Plant Science 170 (2006) 1059–1067 www.elsevier.com/locate/plantsci

Effect of elevated CO2, temperature and drought on dry matter partitioning and photosynthesis before and after cutting of nodulated alfalfa Gorka Erice a, Juan J. Irigoyen a, Pilar Pe´rez b, Rafael Martı´nez-Carrasco b, Manuel Sa´nchez-Dı´az a,* a

Departamento de Biologı´a Vegetal, Facultades de Ciencias y Farmacia, Universidad de Navarra, Irunlarrea s/n, 31008, Pamplona, Navarra, Spain b Instituto de Recursos Naturales y Agrobiologı´a de Salamanca, CSIC, Apartado 257, 3701 Salamanca, Spain Received 29 September 2005; received in revised form 23 December 2005; accepted 26 December 2005 Available online 3 February 2006

Abstract The rising atmospheric CO2 concentration resulting from industrial development may enhance photosynthesis and plant growth. However, there is a lack of research concerning the effect of combined factors such as CO2, temperature and water availability on plant regrowth following cutting or grazing, which represent the usual methods of managing forage legumes like alfalfa. Elevated CO2, temperature and drought can interact with cutting factors (e.g. cutting frequency or height), and source-sink balance differences before and after defoliation can modify photosynthetic behaviour and dry matter accumulation, as well as dry matter partitioning between above- and belowground organs. The aim of our study was to determine the interactive effect of CO2 (ambient, around 350 mmol mol 1 versus 700 mmol mol 1), temperature (ambient versus ambient + 4 8C) and water availability (well-irrigated versus partially irrigated) on dry matter partitioning and photosynthesis in nodulated alfalfa after vegetative normal growth and during regrowth. At the end of vegetative normal growth, CO2 enhanced dry matter accumulation despite photosynthesis being down-regulated at the end of this period. Photosynthesis was stimulated by elevated CO2 and resulted in greater dry matter accumulation during the regrowth period. Aboveground organs were affected more by drought than belowground organs during the entire experiment, particularly during vegetative normal growth. The higher drought tolerance (greater growth) observed during the regrowth period may be related to higher mass and greater reserves accumulated in the roots of plants. # 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Alfalfa; Regrowth; Climate change; Plant dry matter; Photosynthesis; Temperature gradient greenhouses

1. Introduction Human economic development since the industrial revolution has increased the atmospheric CO2 concentration from 280 mmol mol 1 in 1750 to 372 mmol mol 1 [1]. This increasing concentration may reach 550 mmol mol 1 by the middle of this century and 700 mmol mol 1 by the end of the century [1]. According to the Intergovernmental Panel on Climate Change [2], the global temperature may increase by the middle of this century by 1.4–5.8 8C due to the greenhouse properties of atmospheric carbon dioxide.

* Corresponding author. Tel.: +34 948425600x6227; fax: +34 948425649. E-mail address: [email protected] (M. Sa´nchez-Dı´az). 0168-9452/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2005.12.018

In C3 plants such as alfalfa, photosynthesis is CO2-limited [3] and an increase in this greenhouse gas will enhance photosynthesis and plant growth [3–5]. The magnitude of this beneficial effect on growth and dry matter (DM) accumulation depends on other factors such as temperature, relative humidity, drought and mineral nutrition [6–8]. Elevated CO2 and temperature interact in climate systems, so the combined study of both parameters is particularly relevant. The beneficial effect of elevated CO2 on plant growth is reportedly greater at high temperatures [9]. Alfalfa (Medicago sativa L.) is a temperate crop that usually faces low water availability under growth conditions subjected to a Mediterranean climate. As reported by Chaves et al. [10], water deficit has been recognized as one of the most important environmental factors limiting photosynthesis, plant growth

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and production in the Mediterranean climate. Furthermore, several experiments have revealed that plant production response to elevated CO2 can be affected significantly by soil water availability [11–13]. Alfalfa is a forage crop and its management includes defoliation by periodic cutting and removal of shoots. CO2 enrichment may enhance regrowth by increasing the photosynthetic rate and development of aboveground organs. CO2induced effects on alfalfa productivity may also interact with the effects of management practices such as fertilizer input, cutting frequency or stocking rate [14]. The study of these interactions is an important element in adapting land management strategies to a future world containing high atmospheric carbon dioxide [15]. Although some studies have shown no significant interaction between CO2 and cutting factors [16], others have found differences in responses to elevated CO2 when comparing cut and uncut plots [11]. This interaction depends on the species, and large changes in the community structure due to the cutting frequency in temperate multi-species grasslands may even lead to changes in the digestibility of plant tissues [17]. This experiment was carried out to accomplish two main objectives. The first objective was to determine the effect of the interaction between elevated CO2, temperature and water availability on dry matter (DM) partitioning and total green area in the different organs of nodulated alfalfa in one cutregrowth cycle. The second objective was to study the behaviour of photosynthesis activity (A) and internal carbon dioxide concentration (Ci) in response to these factors during alfalfa regrowth. The present study was performed using temperature gradient greenhouses or greenhouses to create and maintain CO2 and temperature at desired levels, and to simulate likely future values. 2. Material and methods 2.1. Plant material Alfalfa (Medicago sativa L. cv. Arago´n) seeds were sterilized in a solution of HgCl2 (0.1%, w/v) and germinated in Petri dishes. One week later, seedlings were planted in pots (20 plants per pot) containing a mixture of perlite–vermiculite (2:1, v/v). Pots with a capacity of 13 l were used to avoid possible potbounding and subsequent acclimation to elevated CO2. Plants were grown in a greenhouse at 25/15 8C, 50% RH, with a 14 h photoperiod under natural daylight supplemented with fluorescent lamps (Sylvania DECOR 183, Professional-58 W, Germany) providing a PPFD of 300–400 mmol m 2 s 1. During this month plants were inoculated three times with Sinorhizobium meliloti strain 102F78 (The Nitragin Co. Milwaukee, WI, USA) and irrigated alternatively with Evans N-free solution [18] and distilled water to avoid salt accumulation in the substrate. 2.2. Experimental design and description of temperature gradient greenhouses (TGT) Thirty-day-old plants were placed into two temperature gradient greenhouses in an alfalfa field at the Mun˜ovela farm of

the Instituto de Recursos Naturales y Agrobiologı´a (CSIC, Salamanca, Spain). The design of the temperature gradient greenhouses was similar to that described by Rawson [19], Rawson et al. [20], Pe´rez et al. [21] and Aranjuelo et al. [22]. CO2 concentration, temperature, relative humidity, and solar radiation levels inside and outside the greenhouses were continuously monitored and controlled by a computerized system. Plants were divided into eight treatments comprising a combination of two CO2 levels (ambient, around 350 and 700 mmol mol 1), two temperature regimes (ambient and ambient + 4 8C), and two water availability conditions (wellirrigated, control and partially irrigated, drought). One greenhouse was maintained at an ambient CO2 concentration level (400 and 373 mmol mol 1 CO2 for years 2002 and 2003, respectively), and the other at an elevated CO2 level (722 and 684 mmol mol 1 CO2 for years 2002 and 2003, respectively). Greenhouses were 9.6 m long (including a 0.6 m outlet compartment), 2.2 m wide and 1.7 m high at the edge with a modular design. Each greenhouse was divided into three modules, thereby providing different temperature values. The middle module was considered the transition module and no experimental plants were included. In each greenhouse, two inlet fans mounted on the inlet module and an outlet fan mounted in the roof of the outlet compartment continuously circulated air through the greenhouse at the speed required to maintain a difference of 4 8C between the two extreme modules. Three small fan heaters, placed above the plant level in the outlet compartment and facing the greenhouse interior, were used to help maintaining the temperature difference at night and whenever solar radiation was insufficient to raise temperature. Inside the greenhouses, pots were placed in holes made in the soil to ensure temperature conditions in the root zone approximated those of field conditions [8]. The pots were rotated daily, within the corresponding greenhouse compartment, to avoid edge effects. The maximum soil volumetric water content (uv), corresponding to the well-irrigated treatments, was around 0.4 cm3 cm 3. Drought treatments were kept at 50% uv (around 0.2 cm3 cm 3). These uv levels were maintained throughout the experiment by measuring daily transpired water (calculated by weighing the pots) and replenishing the water that was lost. In order to reduce soil evaporation, pots were covered with plastic sheets perforated with very small holes to allow stems to pass through. The desired drought level was reached around 15 days after the beginning of treatment, when plants were 45 days old. Fully irrigated plants were alternatively watered with Evans N-free nutrient solution [18] and distilled water, whereas plants subjected to drought were always watered with Evans solution in order to supply all treatments with the same amount of nutrients. Experimental plants (in pots) were surrounded by natural alfalfa plants, and shading of natural over experimental plants was avoided by regular cutting to keep all plants at a similar height. Three harvests were carried out during the experiment. The first harvest was performed after 1 month in the greenhouses (60-day-old plants) (vegetative normal growth), and plants were cut to a 5 cm stem height. The second harvest was

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performed 15 days after cutting (15-day regrowth), and the third 30 days after cutting (30-day regrowth). In each harvest two pots (40 plants) were harvested and the following measurements were obtained: leaf, stem, root and nodule dry matter (DM), total leaf area and specific transpiration, and gas exchange parameters.

relative humidity condition for all treatments was 60%. Four replicates were measured for each treatment. Gas exchange parameters were calculated as described by von Caemmerer and Farquhar [24].

2.3. Growth parameters and transpiration

The experiment was repeated for two consecutive years. Growth conditions inside and between the greenhouses were alternated for the second year. The greenhouse maintained at an ambient CO2 level during the first year was kept at an elevated CO2 level for the second year, and vice versa. Additionally, modules maintained at ambient temperature conditions during the first year were maintained at an elevated temperature during the second year, and vice versa. In summary, these experiments provided a total of 16 plots comprising a combination of two CO2 levels, two temperature regimes, two water availability conditions and two replicates. The effect of CO2, temperature (T) and water availability (H2O) was tested by a split–split-plot multiple analysis of variance (ANOVA) with three factorial treatments (CO2, temperature and water availability) [25]. CO2 treatment was the main plot factor, temperature was the split-plot treatment, and water level was considered the split–split-plot treatment. The main plot analysis contains the sum of squares for CO2 divided by main plot error sum of squares. The split-plot test included temperature and CO2  T mean square and subplot error sums of squares. Finally, the split–split-plot test included H2O, H2O  CO2, H2O  T and H2O  CO2  T corresponding mean square divided by split-subplot error. The results were considered significant at P < 0.05.

Harvested plants were separated into leaves, stems, roots and nodules. Total leaf area was measured using an automatic leaf area meter (Li-3000, LiCor, NE, USA). Plant dry mass was obtained after drying at 85 8C for 48 h. Specific transpiration was calculated as the transpired water, measured by weighing of the pots, throughout the day of harvest per leaf area unit. 2.4. Water status The water status of the plants was evaluated by measuring the midday leaf water potential (Cw) [23] and leaf relative water content (RWC) [24] in the youngest fully expanded leaves. Four leaves were chosen for each treatment and year. 2.5. Gas exchange Gas exchange parameters were measured in fully expanded young leaves using a CIRAS-2 portable photosynthesis system (PP Systems, UK). The leaves were measured under their respective CO2 atmospheric concentrations during growth (350 or 700 mmol mol 1 CO2) and cuvette temperature (25 or 29 8C for ambient or elevated temperature treatment) at 1500 mmol m 2 s 1 PPF provided by an LED light. Cuvette

2.6. Statistical analysis

Fig. 1. Effect of CO2, temperature and water availability on aboveground organ (leaf and stem) DM in nodulated alfalfa at the end of the vegetative normal growth period (A) and (D), after 15 days of regrowth (B) and (E), and after 30 days of regrowth (C) and (F). Values represent the mean  S.E.; n = 8.

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3. Results At the end of the vegetative normal growth period, elevated CO2 significantly enhanced leaf (F = 120.20, P < 0.001) and stem (F = 98.43, P < 0.001) dry matter production (DM) in ambient and ambient + 4 8C treatments (Fig. 1A and D). The positive effect of elevated CO2 was also observed in drought treatments (leaf: F = 20.63, P = 0.01; stem: F = 12.27, P = 0.04); however, leaf and stem DM values were clearly lower than corresponding values of control treatments (around 50% lower) (leaf: F = 164.69, P < 0.001; stem: F = 233.97, P < 0.001). Belowground (root and nodule) organ DM was not affected by elevated CO2 (Fig. 2A and D), but increased with temperature (root: F = 37.78, P = 0.001; nodule: F = 20.90, P = 0.004), especially in roots grown under control conditions (F = 11.17, P = 0.006) and under elevated CO2, and a similar response was observed under conditions of drought in the case of nodules (F = 38.17, P < 0.001). The negative effect of drought on DM production was also observed in belowground organs (root: F = 16.29, P = 0.002; nodule: F = 38.17, P < 0.001), though the effect was less marked (around 16% lower) than that observed for aboveground organs. After 15 days of regrowth, high CO2 stimulated aboveground organ production (Fig. 1B and E) under high temperature conditions (leaf: F = 33.23, P = 0.001; stem: F = 18.54, P = 0.005). Drought significantly reduced leaf and stem DM by an average of 43% (leaf: F = 52.26, P < 0.001; stem: F = 194.10, P < 0.001). Root DM production (Fig. 2B) increased in response to elevated CO2 in control conditions (F = 11.25, P = 0.006) and elevated temperature (F = 5.60, P = 0.036). In turn, nodule DM production (Fig. 2D)

increased in response to elevated temperature in control treatments (F = 11.06, P = 0.006). Drought significantly reduced belowground organ DM production by an average of 23% (root: F = 24.21, P < 0.001). After 30 days of regrowth, aboveground organ DM production (Fig. 1A and D) increased in response to elevated CO2 in the case of leaves for both temperature treatments (F = 24.15, P = 0.003), and only at the elevated temperature (ambient + 4 8C) in the case of stems (F = 10.81, P = 0.017). Low water availability significantly decreased aboveground organ DM production (Fig. 1C and F) by an average of 28% (leaf: F = 34.85, P < 0.001; stems: F = 36.95, P < 0.001). Root DM production increased under conditions of elevated CO2 in all cases except control treatment plants grown at an ambient temperature (F = 11.34, P = 0.006) (Fig. 2C). Nodule DM production increased in response to elevated CO2 only in combination with ambient temperature and drought (F = 14.80, P = 0.002) (Fig. 2F). Drought decreased belowground organ DM production by an average of 18%, with no significant differences between treatments. At the end of vegetative normal growth, leaf area (Fig. 3A) was not affected by elevated CO2 but was reduced significantly under conditions of drought by an average of 52% (F = 167.84, P < 0.001). The specific transpiration rate (Fig. 3D) was reduced by elevated CO2 (F = 493.61, P < 0.001). Drought increased specific transpiration under conditions of ambient CO2 (F = 86.84, P < 0.001). After 15 days of regrowth, leaf area (Fig. 3B) increased in response to the combined effects of elevated CO2 and temperature under control conditions (F = 11.52, P = 0.005), and drought decreased leaf area significantly by an average of 36% (F = 89.64, P < 0.001).

Fig. 2. Effect of CO2, temperature and water availability on belowground organ (root and nodules) DM in nodulated alfalfa at the end of the vegetative normal growth period (A) and (D), after 15 days of regrowth (B) and (E), and after 30 days of regrowth (C) and (F). Otherwise as for Fig. 1.

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Fig. 3. Effect of CO2, temperature and water availability on leaf area and specific transpiration in nodulated alfalfa at the end of the vegetative normal growth period (A) and (D), after 15 days of regrowth (B) and (E), and after 30 days of regrowth (C) and (F). Otherwise as for Fig. 1.

In control treatments, the specific transpiration rate (Fig. 3E) was reduced by elevated CO2 under high temperature conditions 15 days after cutting (F = 32.11, P = 0.001). After 30 days of regrowth, leaf area (Fig. 3C) was not affected by either elevated CO2 or increased temperature in well-watered treatments (control). Leaf area was reduced significantly by an average of 31% under drought conditions (F = 89.48, P < 0.001). Specific transpiration (Fig. 3F) was reduced by elevated CO2 only in drought treatments grown at an ambient temperature (F = 25.50, P < 0.001).

At the end of vegetative normal growth, leaf water potential (Cw) (Table 1) was decreased by elevated temperature (F = 8.54, P = 0.033) and unnaffected by CO2 or water availability but, at the end of regrowth period, was not affected by the studied parameters (CO2, temperature or drought). No significant differences in leaf relative water content (RWC) (Table 1) were found between the different treatments for both periods, vegetative normal growth and regrowth. At the end of vegetative normal growth, the net photosynthetic rate (A) measured under CO2 growth conditions (350

Table 1 Effect of the interaction between CO2, temperature and water availability on midday leaf water potential (Cw) and relative water content (RWC) in nodulated alfalfa at the end of vegetative normal growth (VNG) and after 1 month regrowth (RG) Treatments (H2O  CO2  T)

VNG Cw (MPa)

VNG RWC (%)

RG Cw (MPa)

RG RWC (%)

Control Control Control Control

Amb 700 Amb 700

Amb Amb Amb + 4 8C Amb + 4 8C

1.48  0.09 1.58  0.03 1.67  0.10 1.67  0.01

84.4  4.5 88.7  2.7 82.0  1.4 87.5  2.0

1.95  0.09 1.78  0.06 1.85  0.18 1.79  0.09

93.0  1.0 91.2  0.2 86.6  3.4 90.4  1.0

Drought Drought Drought Drought

Amb 700 Amb 700

Amb Amb Amb + 4 8C Amb + 4 8C

1.63  0.12 1.68  0.06 1.90  0.04 1.64  0.01

87.1  2.1 90.5  2.6 91.3  0.1 88.7  0.8

1.79  0.02 1.82  0.13 1.71  0.04 1.80  0.04

87.1  2.1 92.8  0.4 91.3  0.1 88.7  0.8

H2O T CO2 H2O  T H2O  CO2 T  CO2 H2O  T  CO2

NS *

NS NS NS NS NS

NS NS NS NS NS NS NS

NS NS NS NS NS NS NS

NS NS NS NS NS NS *

Each value represents mean  S.E. of n = 8. The meaning of the symbols used in the split–split-plot ANOVA are: NS, not significant differences. * Significant difference at 5%.

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Fig. 4. Effect of CO2, temperature and water availability on net photosynthesis rate (A) and intracellular CO2 concentration (Ci) at the end of the vegetative normal growth period (A) and (D), after 15 days of regrowth (B) and (E), and after 30 days of regrowth (C) and (F). Otherwise as for Fig. 1.

ambient or 700 mmol mol 1 elevated) (Fig. 4A) was unaffected by elevated CO2, temperature or water availability, while internal leaf CO2 concentration (Ci) (Fig. 4D) increased significantly in response to elevated CO2 (F = 573.76, P < 0.001). Fifteen days after cutting (Fig. 4B), plants grown under elevated CO2 had significantly higher A levels than plants grown under an ambient CO2 concentration (F = 50.52, P < 0.001), while no effect was observed under conditions of drought. Ci increased significantly under conditions of elevated CO2 in all treatments (F = 871.49, P < 0.001) (Fig. 4E). After 30 days of regrowth (Fig. 4C and F), A and Ci increased significantly in response to CO2 (F = 36.38, P = 0.001 and F = 500.98, P < 0.001, respectively), but were unaffected by water availability. 4. Discussion Differences in response to experimental factors were observed between aboveground and belowground organs at certain growth periods, and between vegetative and early and late regrowth periods of alfalfa. Thus, at the end of 1 month of vegetative normal growth, elevated CO2 increased aboveground organ DM (leaf and stem) (Fig. 1A and D), but not that of belowground components (root and nodule) (Fig. 2A and D). Similar results were obtained by Harmens et al. [26], who observed a transient increase in dry matter partitioning into the shoot during the early stages of Dactylis glomerata growth under low N supply at an elevated CO2 concentration. In our experiments, the DM increase during vegetative normal growth was not explained by the differences in final A or leaf area. Failure of A to increase under conditions of elevated CO2 at the end of

vegetative normal growth suggests a photosynthetic downregulation or acclimation to high CO2 [27], as reported in C3 short-grass species [16,28] in the Mojave Dessert [29,30], pastures of New Zealand [31], and in alfalfa [32]. The lower stomatal conductance induced by high CO2 did not limit CO2 supply to mesophyll cells, as evidenced by the higher Ci (Fig. 4A and D), implying that down-regulation of photosynthesis was due to a limitation in the photosynthetic apparatus. This occurs when the sink capacity of the plant is insufficient to accept the photosynthetic products supplied by the sources [33–35]. A previous study revealed a 34% reduction of Rubisco protein levels in alfalfa during vegetative normal growth under elevated CO2 [32]. The increased growth and decreased photosynthesis at the end of vegetative normal growth under conditions of elevated CO2 points to an age-dependent effect of CO2 enrichment; the growth rate is increased in young, but not older plants [36], and the larger final DM reflects a faster photosynthesis and growth at an earlier period. As in the preceding growth period, 15 and 30 days after cutting, an elevated level of CO2 enhanced DM accumulation in aboveground organs (Fig. 1). However, in contrast with that period, root (excluding nodule) DM (Fig. 2) also increased, especially under elevated temperature conditions [8,20], and shows a positive interaction between these two factors. It is well known that temperature modifies the CO2 effect [37], which is greater at higher temperatures or even negative at low temperatures [20,38–40]. In contrast to the results at the end of vegetative normal growth and 30 days after cutting, the leaf area after 15 days of regrowth (Fig. 3) increased in response to the combination of elevated CO2 and elevated temperature in well-watered plants, although no effect due to CO2 on leaf area

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was observed in other treatments. The early and transient nature of this leaf area increase during regrowth is consistent with the results of the vegetative normal period and previous reports [36]. The enhancement of DM accumulation by elevated CO2 can be partly explained by this increase in leaf area and it was more generally explained by the increase in A under CO2 enrichment (Fig. 4). Thus, a remarkable difference in the response of photosynthesis to elevated CO2 was observed before and after cutting, with a disappearance during regrowth of the photosynthetic down-regulation found at the end of vegetative normal growth. The reason for this contrasting response to CO2 enrichment may be that leaf removal from cutting decreased the source/sink ratio; moreover, the larger shoot and root mass during later regrowth stages provided an increased sink for assimilates in comparison with earlier periods of growth. This enhanced capacity for the utilization of photosynthate leads to increased photosynthesis under conditions of elevated CO2 [41]. The reactivation of photosynthesis can restore bacterial feeding after cutting and, as a consequence, symbiotic N2-fixation [42]. Therefore, plant dry matter under conditions of elevated CO2 can be enhanced and recover more rapidly from the effects of cutting. However, nodule DM showed no positive response to elevated CO2 (Fig. 2), and is consistent with the smaller responsiveness of root mass to CO2 enrichment in nodulated rather than nonnodulated soybean plants [43]. Additional carbohydrate produced under elevated CO2 may have been used, however, to increase specific nodule N-fixing activity. Further analysis of plant N content can provide information on this possible response to CO2 enrichment. At the end of vegetative normal growth, as well as 15 and 30 days of regrowth after cutting, drought reduced both the aboveground and belowground DM, with a greater decrease exhibited by the aboveground components. Shoot and root dry matter accumulations are affected differently by drought, with the shoots generally being more prone than the roots [44,45] because the transpiring parts of the plant usually develop greater and longer water deficits [46,47]. While aboveground DM decreased with drought in a similar manner at the end of vegetative normal growth and after 15 days of regrowth, and root DM decreases were roughly similar in all three periods, the reduction by low water availability of DM in aboveground organs (Fig. 1) was less marked after 30 days of regrowth (28%) than previous periods (50% and 43% at the end of vegetative normal growth and 15 days of regrowth, respectively). This suggests a better acclimation of nodulated alfalfa to conditions of low water availability during the regrowth period. It may be that larger roots and increased root reserves (mainly carbohydrates and proteins) made alfalfa more drought tolerant. The reduction in DM caused by low water availability was not related to declining A [32,48] (Fig. 4), but to a decrease in leaf area (Fig. 3). One of the best known effects of elevated CO2 is the reduction of stomatal conductance (gs) [49], which explains the results obtained at the end of vegetative normal growth period revealing that specific transpiration was reduced by elevated CO2 (Fig. 3). With an unaltered leaf area, this response can lead to reduced canopy evapotranspiration and higher soil water content in natural communities, as observed by Field et al. [50].

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This indirect effect of elevated CO2 may be especially important in drier ecosystems or when soil water is limiting growth [51]. After 15 days of regrowth, specific transpiration was also reduced by elevated CO2, except for an increase observed in drought, ambient temperature plants that coincided with a decreased leaf area. Similarly, the overall lack of an effect due to elevated CO2 on specific transpiration 30 days after cutting (Fig. 3) coincided with a decrease in this parameter and a noticeable increase in leaf area in comparison to values recorded 15 days earlier. This shows that changes in leaf area can modify the effect of CO2 enrichment on specific transpiration at any given soil water potential. It is expected that decreased stomatal conductance under conditions of elevated CO2 may to lead to a better plant water status [52]. However, the water potential (cw) and relative water content during the entire experiment showed no significant differences among treatments (Table 1), suggesting that plants acclimate their growth (Figs. 1 and 2) to the available water in the root medium. In conclusion, DM during the first month of vegetative normal growth increased with exposure to elevated atmospheric CO2 concentration due to an earlier and transient enhancement of photosynthesis and growth rates. Photosynthesis at the end of this growth period was down-regulated under elevated CO2. Photosynthesis was stimulated by elevated CO2 during the entire regrowth period, resulting in a greater DM accumulation. CO2 enhanced DM only in aboveground organs during vegetative normal growth, but in both above- and belowground organs during regrowth, indicating that the regrowth period was more responsive to CO2 enrichment than the vegetative normal period. The DM of aboveground organs in all treatments was affected by drought to a greater degree than the DM of belowground organs. The higher drought tolerance of nodulated alfalfa during the regrowth period may be related to higher plant mass and greater reserves accumulated in the roots. Plant growth was acclimated to the water available in the root medium so that the water potential (Cw) and relative water content remained unaltered by atmospheric CO2, temperature and soil water availability. Acknowledgements This work was supported by the Spanish Science and Technology Ministry (BFI2000-0154 and BFU-2004-05096/ BFI) and Fundacio´n Universitaria de Navarra. G. Erice Soreasu was the recipient of a research grant from the Basque Government (BFI-0294). The temperature gradient greenhouses used in this study were funded by the Spanish Commission of Science and Technology (AMB96-0396). The authors wish to thank Mr. A. Urdiain for his technical assistance and H. Santesteban, AL. Verdejo, A. Alonso and S. Kostadinova for their cooperation during harvests. References [1] I. Prentice, G. Farquhar, M. Fasham, M. Goulden, M. Heinmann et al., The carbon cycle and atmospheric carbon dioxide, in: J.T., Houghton, Y., Ding,

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