water use, i.e., maximum value in the rainy season and maximum and minimum values in the dry season, were automatically selected by analyzing a moving average over a 5-day period applied to each year. To analyze annual and seasonal effects on Ψ pd and daily water use, characteristic values, e.g., maximum value in the rainy season and maximum value in the dry season, were analyzed by a fixed factor analysis of variance with the factors being season, year and tree.

Results

Evaporative demand, rainfall and soil water availability

Reference PET showed seasonal variation (Figure 1a). Values were maximal (7-9 mm day -1 ) in the hot dry season from March to May. During the wet season, from July to September, values were intermediate (5-7 mm day -1 ). The lowest values of 4-5 mm day -1 were recorded in the cool dry season from December to February. Daily maximum values of vapor pressure deficit (D max ) ranged from 7 kPa in the hot dry season to 2 kPa in the wet season. Annual PET averaged 2260 mm with a minimum of 2181 mm in 1999 and a maximum of 2331 mm in 1997 (Table 1). Over the 4-year study, annual rainfall averaged 235 mm with a minimum of 146 mm in 1997 and a maximum of 367 mm in 1999 (Table 1, Figure 1a). The interannual variation (CV = 37%) was similar to the long-term variability for the area. Rains occurred mainly between July and September. The dates for the onset and end of rains were variable, i.e., between June and July and between September and October, respectively. August and September were the wettest months. Upper soil SWA had low and constant values and was below 5% in the hot dry season (Figure 1b). Soil water availability then increased with rainfall and with the irrigation applied in 1998. However, two months after the rainy season, SWA decreased to low values similar to those recorded in other years. The water table depth was 31 m (Figure 2). Between the uppermost soil layer and the water table, SWA was limited as shown by the actual water contents, which were equivalent to a water potential of -1.6 MPa (Figure 2). Living root fragments of A. tortilis were collected at depths of 6 and 25 m, confirming that these trees explored the soil profile to the depth of the water table (Figure 2).

Predawn leaf water potential, canopy phenology and cambial growth

Predawn leaf water potential may reflect SWA from either superficial or deep soil water resources. Changes in Ψ pd were strongly seasonal (Figure 1c). Maximum values occurred in the wet season, varying between -1.2 MPa in 1996 and -0.4 MPa in 1998 with irrigation (year effect: P < 0.001). The lowest values were recorded at the end of the cool dry season and were similar in each year (-2.0 MPa, SD = 0.9). The seasonal effect, determined by comparing extreme values from the rainy season and the cool dry season, was highly significant (P < 0.001). There was an increase in Ψ pd in the middle of the hot dry season, which reached similar values each year (-1.3 MPa, SD = 0.9).

Canopy fullness showed an annual cycle with some variation between years (Figure 1d). The dynamic was similar to the mean pattern recorded for 39 individuals studied in the protected area [START_REF] Do | Environmental influence on canopy phenology in the dry tropics[END_REF]. The defoliation phase was short and centered around the hot dry season. Defoliation was rarely complete, except in 1997. Leaf flushing occurred in the transition phase between the hot dry season and the rainy season and was particularly pronounced before the first rains in 1999 (Figure 1d). The full canopy stage (CF index = 3) lasted between 5 and 8 months, from July to February, and broadly included the rainy and cool dry seasons (Figure 1d).

Total leaf area of the experimental trees, estimated at the end of the full canopy stage in 2000, averaged 14 m 2 (SD = 6.2). According to the projected canopy area of 30 m 2 , a low clumped leaf area index of 0.5 per tree was calculated. The ratio of xylem cross-sectional area to leaf area (Huber value) was 13.6 × 10 -4 , which is in the highest range of values reviewed by [START_REF] Eamus | Ecophysiology of trees of seasonally dry tropics: comparisons among phenologies[END_REF]. Leaf density in January was less than half that at the beginning of the full canopy stage in September. However, the Huber value of 7 × 10 -4 is still within the highest range of reported values.

Daily variations in branch diameter (measured at 0600 h) are shown in Figure 1e. Increases in branch diameter were observed only in the rainy season and were followed by a slight decrease in the dry season. Cambial growth of branches showed large variation between years. It was negligible in 1997 and 1999, but represented a 6% increase in diameter in 1998, i.e., diameter increased from 66.5 to 70.5 mm. Girth change measured by continuous recordings showed large variation among the three sample branches (CV = 50%), which was confirmed by manual measurements made each month (Figure 1e). The growth in trunk diameter at breast height showed a similar final increase of 5.5% (SD = 1.4), i.e., + 9.6 mm.

Seasonal water use

Daily water use measured by xylem sap flow showed a similar seasonal pattern between years where links with canopy phenology and Ψ pd were evident (Figure 1d). The seasonal pattern could be split schematically into four time periods. The first period was characterized by the lowest daily water use, in the middle of the hot dry season between April and May at the time of maximum defoliation. Minimum values of daily water use, calculated over 5-day periods, averaged 8.8 l day -1 (SD = 1.9; Figure 3). Differences between years were significant (P < 0.001). The second period corresponded to the increase in water use in the dry-wet transition period, from May to July (Figure 1d). The third period was characterized by the mainte- nance of relatively high water use (> 25 l day -1 ) for about 6 months, between August and January, corresponding to the phenological stage of full canopy development. Maximum water use (40 l day -1 ; SD = 6.0) was recorded at the end of the rainy season, between September and October, when upper SWA and Ψ pd were also maximal. Differences between years were significant (P < 0.001), and this effect was mainly due to a value recorded in 1998, which was 20% higher than the mean (Figure 3). The maximum water use recorded in 1998 of 48 l day -1 for a stem with a mean diameter of 17 cm corresponded to a daily sap flow density of 28 l dm -2 day -1 and a maximum hourly density of 3 l dm -2 h -1 . The fourth and last period was defined by a decrease in water use during the climatic transition between the cool dry and hot dry seasons, from February to March. Phenological cycles are identified by the year in which the rainy season occurred. Maximum daily water use in the dry season occurred at the end of the full canopy stage in January just before the period of leaf shedding. Differences between years and seasons were significant (P < 0.001 in each case). Values averaged 29.0 l day -1 (SD = 4.4), and this mean value was 27% lower than the maximum value recorded at the end of the rainy season.

Seasonal and interannual comparison of water use per sapwood area, or sap flow density (SFD), yielded the same results as water use and total flow analysis. Continuous measurement of SFD for all years permitted the estimation of annual water use for the phenological cycles of 1996 and 1998 (Figure 1d, Table 1). Calculation started on the first of July to focus on the potential influence of contrasting rainy seasons. Between 1996 and 1998, annual SFD or water use per sapwood area was stable at around 5600 l year -1 dm -2 . Total water use was close to 10,000 l year -1 (Table 1). The value was 9% higher in 1998 than in 1996, the difference being fully explained by the increase in conductive sapwood area. The variation in annual water use was small despite large differences in cambial growth and environmental conditions in the rainy season. A broad estimate of water use yielded 90 mm year -1 for an A. tortilis stand, assuming a stand density of 90 trees ha -1 .

Relationships between daily water use, canopy phenology, evaporative demand and predawn leaf water potential

As suggested by Figure 1d, daily SFD was significantly related to canopy fullness (r 2 = 0.49, n = 46; Figure 4). All measurements from the 4-year dataset were within the same range. Paradoxically, daily SFD was not zero on any measurement occasion during the period of complete defoliation (Figure 4); however, phenological assessment is qualitative and trees could have had a small proportion of green leaves, and measurement error is high when SFD values are lower than 0.5 l dm -2 h -1 . The relationship between canopy phenology and tree water use reflects the relationship between canopy fullness index and total leaf area, particularly during the period of defoliation in the hot dry season. Leaf phenology is well known as the main driver of seasonal changes in transpiration. However, the strength of the relationship is surprising, with measurements for the entire 4-year period falling within the same pattern. Canopy fullness index is derived from a qualitative phenological assessment, and the same Stage 3 of full canopy may correspond to different actual leaf areas. Moreover, in a particular season, evaporative demand varies from day-today and year-to-year. The relationship between canopy phenology and tree water was not improved by the use of SFD averaged over 10-day periods (data not shown, r 2 = 0.50) or by a normalization by Penman PET (data not shown, r 2 = 0.54).

The relationship between water use and canopy phenology is another indication of the marked control of daily water use.

The relationships between SFD and evaporative demand were analyzed specifically for three variables: Penman PET and its main components, global radiation (R g ) and vapor pressure deficits (daily mean, minimum and maximum; D). Considering all data points and pooling values from all years, there was a weak negative correlation between SFD and PET (data not shown, n = 812, r 2 = 0.19) and D max (data not shown, r 2 = 0.15). This relationship is largely explained by the peak in defoliation during the hot dry season (Figure 1d). No global relationships were obtained with any variable when all the data corresponding to full canopy stages were considered (data not shown; n = 341).

The relationship between daily SFD and Ψ pd was weak but significant over all seasons and years (r 2 = 0.40, n = 46; Figure 5a). It was significantly strengthened when only the measurements corresponding to full canopy stages were considered (CF = 3, r 2 = 0.50, n = 26; Figure 5b). For large portions of the dry season, Ψ pd is unlikely to be in equilibrium with SWA. The rapid increase in Ψ pd of between 0.5 and 1.0 MPa, noticed at the time of maximal defoliation, supports this hypothesis. This increase in Ψ pd was not explained by an increase in osmotic potential, which was 0.1 MPa at the time of maximal defoliation (data not shown). Defoliation may facilitate nocturnal rehydration. The relationship between daily SFD and Ψ pd was particularly clear for 1998 (Figure 5b) and variation in SWA could largely explain the daily variation in water use for both the rainy and post-rainy seasons. For 1998, daily SFD increased twofold, from 15 to 30 l day -1 dm -2 , when Ψ pd decreased from -2.0 to -0.5 MPa.

Hydraulic conductance

Daily maximum SFD versus Ψ pd is plotted for full canopy stages in Figure 6a, distinguishing wet and dry seasons. In agreement with the conclusion of stable water use, the mean value in the dry season (1.7 l dm -2 h -1 , SD = 0.3) was not significantly lower than that recorded in the rainy season (1.8 l dm -2 h -1 , SD = 0.5), although Ψ pd averaged -1.7 and -1.0 MPa, respectively. The gradients in water potentials were also similar between the dry season (1.3 MPa, SD = 0.3) and the rainy season (1.3 MPa, SD = 0.2; Figure 6b). Consequently, the mean hydraulic conductances did not differ between the dry season (1.4 l dm -2 h -1 MPa -1 , SD = 0.5) and the rainy season (1.4 l dm -2 h -1 MPa -1 , SD = 0.3; Figure 6c). These results were obtained when mean D max values for the same period were 4.8 kPa in the dry season and 3.3 kPa in the rainy season, respectively (Figure 6d). Such results suggest a strong regulation of canopy conductance, through changes in total leaf area or stomatal conductance, or both, that compensate for the increase in evaporative demand.

Discussion

Seasonal and interannual pattern of tree water use

Annual dynamics revealed that A. tortilis transpires readily over the whole year, except at the peak of the dry season during maximum defoliation. Throughout the dry season during the full canopy stage, there was only a modest decline in tree water use despite the exhaustion of soil water in the uppermost soil layers. An increase in SWA due to rainfall and irrigation did not greatly increase maximum daily water use or annual water use. Moreover, water use would likely have been increased more through irrigation than by natural rainfall alone, as the latter would normally be accompanied by reduced evaporative demand. The relatively uniformity in pattern and amount of tree water use despite environmental fluctuations was striking. These findings support and extend the results of recent studies showing that, in less arid environments, tree water use decreases little [START_REF] Cook | Water balance of a tropical woodland ecosystem, Northern Australia: a combination of micro-meteorological, soil physical and groundwater chemical approaches[END_REF][START_REF] Roupsard | Reverse phenology and dry-season water uptake by Faidherbia albida (Del.) A. Chev. in an agroforestry parkland of Sudanese west Africa[END_REF] or not at all [START_REF] Meinzer | Atmospheric and hydraulic limitations on transpiration in Brazilian cerrado woody species[END_REF][START_REF] Eamus | Dry season conditions determine wet season water use in the wet-dry tropical savannas of northern Australia[END_REF][START_REF] Hutley | Monsoonal influences on evapotranspiration of savanna vegetation of northern Australia[END_REF][START_REF] Bucci | Mechanisms contributing to seasonal homeostasis of minimum leaf water potential and predawn disequilibrium between soil and plant water potential in neotropical savanna trees[END_REF] during the dry season.

Daily water use during the full canopy stage ranged between 22 and 50 l day -1 for trees of average size (DBH =17 cm). Ex-pressed per sapwood area, tree water use ranged from 12 to 30 l dm -2 day -1 . These values are within the range reported for other drought-tolerant species in the dry tropics, e.g., [START_REF] Allen | Measurements of transpiration from savannah shrubs using sap flow gauges[END_REF] for Guiera senegalensis JF Gmel. in Sahelian Africa, [START_REF] Roupsard | Reverse phenology and dry-season water uptake by Faidherbia albida (Del.) A. Chev. in an agroforestry parkland of Sudanese west Africa[END_REF] for Faidherbia albida (Del.) A. Chev. in Soudano-Sahelian Africa, Meinzer et al. (1999) and [START_REF] Eamus | Dry season conditions determine wet season water use in the wet-dry tropical savannas of northern Australia[END_REF] in the Australian savannah. More generally, our results for whole-plant water use fall within the low range given in the review by [START_REF] Wullschleger | A review of whole-plant water use studies in trees[END_REF], which included trees from temperate and tropical humid zones.

Deep water uptake

One explanation for year-round water use could be that the investigated trees, like riparian vegetation, experienced no soil water constraint. Predawn water potential, which varied between -0.4 and -1.5 MPa in the wet season, depending on rainfall or irrigation, decreased to around -2.0 MPa in the dry season, indicating that SWA was limited. However, the values of predawn leaf water potential recorded in the dry season were similar to values reported for other tree and shrub species in less stressful environments [START_REF] Duff | Seasonal patterns in soil moisture, vapor pressure deficit, tree canopy cover and predawn water potential in a northern Australian savanna[END_REF][START_REF] O'grady | Transpiration increases during the dry season: patterns of tree water use in eucalypt open-forests of northern Australia[END_REF]. Furthermore, the operating range of leaf water potential is species-dependent. In the same area, [START_REF] Berger | The water status of six woody species coexisting in the Sahel (Ferlo, Senegal)[END_REF] reported in A. tortilis a minimum value of predawn leaf water potential that was similar to ours compared with -5.0 MPa for the co-occurring evergreen shrub Boscia senegalensis.

The finding that trees transpired during all but one month of the year, although water availability in the upper soil layers was extremely low during 8 to 10 months of the year, implies extensive use of deep soil water. However, the deep soil water profile showed that SWA was low all the way to the water table at a depth of 31 m. A hydrogeological study in the area has confirmed this deep soil water profile and shown a slow downward movement of soil water, which takes 50 years to reach the water table from the surface [START_REF] Gaye | Groundwater recharge estimation using chloride, stable isotopes and tritium profiles in the sands of north-western Senegal[END_REF]. This situation differs from that observed by [START_REF] Cook | Water balance of a tropical woodland ecosystem, Northern Australia: a combination of micro-meteorological, soil physical and groundwater chemical approaches[END_REF] and [START_REF] Roupsard | Reverse phenology and dry-season water uptake by Faidherbia albida (Del.) A. Chev. in an agroforestry parkland of Sudanese west Africa[END_REF] where the high availability of water in the top 10 m was clearly sufficient to maintain transpiration during the dry season. We found living roots at 25 m depth, confirming that the trees had access to deep soil water [START_REF] Lhote | Au sujet de l'arbre du Ténéré[END_REF][START_REF] Deans | Nitrogen in interstitial waters in the Sahel: natural baseline, pollutant or resource?[END_REF].

Control of water losses

Whole-tree hydraulic conductance did not differ between the wet and dry seasons during the full canopy stage, but remained at around 1.4 l dm -2 MPa -1 h -1 . This supports the idea that xylem conductivity, determined by dry season conditions, limits wet season water use [START_REF] Eamus | Dry season conditions determine wet season water use in the wet-dry tropical savannas of northern Australia[END_REF][START_REF] Hutley | Monsoonal influences on evapotranspiration of savanna vegetation of northern Australia[END_REF]). However the observed values of sapwood-specific hydraulic conductance were not particularly low relative to values reviewed by [START_REF] Eamus | Ecophysiology of trees of seasonally dry tropics: comparisons among phenologies[END_REF] for trees in more humid environments. Moreover, our data showed that cambial growth of the trunk and branches of A. tortilis occurred at the end of the wet season. So in our trees, it is unclear whether the dry season conditions affect xylem architecture. However, SWA and evaporative demand during the wet season in the northern Sahel are already limiting, with upper SWA rarely exceeding 50% and D max averaging 3.3 kPa.

Our observations of near uniform year-round water use and stable whole-tree conductance imply that canopy conductance is strongly regulated during the dry season to compensate for increased atmospheric evaporative demand. We can assume from the stand characteristics and the environment that a decrease in leaf area and stomatal conductance have a direct impact on tree transpiration, i.e., transpiration is strongly coupled to the atmosphere as defined by [START_REF] Jarvis | Stomatal control of transpiration: scaling up from leaf to region[END_REF]. The high Huber value found at the end of the full canopy stage in the dry season demonstrates a low leaf area compared with sapwood area for these trees. This feature is a well known adaptation to high evaporative demand [START_REF] Mencuccini | Climate influences the leaf area/sapwood area ratio in Scots pine[END_REF]. Moreover, twig-level studies by [START_REF] Goudiaby | Impact d'un déficit pluviométrique sur la feuillaison d'Acacia tortilis (Forsk.) Hayne subsp. raddiana (Savi) Brenan var. raddiana dans le Nord-Ferlo au Sénégal[END_REF] and [START_REF] Diagne | Impact d'un déficit pluviométrique sur le fonctionnement hydrique foliaire de Acacia tortilis en zone semi-aride (Ferlo-Nord, Senegal)[END_REF] on the same trees confirmed a large decrease in both leaf area and stomatal conductance during the full canopy stage in the dry season. [START_REF] Goudiaby | Impact d'un déficit pluviométrique sur la feuillaison d'Acacia tortilis (Forsk.) Hayne subsp. raddiana (Savi) Brenan var. raddiana dans le Nord-Ferlo au Sénégal[END_REF] observed pronounced inter-annual and seasonal variations in leaf area on terminal branches. Maximal leaf area occurred toward the end of the rainy season (September), with wet-year values (200 cm 2 per linear meter of leafy twigs) that were twice dry-year values. Similarly, at the end of the full canopy stage in the dry season (January), leaf area was threefold ("wet" year) or twofold ("dry" year) lower than in September, respectively. [START_REF] Diagne | Impact d'un déficit pluviométrique sur le fonctionnement hydrique foliaire de Acacia tortilis en zone semi-aride (Ferlo-Nord, Senegal)[END_REF] observed a wide range of stomatal conductance values during the year, with maximum values of 100-200 mmol m -2 s -1 occurring during the rainy season, minimal values of 10-50 mmol m -2 s -1 occurring toward the end of the full canopy stage from November to February, and intermediate values of 50-100 mmol m -2 s -1 occurring at the time of maximal defoliation in the hot dry season. Such responses contribute to seasonal homeostasis of water use and water status by strong stomatal control and decreased leaf area per sapwood area, as has been described by [START_REF] Bucci | Mechanisms contributing to seasonal homeostasis of minimum leaf water potential and predawn disequilibrium between soil and plant water potential in neotropical savanna trees[END_REF] for several Cerrado woody species. In addition, for A. tortilis, [START_REF] Do | Environmental influence on canopy phenology in the dry tropics[END_REF] have shown that interannual variation (up to 2 months) in phenology "optimizes" the onset and duration of the full canopy stage relative to evaporative demand. Such phenological behavior contributes to the control of water use through regulation of canopy conductance. The between-year consistency in minimum Ψ pd (-1.7 to -2.0 MPa), despite a wide range of environmental and growth conditions, also reflects effective regulation of water loss by A. tortilis, resulting from a combination of features, of which deep rooting that provides access to deep water and a strong regulation of canopy conductance are the most obvious.

Tree growth

Irrigation strongly promoted cambial growth of trunks and main branches although it had little effect on tree water use. The complete lack of cambial growth except during the year of irrigation was unexpected; however, the finding was in accordance with the dynamics of Ψ pd , which exceeded -0.8 MPa only in the year of cambial growth. This result does not mean that these trees do not usually grow: a percentage of new green twigs inside the canopy was observed each year. Moreover, cambial growth may have taken place in the root system when it was absent in the branches. cal assistance. We are grateful to Michel Grouzis for initiating the research program. The research was partly funded by the European Commission program INCO-DC "Minimizing competition in dryland agroforestry Contract no. ERBIC 18CT 98 0322" (coordinator: J.D. Deans). We thank Thomas Verron for custom-made software support.

Figure 1 .

 1 Figure 1. Seasonal variation in environmental conditions and tree water use in a natural grove of Acacia tortilis (three mature individuals) growing in north Senegal. (a) Daily rainfall (bars), 15-day moving mean of Penman potential evapotranspiration (PET) and maximum vapor pressure deficit of the air (D max ). Irrigation was provided during August and September 1998. (b) Soil water availability (SWA) in the upper layer (0-1 m) (bars are 1 SD; n = 3). (c) Predawn and minimum leaf water potentials (Ψ) (means for three trees, bars are 1 SD; n = 6). (d) Canopy fullness index (n = 3) and daily tree water use measured by sapflow sensors (n = 9). (e) Branch diameter changes, expressed in ‰ of initial diameters (66.5, SD = 1 mm; bars are 1 SD; n = 3).

Figure 2 .

 2 Figure2. Deep profiles of soil water content in the experimental plot: actual soil water content and that equivalent to a water potential of -1.6 MPa ("wilting point"). Characteristic profiles used to compute soil water availability in the upper layer (0-4 m) are also shown: driest profile with soil water contents slightly lower than those equivalent to -1.6 MPa and the profile at field capacity for the 0-2 m layer. The large filled dots show the depths at which living roots of Acacia tortilis were found in the soil cores.

Figure 3 .

 3 Figure 3. Characteristic values of daily water use for four annual patterns: minimum value recorded in the hot dry season (open bar), between April and May; maximum value recorded at the end of the rainy season (black bar), between September and October; and maximum value recorded in January in the cool dry season (gray bar). Phenological cycles are identified by the year in which the rainy season occurred. Values are averaged over 5-day periods (n = 3 sensors × 3 trees × 5 days = 45). Bars indicate confidence intervals at 95% of probability. Each annual pattern of water use is labeled by the year.

Figure 4 .

 4 Figure 4. Relationships between daily sap flow density per sapwood area (SFD) and canopy fullness index, including four annual patterns (for labels same remark as in Figure 3), n = 46.

Figure 6 .

 6 Figure 6. Plots of predawn leaf water potential (Ψ pd ) versus (a) daily maximum sap flow density per sapwood area (SFD), (b) maximum gradient of leaf water potential (grad Ψ), (c) whole hydraulic conductivity (K h ) and (d) maximum vapor pressure deficit of the air (D max ). Measurements were made for the full canopy stages during wet (᭜; August-September, n = 16) and dry seasons (᭺; October-January, n = 10).

Figure 5 .

 5 Figure 5. Relationship between daily sap flow density per sapwood area (SFD) and predawn leaf water potential (Ψ pd ). (a) The dataset includes all measurements of the four annual patterns, n = 46. (b) Dataset includes selected measurements corresponding to the full canopy stages, n = 26.

Table 1 .

 1 Cumulated annual Penman potential evapotranspiration (PET), annual rainfall (irrigation was provided in 1998), cumulated sap flow density per sapwood area (SFD) and cumulated total flow or tree water use based on the density of trees in the area (90 ha -1 ). Cumulated values were computed from July 1 to June 30 to determine the potential effect of wet season conditions (July-September) on the annual pattern of tree water use.

	100	DO, ROCHETEAU, DIAGNE, GOUDIABY, GRANIER AND LHOMME

Acknowledgments

We thank Youm Mbaye, Hyacinthe Tendeng and Baïdi Li for techni-TREE PHYSIOLOGY ONLINE at http://heronpublishing.com ANNUAL PATTERN OF WATER USE BY ACACIA TORTILIS


Séraphine Vizoso

Dominique Gerant

J.-M Guehl

Richard Joffre

Michel M Chalot

Patrick Gross

Pascale Maillard

Seraphine Vizoso

Jean Marc Guehl

Do elevation of CO2 concentration and nitrogen fertilization alter storage and remobilization of carbon and nitrogen in pedunculate oak saplings ?

Keywords: 13 C, 15 N, carbon remobilization, carbon storage, elevated CO 2, nitrogen remobilization, nitrogen storage, Quercus robur

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

Introduction

Elevation of atmospheric carbon dioxide concentration ([CO 2 ]) can enhance plant growth, thereby increasing the demand for mineral nutrients [START_REF] Ceulemans | Effects of elevated atmospheric CO 2 on woody plants[END_REF]Mousseau 1994, Stitt and[START_REF] Stitt | The interaction between elevated carbon dioxide and nitrogen nutrition: the physiological and molecular background[END_REF]. Responses to elevated [CO 2 ] vary depending on nutrient availability and species. Given the importance of nitrogen limitation on the growth responses of temperate forests and the increase in N deposition [START_REF] Bauer | Regenerating temperate forests under elevated CO 2 and nitrogen deposition: comparing biochemical and stomatal limitation of photosynthesis[END_REF], the effect of soil N availability on tree responses to increasing atmospheric [CO 2 ] is of particular interest. Positive effects of elevated [CO 2 ] on carbon assimilation and plant growth are often limited on N-poor soils, as shown in Quercus virginiana Mill., Quercus ilex L. and Quercus robur L. (Tognetti and Johnson 1999a, 1999b[START_REF] Maillard | Interactive effects of elevated CO 2 concentration and nitrogen supply on partitioning of newly fixed 13 C and 15 N between shoot and roots of pedunculate oak seedlings (Quercus robur)[END_REF]. In contrast, increasing N supplies can significantly enhance growth responses of trees to elevated [CO 2 ] and modify C and N dynamics [START_REF] Bauer | Regenerating temperate forests under elevated CO 2 and nitrogen deposition: comparing biochemical and stomatal limitation of photosynthesis[END_REF][START_REF] Maillard | Interactive effects of elevated CO 2 concentration and nitrogen supply on partitioning of newly fixed 13 C and 15 N between shoot and roots of pedunculate oak seedlings (Quercus robur)[END_REF][START_REF] Dyckmans | Influence of tree internal nitrogen reserves on the response of beech (Fagus sylvatica) trees to elevated atmospheric carbon dioxide concentration[END_REF].

Unlike annual plants, which respond to a CO 2 -enriched atmosphere without carry-over effects of an earlier lower [CO 2 ], except as mediated by an effect on seeds, perennial organs of trees may show cumulative effects of elevated [CO 2 ] [START_REF] Körner | Plant CO 2 responses: an issue of definition, time and resource supply[END_REF]. Internal reserves of stems and roots play an essential role during winter and early spring by supplying C and N for both maintenance metabolism and new growth, and their seasonal dynamics are central to the responses of trees to elevated [CO 2 ].

Nitrogen availability during growth has marked effects on development and N storage capacity of temperate trees [START_REF] Stephen | The influence of nitrogen supply on growth and internal recycling of nitrogen in young Nothofagus fusca trees[END_REF]. Although the effects of elevated [CO 2 ] on soil-N uptake during the growing season have been documented, there have been few studies on N remobilization in trees growing in an elevated [CO 2 ] atmosphere. Increased growth and storage sink capacity in response to elevated [CO 2 ] may increase the extent of N storage and remobilization in several tree species (Temperton et al. 2003, Dyckmans and[START_REF] Dyckmans | Partitioning of remobilised N in young beech (Fagus sylvatica L.) is not affected by elevated [CO 2 ][END_REF]. Changes in soil fertility may also alter the seasonal dynamics of storage and remobilization of C and N in forest trees and play an important role in the long-term responses to ele-vated [CO 2 ].

Seasonal dynamics of C [START_REF] Barbaroux | Distribution of aboveground and below-ground carbohydrate reserves in adult trees of two contrasting broad-leaved species (Quercus petraea and Fagus sylvatica)[END_REF] and N (Cheng andFuchigami 2002, Gomez and[START_REF] Gomez | Contribution of vegetative storage proteins to seasonal nitrogen variations in the young shoots of peach trees[END_REF] reserves have usually been studied separately, although C and N have interactive effects on vegetative development [START_REF] Sauter | Storage, mobilization and interrelations of starch, sugars, protein and fat in the ray storage tissue of poplar trees[END_REF][START_REF] Bollmark | Seasonal dynamics and effects of nitrogen supply rate on nitrogen and carbohydrate reserves in cutting-derived Salix viminalis plants[END_REF][START_REF] Dyckmans | Influence of tree internal N status on uptake and translocation of C and N in beech: a dual 13 C and 15 N labeling approach[END_REF][START_REF] Cerasoli | Carbon and nitrogen winter storage and remobilisation during seasonal flush growth in two-year-old cork oak (Quercus suber L.) saplings[END_REF]). Furthermore, there have been few studies on the dynamics of C and N reserves in forest trees in response to climate change.

Our aim was to examine the effects of N availability on storage and remobilization of C and N in Quercus robur saplings grown for 2 years in elevated [CO 2 ]. Saplings were subjected to long-term dual labeling with 13 C and 15 N isotopes at the end of the first growing season, allowing quantification of the effects of N availability and elevated [CO 2 ] on the accumulation of C and N compounds during the autumn, and their remobilization in spring.

Materials and methods

Plant material and cultivation

Two hundred acorns (Quercus robur L., Heuilly sur Saône, NE France) were sown in 5-l plastic containers filled with vermiculite. Half of the containers were placed in a 50-µm-thick transparent polypropylene tunnel (5 × 3 × 2.3 m) and exposed continuously to ambient atmospheric [CO 2 ] (A, 390 ± 30 µmol mol -1 ). The other containers were placed in an identical tunnel, but exposed to elevated atmospheric [CO 2 ] (E, 700 ± 50 µmol mol -1 ). The tunnels (A, E) were located in a greenhouse at INRA-Nancy, France. Seedlings in the A and E treatments were subdivided into two groups that were watered to field capacity twice daily with a complete nutrient solution (Le Blevennec 1986) containing either 6.1 (High N: HN) or 0.61 mmol N l -1 (Low N: LN). The seedlings were assigned to the N treatments in a full factorial design (A LN, A HN, E LN, E HN). To reduce the effect of environmental heterogeneity, plants were randomized within the tunnels throughout the experiment. Atmospheric [CO 2 ] in each tunnel was continuously monitored by infrared gas analyzers (ADC 225 MK3, U.K.) and controlled by an automated regulation system [START_REF] Vivin | Whole-plant CO 2 exchange, carbon partitioning and growth in Quercus robur seedlings exposed to elevated CO 2[END_REF][START_REF] Maillard | Interactive effects of elevated CO 2 concentration and nitrogen supply on partitioning of newly fixed 13 C and 15 N between shoot and roots of pedunculate oak seedlings (Quercus robur)[END_REF]. Photosynthetic photon flux in the tunnels was about 60% of full sunlight and never exceeded 1400 µmol m -2 s -1 . Air temperature ranged from 15 to 25 °C and was maintained above 8 °C during cold periods. Vapor pressure deficit ranged from 1.01 to 2.02 kPa.

C and 15 N labeling

Ten saplings were randomly selected from each treatment and labeled during three 2-day cycles over one month [from October to November (Year 1)] with 13 CO 2 and 15 NH 4 NO 3 . The selected saplings were placed in a VTPH 5/1000 controlled environment chamber (Vötsch Industrietechnik GmbH, Reiskirchen-Lindenstruth, Germany) operated as a semi-closed system. The following day, each plant container was supplied with 20 ml of 10 mM 15 NH 4 NO 3 solution (Eurisotop, Gif sur Yvette, France). Saplings were exposed for two consecutive days to 13 CO 2 -enriched air (4.4 atom% 13 C) with a [CO 2 ] of either 390 or 700 µmol mol -1 . Chamber temperature was 20 ± 1 °C and relative humidity was 97%. Three high-pressure sodium vapor discharge lamps (SONT, Philips Electronics N.V., Amsterdam) provided a photosynthetic photon flux of 350 µmol m -2 s -1 at plant level. After each labeling cycle, labeled plants were returned to their respective tunnel.

At the end of the labeling period, roots of labeled saplings were washed to eliminate unabsorbed 15 N. Roots of unlabeled saplings were washed in the same way. All saplings were repotted before winter (Year 1) in 7-l plastic containers to prevent root confinement, and returned to their respective tunnel.

Sampling and growth measurements

For each treatment, half of the labeled saplings were harvested during dormancy (January of Year 2) and the remaining saplings were harvested in April of Year 2 when leaves of the new shoots had reached maturity. On each sampling date, three unlabeled saplings were harvested to assess baseline abundance of 13 C and 15 N. Harvested saplings were separated into old stem, large (diameter > 2 mm) and small (diameter < 2 mm) roots, and in April only, new leaves and new stems. Roots were washed to remove vermiculite and nutrient solution. In April, numbers and lengths of new shoots were measured. Leaf area was measured with a planimeter (Delta-T Devices, Cambridge, U.K.). To assess leaf N retranslocation efficiency, mature leaves and litter of eight unlabeled saplings were harvested in November (Year 1) and in January (Year 2), respectively. Plant material was frozen in liquid nitrogen, freeze-dried, weighed and ground to a fine powder with a laboratory mill (Tecator, Cyclotec 1093, Höganäs, Sweden) for C and N analyses.

Isotopic analyses

Total C and N contents and concentrations, and 13 C/ 12 C and 15 N/ 14 N isotopic ratios of plant tissues were measured with an elemental analyzer (NA 1500 NCS, Carlo Erba, Milan, Italy) coupled to a Delta-S isotopic ratio mass spectrometer (Finnigan-Mat, Thermoquest Corp., San Jose, CA).

Isotopic abundance (atom% or A%) for C and N was computed as: The contributions of C and N assimilated during spring growth (Year 2) and C and N remobilized from perennial organs were calculated with equations of isotopic dilution [START_REF] Deléens | Using 13 C and 15 N label near natural abundance for monitoring carbon and nitrogen partitioning[END_REF]. For simplicity, only variables related to C are shown. The corresponding variables for N can be defined by substituting 13 C and 12 C with 15 N and 14 N, respectively. Atom excess % is defined as the difference between the isotopic abundance of a given plant organ following administration of the 13 C or 15 N tracers (A C,lab %), and the 13 C or 15 N natural abundance measured for the same component of an unlabeled plant (A C,unlab %). 13

+ Y C = 1: A X Y X A Y A C,lab, org C C C C,lab, pl Jan C C,unlab, o %( + ) = ( % ) + ( rg %)
where X C , the proportion of old C, was estimated for a given plant as:

X A A A A C C,lab, org Apr C,unlab, org C,lab, pl Jan C,un = % - % % - lab, org %
Isotopic abundance for 13 C or in labeled plants (A C,lab,pl ) in January is assumed to correspond to the amount of C available for remobilization. We did not assess such values but assumed that they were equal to those of bulk plant material [START_REF] Pellicer | Carbon and nitrogen mobilization in Larix × eurolepis leafy stem cuttings assessed by dual 13 C and 15 N labelling: relationships with rooting[END_REF], Maillard et al. 2004).

Total C content (C tot ) comprised old C (C old , incorporated before January) and new C (C new , incorporated between January and April). Old C was incorporated in perennial organs of the labeled plant, and C new was incorporated in newly formed leaves of the labeled plant. Thus, C tot = C old + C new , where C old = X C C tot and C new = (1 -X C )C tot .

Retranslocation efficiency from senescing leaves

Autumnal N retranslocation efficiency (RE N , %) from senescing leaves was calculated according to [START_REF] Wang | Seasonal changes in element contents in mangrove element retranslocation during leaf senescence[END_REF]:

RE = N old Nov litter Jan old Nov N N N - 100
where N old is N content in old leaves, and N litter is N content in litter.

Carbon and nitrogen remobilization

In perennial organs, the difference between total C (or N) content (C org ) in January and old C (

Near-infrared spectroscopic measurements

Concentrations of soluble sugars, starch, total free amino ac-ids, total soluble proteins, C and N of all samples of organs with a total dry mass of more than 1 g, and for each date, were determined by near-infrared spectroscopy (NIRSystems 6500, Foss NIRSystems, Raamsdonksveer, The Netherlands) as described by [START_REF] Joffre | The use of near-infrared reflectance spectroscopy in litter decomposition studies[END_REF]. Each sample was milled to a fine powder (Cyclotec 1093 Sample Mill, Tecator, Höganäs, Sweden) and scanned by NIRS. Three distinct populations of NIR spectra, corresponding to leaves, stems and roots, were considered. Independent calibration equations between spectral and chemical data were built for each population with a partial least squares (PLS) algorithm [START_REF] Martens | Multivariate calibration[END_REF]Naes 1989, Shenk andWesterhaus 1991a). About 60 samples were selected per population, based on Mahalanobis distances between all pairs of spectra (Select procedure of ISI software, see Shenk and Westerhaus 1991b), and analyzed for soluble sugars, starch, total free amino acids, total soluble proteins, carbon and nitrogen as described below. Chemical concentrations of all samples were obtained with standard errors for leaves, stems and roots, respectively, of 0.59, 0.45 and 0.75% for soluble sugars; 0.61, 0.70 and 0.70% for starch; 0.32, 1.07 and 1.07% for total free amino acids; 1.68, 1.12 and 1.12% for total soluble proteins; 0.41, 0.72 and 0.66% for carbon; and 0.10, 0.05 and 0.04% for nitrogen.

Carbohydrate analysis for NIRS calibration

Soluble sugars were extracted twice from 5-10 mg of ground sample by incubating in 80% boiling ethanol (1 ml) for 30 min and centrifuging for 10 min at 12,620 g. The pellet was retained, and the supernatants were combined and dried overnight in a vacuum-evaporator to eliminate ethanol. Dried extracts were solubilized in 0.5 ml of 0.02 N NaOH, and the fructose and sucrose converted to glucose equivalents [START_REF] Boehringer | Methods of enzymatic food analysis using single reagents[END_REF]. Total glucose was quantified at 340 nm by an enzymatic method (hexokinase (EC 2.7.1.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49;[START_REF] Boehringer | Methods of enzymatic food analysis using single reagents[END_REF]. Starch was extracted from the pellet after extraction of soluble sugars by boiling for 1 h in 0.02 N NaOH, and hydrolyzed to glucose with α-amyloglucosidase (EC 3.2.1.3, Boehringer Manheim Biochemicals, Mannheim, Germany) in 0.32 M citrate buffer, pH 4.2 at 48 °C for 30 min. Glucose was assessed as described by [START_REF] Boehringer | Methods of enzymatic food analysis using single reagents[END_REF].

Total soluble protein analysis for NIRS calibration

Total soluble proteins were extracted from 30 mg of ground tissue of each organ at 4 °C in 4 ml of 0.1 M phosphate buffer (pH 7.38) containing 60% (w/w) polyethylene glycol (PEG 20,000), polyvinyl-pyrrolidone (PVP) and 5 mM dithiothreitol. Concentrations of PVP varied with plant organ: 7% (w/w) PVP 40,000 for stems and large and small roots, and 3.75% (w/w) PVP 40,000 for leaves. Samples were sonicated (Bransonic 2510, Branson Ultrasonic Corporation, USA) for 30 min and centrifuged for 15 min at 12,620 g. Each supernatant was kept and the extraction was repeated twice on the pellet. The three supernatants were combined and total soluble protein concentration assessed colorimetrically at 595 nm according to [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF], using Coomassie Blue G 250 TREE PHYSIOLOGY ONLINE at http://heronpublishing.com (Bio-Rad, 500-0006), with bovine serum albumin as the standard. Assuming that proteins contain about 22.6% N [START_REF] Yeoh | Leaf protein contents and nitrogen-to-protein conversion factors for 90 plant species[END_REF], results were expressed as mg soluble protein N (100 g DM ) -1 .

Total amino acid analysis for NIRS calibration

Free amino acids were extracted from 10 mg of ground tissue for 10 min at 4 °C in 650 µl of 7:3 (v/v) methanol:water (MW). The mixture was centrifuged for 5 min at 12,620 g. The supernatant was retained, and the pellet was rinsed twice with 2 ml of MW and collected each time by centrifuging at 12,620 g for 15 min. The supernatants were combined and the free amino acid concentration was determined colorimetrically at 570 nm by the ninhydrin method [START_REF] Yemm | The determination of aminoacids with ninhydrin[END_REF], with leucine as the standard. Assuming that mean N concentration of free amino acids is 13%, results were expressed as mg amino acid N (100 g DM ) -1 .

Statistical analysis

The experimental design was a full factorial with two factors: [CO 2 ] (ambient and elevated) and N fertilization (LN and HN). Treatment effects were assessed by a two-way analysis of variance (ANOVA) followed by Tukey's test.

Results

Nitrogen mobilization at leaf fall

By November of Year 1, HN increased total leaf N content 400%, whereas [CO 2 ] had no effect on leaf N content (Figure 1a). Leaf N concentration was reduced by both elevated [CO 2 ] and LN (Figure 1b), but there was no [CO 2 ] × N interaction. Leaf N represented about 30% of sapling N in all treatments (Figure 1c).

In January, litter N content, representing N lost during leaf fall, was higher in the HN treatment than in the LN treatment. Nitrogen retranslocation efficiency from senescing leaves was higher in LN saplings (60%) than in HN saplings (35%) (Figure 1a). However, in all treatments, N lost in litter equaled about 15% of sapling N (Figure 1c).

Carbon and nitrogen storage and remobilization

Accumulations of C and N were strongly enhanced by HN, with elevated [CO 2 ] having an additive effect (Figures 2a and3a) in all perennial organs (Figures 2b,2d,2f,3b,3d and 3f). Total C and N contents of saplings (Figures 2a and3a) and total C content of perennial organs did not change significantly from January to April (Figures 2b, 2d, and2e). In contrast, total N content of old stems and large roots decreased between January and April (Figures 3b and3d) but remained constant in fine roots (Figure 3f). In April, about 15% of total C and 40% of total N were allocated to new leaves (Figures 2e and3e).

Old C was mobilized rapidly between January and April in perennial organs, especially of E HN saplings (Figure 2b and2d). In old stems, remobilization accounted for 50-58% of C reserves, and up to 75% in old stems of E HN saplings. In large roots of HN saplings, 55-68% of the old C was mobilized. Old N was mobilized strongly in perennial organs and at a similar rate in all treatments (Figures 3b and3d). Nitrogen remobilization accounted for 65-79% in old stems, and for 56-69% in large roots.

Perennial organs of HN saplings had high N concentrations (Table 1). Nitrogen concentrations of old stems and large roots decreased in response to elevated [CO 2 ] with a [CO 2 ] × N interaction for large roots (Table 1). Nitrogen concentrations of old stems and large roots declined sharply from January to April (Table 1). Elevated [CO 2 ] significantly reduced N concentrations in leaves and new stems of LN saplings (Table 1). In January, starch concentration was greatest in large roots (35-50% DM), followed by small roots (10-20%) and old stem (10%) (Table 2). Elevated [CO 2 ] had no effect on starch concentrations of perennial organs, whereas LN increased the starch concentration in large roots (A: +17%, E: +31%), decreased it in small roots (A: -35%, E: -43%) and had no effect on starch concentration in old stems (Table 2). Soluble sugar concentrations were similar in all treatments, except in old stems of E LN saplings which had increased soluble sugar concentrations (Table 2).

From January to April, starch concentrations decreased in large and small roots in all treatments, and in old stems of HN saplings (Table 2). Soluble sugar concentrations decreased in old stems of LN saplings and strongly increased in large roots of HN saplings (Table 2). In April, starch concentration of new leaves was markedly increased by both elevated [CO 2 ] and LN (Table 2), and a similar but less pronounced effect was observed in new stems. The HN treatment increased the proportion of soluble sugars in new organs with no [CO 2 ] effect (Table 2).

[CO 2 ] and N supply and N storage and remobilization

Soluble protein and amino acid N concentrations represented less than 0.25% of organ dry mass and about 20 and 13% of their total N, respectively (Table 1). In January, HN greatly increased soluble protein and amino acid N concentrations. Elevated [CO 2 ] decreased soluble protein N (on average 17%) and amino acid N (37%) concentrations in HN saplings but had no effect in LN saplings (Table 1).

From January to April, soluble protein N concentrations decreased significantly in old stems in all treatments. Amino acid N concentrations were decreased in both old stems and large roots in all treatments (Table 1). In April, soluble protein N concentration in new leaves was higher than in perennial organs. The HN treatments greatly increased soluble protein and amino acid N concentrations in new organs, and this effect was lessened by elevated [CO 2 ] (Table 1).

Carbon dioxide concentration, nitrogen supply and spring growth

Spring growth was markedly increased by HN leading to large changes in development and organogenesis of axillary organs (Table 3). Lengths of new axes and numbers of branches and leaves were significantly increased in HN saplings compared with LN saplings (Table 3). In contrast, the N treatments had no effect on specific leaf area (SLA). Sapling morphology was modified by elevated [CO 2 ] (Table 3): branch length, main leaf area and SLA were markedly decreased, whereas numbers of branches and axillary leaves were strongly increased. The root:shoot ratio was strongly increased by LN, particularly in the ambient [CO 2 ] treatment (Table 3).

[CO 2 ] and N supply and partitioning of newly assimilated C and N between new and old organs

In April, new C and N uptake in whole saplings depended more on N availability than on [CO 2 ] (Figures 2a and3a). At the end of leaf expansion, 50-60% of the C in HN saplings originated from assimilation compared with only 10-20% in LN saplings (Figure 2a). New N was incorporated in HN saplings only and represented less than 20% of total N (Figure 3a). New leaves contained 50-60% of new C, except in A LN saplings, where new C represented only 15% of total C (Figure 2e). The fraction of new C in new stems was low and never exceeded 20% of total C (Figure 2c) in HN saplings. New organs were built mostly with old N (75-95% of total N) (Figures 3c and3e). New C and N were found in perennial organs (Figures 2b,2d, 2f, 3b, 3d and 3f), with the largest accumulation occurring in old stems with about 45% of total new C and 33-49% of new N (Figures 2b and3b).

Discussion

Elevated [CO 2 ] and N fertilization increased second-year growth of Quercus robur saplings, with N treatment having the greatest effect. This finding is in agreement with earlier results for Quercus virginiana, Quercus ilex (Tognetti andJohnson 1999a, 1999b) and Quercus robur [START_REF] Maillard | Interactive effects of elevated CO 2 concentration and nitrogen supply on partitioning of newly fixed 13 C and 15 N between shoot and roots of pedunculate oak seedlings (Quercus robur)[END_REF]). Elevated [CO 2 ] induced similar changes in several other tree species [START_REF] Saxe | Tree and forest functioning in an enriched CO 2 atmosphere[END_REF]), but the physiological mechanisms controlling these changes remain poorly understood. Cell division and cell expansion may be affected, driven mainly by increased substrate availability and by differential expression of genes involved in cell cycling or cell expansion mediated by plant hormones [START_REF] Pritchard | Elevated CO 2 and plant structure: a review[END_REF]. Low N supply typically induces a higher root:shoot ratio, an adjustment in biomass allocation in response to an imbalance of exogenous resources that tends to increase the supply of the limiting resource [START_REF] Marschner | Mineral nutrition of higher plants[END_REF][START_REF] Hermans | How do plants respond to nutrient shortage by biomass allocation?[END_REF]).

Although elevated [CO 2 ] and HN stimulated growth of new shoots (Table 3) at the sapling level (Figures 2a and3a), we observed no differences in total C and N contents between winter and spring, indicating that new C assimilation and N uptake were not achieved or were compensated by respiratory losses, and that initial new growth measured in April depended on C and N stored in perennial organs. We hypothesized that elevated [CO 2 ] and N fertilization increased the storage of both C and N compounds during the previous year and their remobilization at the beginning of the following spring.

In January, all perennial organs, especially those of HN saplings, had accumulated more C (Figures 2b, 2d and2e) and more N (Figures 3b,3d ] with low (LN) or high (HN) nitrogen fertilization, sampled in winter (January, Year 2) and after the new spring flush was expanded (April, Year 2). Means ± SE (n = 8). For each organ, the significance of treatment effects and their interaction on total N are indicated by different letters (lowercase for January, uppercase for April) (two-way ANOVA followed by the Tukey test, P < 0.05). For whole saplings (a) and perennial organs (b, d, f), significant differences in total N content between January and April are indicated at the top of each figure (ns, non significant; *, P < 0.05).

[CO 2 ] treatment indicated that the additional increase in C was higher than the increase in N. [START_REF] Maillard | Interactive effects of elevated CO 2 concentration and nitrogen supply on partitioning of newly fixed 13 C and 15 N between shoot and roots of pedunculate oak seedlings (Quercus robur)[END_REF] reported that, in 1-year-old oak saplings, elevated [CO 2 ] and HN increased net CO 2 assimilation more than 15 N uptake, creating a C:N ratio imbalance in summer that was maintained after leaf shedding. Moreover, during leaf shedding the previous year, HN saplings recovered N from senescing leaves less efficiently than LN saplings (Figure 1a). Similar behavior was observed in N-deficient Malus domestica Borkh. and Fagus sylvatica L. [START_REF] Millard | Ecophysiology of the internal cycling of nitrogen for tree growth[END_REF]Thomson 1989, Dyckmans and[START_REF] Dyckmans | Influence of tree internal N status on uptake and translocation of C and N in beech: a dual 13 C and 15 N labeling approach[END_REF]. These findings led us to hypothesize that N recovery from old leaves is more essential for survival and spring growth in N-deficient trees than in well-fertilized trees.

In January, oak saplings stored C as starch mainly in large roots, and starch concentrations were higher in LN saplings than in HN saplings with no effect of elevated [CO 2 ] (Table 2). Nitrate limitation typically leads to large increases in starch concentration in plants [START_REF] Stitt | The interaction between elevated carbon dioxide and nitrogen nutrition: the physiological and molecular background[END_REF]Krapp 1999, Cheng and[START_REF] Cheng | Growth of young apple trees in relation to reserve nitrogen and carbohydrates[END_REF]. It has been assumed that starch accumulation is a passive response to decreased rates of growth. Conversely, in well-fertilized oak saplings, lower starch concen-trations, even under elevated [CO 2 ] conditions, imply that structural compounds account for a larger proportion of total biomass. Reduced starch accumulation could be related both to increased carbon requirements for the assimilation of inorganic N and to rapid consumption for the synthesis of structural compounds that occurred during the first year, as also shown in Pinus nigra L. [START_REF] Luo | Carbon partitioning to mobile and structural fractions in poplar wood under elevated CO 2 (EUROFACE) and N fertilization[END_REF].

In January, elevated [CO 2 ] decreased N concentrations in all perennial organs only in the HN saplings (Table 1). This effect has been interpreted in different ways: higher nitrogen-use efficiency, higher growth under an elevated [CO 2 ], accelerated senescence, and inadequate N fertilization or N uptake [START_REF] Stitt | The interaction between elevated carbon dioxide and nitrogen nutrition: the physiological and molecular background[END_REF]. Nitrogen was stored in both soluble proteins and amino acids in all perennial organs. The HN treatment typically increased soluble protein and amino acid N storage in perennial organs (Table 1), but this effect was reduced by elevated [CO 2 ]. The HN-induced increase in amino acid N concentration was greater than the increase in soluble protein N concentration with the result that the ratio of soluble protein N to amino acid N was reduced by fertilization. Preferential storage of additional N in free amino acids rather than proteins in TREE PHYSIOLOGY ONLINE at http://heronpublishing.com Table 1. Nitrogen (N), soluble protein and amino acid N concentrations (g (100 g DM ) -1 ) in organs of Quercus robur saplings grown in ambient (A) or elevated (E) [CO 2 ] with low (LN) or high (HN) nitrogen fertiliation. Saplings (n = 8) were sampled in winter (January, Year 2) and after the new spring flush had expanded (April, Year 2). Significance of treatment effects and their interaction are indicated by asterisks: ns, non significant; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. Within a row, different lowercase letters indicate significant differences between treatments (two-way ANOVA, P < 0.05). Different uppercase letters indicate significant differences between January and April within a treatment. perennial organs was also shown in apple trees [START_REF] Cheng | Nitrogen storage and its interaction with carbohydrates of young apple trees in response to nitrogen supply[END_REF]. This mechanism provides saplings the advantage that amino acids are readily available when growth resumes in spring [START_REF] Cheng | Nitrogen storage and its interaction with carbohydrates of young apple trees in response to nitrogen supply[END_REF]. Elevated [CO 2 ] decreased both soluble N concentration and total N concentration in fertilized oak saplings. By using 15 N-and 13 C-labeling of reserves before winter, we demonstrated that spring growth of Quercus robur was strongly controlled by the availability of internal resources. About 20% of old C (Figures 2c and2e) and 50% of old N 2c). In contrast, new leaves comprised 60% new C (Figure 2e), this percentage being smaller in LN saplings than in HN saplings. Conversely, [START_REF] Dyckmans | Influence of tree internal N status on uptake and translocation of C and N in beech: a dual 13 C and 15 N labeling approach[END_REF] reported that, 2 weeks after bud burst, 38.9 and 52.7% of leaf C in HN-and LN-pretreated 3-year-old beech trees was derived from new C. However, in our experiment, the total amount of new C in beech leaves was 43% lower in LN trees compared with HN trees, 12 weeks after bud burst. Elevated [CO 2 ] significantly modified branching architecture (i.e., leaf and branch numbers, branch length) of HN saplings (Table 3), resulting in increased leaf area and subsequent C acquisition in an expected positive feedback loop. In fully expanded leaves of E HN saplings, the large amount of new C, which was unaccompanied by starch accumulation (Table 2), may indicate efficient utilization of new C for structural syntheses as reported in leaves of northern red oak seedlings [START_REF] Dickson | Partitioning of current photosynthate to different chemical fractions in leaves, stems, and roots of northern red oak seedlings during episodic growth[END_REF].

Organ

Remobilization of internal C from perennial organs was increased by HN, and additional remobilization from old stems was observed in response to elevated [CO 2 ] (Figure 2). For HN saplings, not all old C mobilized from the perennial organs was allocated to new shoots (Figure 2), reflecting respiratory C losses during the winter (maintenance) and early spring (growth). Respiratory C loss was compensated by new assimilation because the total C content did not change between January and April (Figures 2b, 2d and2f). Thus, HN saplings renewed 50-60% of their total C during April (Figure 2a).

Because allocation of new C occurred in perennial organs and we did not assess 13 C labeling of starch, we were unable to calculate remobilization (old C) and renewal (new C) in the total starch pool. However, differences in starch concentrations were observed between January and April, suggesting that starch remobilization occurred in most perennial organs (Table 2). Spring remobilization of starch often results in its depletion in perennial organs, and the lowest starch concentrations have been observed just before bud break in some deciduous trees [START_REF] Witt | Starch metabolism in poplar wood ray cells during spring mobilization and summer deposition[END_REF][START_REF] Piispanen | Variation of non-structural carbohydrates in silver birch (Betula pendula Roth) wood[END_REF][START_REF] Barbaroux | Distribution of aboveground and below-ground carbohydrate reserves in adult trees of two contrasting broad-leaved species (Quercus petraea and Fagus sylvatica)[END_REF]. The extent of starch remobilization was higher in HN saplings than in LN saplings (Table 2). The decrease in starch concentration in HN saplings was associated with an accumulation of soluble sugars in large roots and no change in old stems. We hypothesized that, in old stems of HN saplings, a significant portion of the soluble carbohydrates formed during starch hydrolysis is promptly converted to structural carbon compounds or exported. In contrast, in the old stems of LN saplings, no starch remobilization occurred in April, resulting in greater starch accumulation compared with HN saplings, which was not observed in January.

Our labeling experiment confirmed that, until April, no major N absorption occurred to supply N for new spring growth (Figure 3). Nitrogen uptake occurred only in HN saplings, and some new N was allocated to new shoots and new leaves (15-20% of total new N). During this period, internal N stores were used for new growth has been shown in Fagus sylvatica L. [START_REF] Dyckmans | Influence of tree internal nitrogen reserves on the response of beech (Fagus sylvatica) trees to elevated atmospheric carbon dioxide concentration[END_REF] and Alnus glutinosa (L.) Gaertn., despite the latter species being an N 2 -fixer [START_REF] Temperton | Does elevated atmospheric carbon dioxide affect internal nitrogen allocation in the temperate trees Alnus glutinosa and Pinus sylvestris?[END_REF]. In oak saplings, the extent of N remobilization was unaffected by elevated [CO 2 ] (Figure 3) (cf. [START_REF] Maillard | Interactive effects of elevated CO 2 concentration and nitrogen supply on partitioning of newly fixed 13 C and 15 N between shoot and roots of pedunculate oak seedlings (Quercus robur)[END_REF]. In contrast, [START_REF] Temperton | Does elevated atmospheric carbon dioxide affect internal nitrogen allocation in the temperate trees Alnus glutinosa and Pinus sylvestris?[END_REF] reported that elevated [CO 2 ] increased N remobilization for leaf growth in A. glutinosa but not in Pinus sylvestris L. When leaves had just completed expansion, N concentrations of all organs were lower in E HN saplings than in A HN saplings (Table 1). This difference may be partly linked to a delay between new CO 2 assimilation and new N uptake, because the new C:new N ratio was higher in E HN saplings than in A HN saplings (Figure 3). Nitrogen remobilization from perennial organs was not compensated by new N accumulation, leading to decreased N content over time (Figures 3b and3d). Several studies have reported that stored N is mobilized and used to support early shoot growth leading to a depletion of stored N in source organs such as branches (Millard 1996, Sauter and[START_REF] Sauter | Seasonal changes in content of starch, protein and sugars in the twig wood of Salix caprea L[END_REF]. Under natural conditions, replenishment of N reserves in perennial organs mainly occurs at the beginning of leaf yellowing during late summer and early fall [START_REF] Sauter | Structure and function of rays: storage, mobilisation, transport[END_REF], so that until this time, N uptake is necessary for further N reserve replenishment to occur. Even if soluble proteins were significantly mobilized to support spring growth (Table 1), our results show that amino acids were the most heavily mobilized N fraction, as observed in Prunus persica L. by [START_REF] Gomez | Contribution of vegetative storage proteins to seasonal nitrogen variations in the young shoots of peach trees[END_REF] who hypothesized that the role of storage proteins is linked to growth initiation in spring.

In conclusion, through primary effects on C and N storage compounds, elevated [CO 2 ] and N fertilization have indirect consequences on remobilization the following spring, which supports new growth of oak saplings. Our results demonstrated that initial spring growth was strongly controlled by the availability of internal N. The contrasting N storage capacities imposed during the first year affected both C and N remobilization and uptake the following year. New spring shoot architecture was modified by elevated [CO 2 ] and N fertilization, leading rapidly to more efficient C acquisition and N uptake. However, immediately following full leaf expansion, the delay between new CO 2 assimilation and N uptake resulted in a decrease in N concentrations in E HN saplings. During this period, internal cycling of N in the tree might help overcome N deficiency under elevated [CO 2 ] conditions. Our data demonstrate the importance of N storage and remobilization for the initial growth of oak saplings.

  or N) content (C old,org ) in April indicated the amount of stored C (or N) mobilized between January and April. Remobilization of C (RM C , %) and N (RM N , %) were calculated as:

Figure 1 .

 1 Figure 1. Nitrogen budget of old leaves (sampled in November, Year 1, filled bars) and litter (sampled after leaf fall in January, Year 2, open bars) of Quercus robur saplings grown in ambient (A) or elevated (E) [CO 2 ] with low (LN) or high (HN) nitrogen fertilization. Significant treatment effects and their interactions are indicated by different letters (two-way ANOVA followed by Tukey's test, P < 0.05). Values are means ± SE (n = 8).

  Figure 3. Old (filled bars) and new (open bars) nitrogen contents in whole saplings and individual organs of Quercus robur grown in ambient (A) or elevated (E) [CO 2] with low (LN) or high (HN) nitrogen fertilization, sampled in winter (January, Year 2) and after the new spring flush was expanded (April, Year 2). Means ± SE (n = 8). For each organ, the significance of treatment effects and their interaction on total N are indicated by different letters (lowercase for January, uppercase for April) (two-way ANOVA followed by the Tukey test, P < 0.05). For whole saplings (a) and perennial organs (b, d, f), significant differences in total N content between January and April are indicated at the top of each figure (ns, non significant; *, P < 0.05).

  Downloaded from https://academic.oup.com/treephys/article/28/11/1729/1671315 by guest on 31 October 2023 [CO 2 ] and N supply and starch and soluble sugar storage and remobilization

Table 3 .

 3 Growth variables of new shoots and biomass root: shoot ratio of Quercus robur saplings grown in ambient (A) or elevated (E) [CO 2 ] with low (LN) or high (HN) nitrogen fertilization. Saplings (n = 8) were sampled after the new spring flush had expanded (April, Year 2). Significance of treatment effects and their interaction are indicated by asterisks: ns, non significant; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. Within a row, different letters indicate significant differences between treatments (two-way ANOVA followed by the Tukey test, P < 0.05).

	1736	VIZOSO ET AL.

Acknowledgments

This work was supported financially by NETCARB (Network for Ecophysiology in Closing Terrestrial CARbon Budget, EU project) coordinated by J. Ghashghaie (UMR 8079, CNRS-Université Paris-Sud, Orsay, France). The authors thank J. Marchand and C. Bréchet (Stable Isotope Laboratory, UMR 1137, INRA Nancy, France) for elementary and mass spectrometry analyses. Prof. S. Thornton (Nancy Université) and Dr. E. Dreyer (INRA, Nancy) are acknowledged for helpful discussions and critical reading of the manuscript.

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