Plant material and growth conditions.

Tall fescue (‘Rembrandt’) sod (10 cm diameter) was obtained on 10 June 2011 from the turf research field at Rutgers University in North Brunswick, NJ, and were established in plastic pots (10 cm diameter and 60 cm deep) filled with a mixture of sand and topsoil (fine, montmorillonitic, mesic, aquic arqui-dolls) (1:3, v/v). Plants were maintained in a greenhouse for 2 months for establishing the canopy and roots with an average temperature at 23/16 °C (day/night) and 760 μmol·m⁻²·s⁻¹ photosynthetically active radiation (PAR). Plants were moved to a growth chamber (GC15; Environmental Growth Chambers, Chagrin Falls, OH) for the acclimation to controlled-environment conditions for 14 d before imposing CO₂ and watering treatments. The growth chambers were set at 11-h photoperiod with 680 μmol·m⁻²·s⁻¹ PAR and day/night temperatures at 23/16 °C. During plant establishment in the greenhouse or growth chamber, plants were trimmed weekly to ≈10 cm, watered every 2 d, and fertilized weekly with half-strength Hoagland’s nutrient solution (Hoagland and Arnon, 1950).

Treatments and experimental design.

The experiment consisted of two CO₂ concentrations and two watering treatments, which were arranged in a randomized complete block split-plot design with CO₂ concentrations as main plots and watering treatments as subplots. Each CO₂ treatment was replicated in four growth chambers. Each watering treatment was replicated in four pots placed inside each growth chamber under each CO₂ treatment.

The CO₂ treatments included ambient CO₂ concentration (400 ± 10 μmol·mol⁻¹) and elevated CO₂ concentration (800 ± 10 μmol·mol⁻¹). Plants were grown at the two CO₂ levels in growth chambers for 14 d before and then during drought (15 d) and rewatering (6 d) treatments for a total of 35 d. The CO₂ controlling setup of growth chambers followed the design as described in Yu et al. (2012a, 2012b). The concentration of CO₂ inside each growth chamber was maintained through an automated, open-chamber CO₂ control system connected to a gas tank containing 100% CO₂ (Airgas, Radnor, PA). The CO₂ levels were continuously monitored through an infrared gas analyzer (LI-820; LI-COR, Lincoln, NE) and controlled using an automatic system consisting of a programmable logic controller unit, solenoid valves, and a laptop computer with monitoring software accurate to within 10 μmol·mol⁻¹.

After 14 d of pre-exposure to CO₂ treatments, plants were then exposed to two levels of water treatments under either ambient or elevated CO₂ concentration: 1) well-watered control by watering plants every 2 d until water drainage from the bottom of the pot to maintain soil volumetric water content (SWC) at the pot capacity (≈25%); 2) drought stress by withholding irrigation for 15 d when SWC decreased to 4.98%; and 3) rewatering for 6 d with irrigating previously drought-stressed plants and SWC resumed to the pot capacity.

Physiological measurements.

Leaf relative water content (RWC) of fully expanded leaves was determined at 0, 5, 10, and 15 d of drought stress and at 2 and 6 d of rewatering based on fresh weight (FW), turgid weight (TW), and dry weight (DW) using the following formula: RWC (%) = [(FW – DW)/(TW – DW)] × 100. Leaf fresh weight was immediately weighed after excised from the plants and then was soaked in deionized water for 6 h at 23 °C. Leaf samples were then blotted dry and immediately weighed for determination of TW. Samples were then dried in an oven at 80 °C for 72 h and weighed again for DW (Bagatta et al., 2008).

Leaf osmotic potential (ψ_S) was measured at 5, 10, and 15 d of drought stress following the method described by Fu et al. (2010). Briefly, leaf samples were soaked in deionized water for 12 h at 4 °C and blotted dry. Leaves were submerged in liquid nitrogen for rapid freezing and then stored at –20 °C until analyzed. Frozen samples were thawed for 30 min and cell sap was pressed using a laboratory press (Fred S. Carver, Wabash, IN). A 10-μL aliquot of the expressed sap was pipetted onto a filter paper disc that was placed in the sampling chamber of an osmometer (Wescor, Logan, UT) for analyzing solute concentration [C (millimoles per kilogram)]. Osmolarity of leaf sap was converted from millimoles per kilogram to megapascals using the formula: MPa = –C × 2.58 × 10⁻³ (Bajji et al., 2001)

Soluble sugar content of leaves was measured at 5, 10, and 15 d of drought stress using the spectrophotometric method described by Pei et al. (2010) with modifications. Briefly, 0.2 g fresh leaves were boiled in 10 mL distilled water for 30 min. The extract was filtered through two layers of cheesecloth. The filtrate (0.5 mL) was mixed with 1.5 mL distilled water and 1 mL of 9% phenol and 5 mL H₂SO₄. Tubes with this mixture were left at 23 °C for 30 min. The absorbance of the solution was measured with a spectrophotometer (Spectronic Genesys 2; Thermo Electron Corp., Madison, WI) at 485 nm. The soluble sugar concentration was determined using a standard curve.

Leaf net photosynthesis (P_n), transpiration rate (T_r), and g_S were determined on the second fully expanded leaves at 0, 5, 10, and 15 d of drought and 2 and 6 d of rewatering using an infrared gas analyzer (LI-6400; LI-COR). Ten individual leaves attached to the plants were taken from each pot and were placed in the leaf chamber with a built-in red and blue light source of the LI-6400, and all measurements were taken on at the level of 800 μmol·m⁻²·s⁻¹ photosynthetic photon flux. The CO₂ concentration settings in leaf chamber were 400 (μmol·mol⁻¹) for ambient CO₂ treatments and 800 (μmol·mol⁻¹) for elevated CO₂ treatments, respectively. Leaf instantaneous water use efficiency (WUE) was calculated as the ratio of P_n to T_r. Stomatal opening and closure in response to elevated CO₂ concentration, drought stress, and rewatering was examined on the second fully expanded from the top of plants from each treatment and photographs were taken using a camera fitted to a microscope (H600L; Nikon Instruments, Tokyo, Japan).

Rubisco extraction and activity assays were conducted using methods described by Hu et al. (2010) with modifications. Samples of fresh leaves (0.2 g) collected at 10 and 15 d of drought and 2 d of rewatering were immediately frozen in liquid nitrogen and kept at –80 °C before extraction. For extraction, the leaf tissue was ground in liquid nitrogen with 3 mL extraction buffer containing 50 mm Hepes-KOH (pH = 7.5 at 25 °C), 10 mm MgCl₂, 2 mm ethylene diamine tetraacetic acid, 10 mm dithiothreitol, 10% glycerol (v/v), 1% bovine serum albumin (w/v), and 1% Triton X-100 (v/v) (Sigma, St. Louis, MO). The supernatant was isolated by centrifugation at 14,000 g_n for 10 min at 4 °C and used immediately for Rubisco activity assays. Rubisco activity was measured by adding RuBP to the assay solution [100 mm Bicine (pH = 8.0), 25 mm KHCO₃, 20 mm MgCl₂, 3.5 mm adenosine-5′-triphosphate, 5 mm phosphocreatine, 5 units glyceraldehyde-3-phosphate dehydrogenase, 5 units 3-phosphoglyceric phosphokinase, 17.5 units creatine phosphokinase, and 0.25 mm NADH] and absorbance measured at 340 nm with a spectrophotometer (Helios Alpha; Thermospectronic, Rochester, NY). Initial activity was measured immediately after adding RuBP and total activity was measured after incubating samples at 25 °C for 5 min. Rubisco activation state was determined by the ratio of initial to total activity. Fifty microliters of leaf extract was used to determine soluble protein concentration (Bradford, 1976). Total soluble protein was estimated from standard curves prepared with bovine serum albumin and used to calculate Rubisco activity expressed as micromoles CO₂ per second per milligram protein.

Statistical analysis.

All data were analyzed using SAS statistics software (Version 9.0; SAS Institute, Cary, NC). The analysis of variance with a fixed model was used to determine differences among treatments in RWC, P_n, g_S, T_r, WUE, ψ_S, soluble sugar content, Rubisco activity, and Rubisco activation state. Means of different treatments were tested with least significant difference at a probability level of 0.05.

Effects of elevated CO₂ concentration on leaf internal water status and water use efficiency under drought stress and rewatering.

Leaf water retention capacity, water use rate, and WUE are important factors controlling plant water status during drought stress and ultimately whole-plant drought resistance. RWC is a good indicator of leaf hydration status and the level of drought tolerance (Flexas and Medrano, 2002). In this study, RWC of tall fescue was maintained at an average of 89.87% in well-watered plants during the experimental period, regardless of the CO₂ treatments, but it declined below the well-watered control level after 10 d of drought stress in both ambient and elevated CO₂ treatments (Fig. 1). The decline in RWC was less severe in the elevated CO₂ with drought treatment than in the ambient CO₂ with drought treatment. A significantly higher leaf RWC in tall fescue was observed in elevated CO₂ with drought-stressed plants than those in ambient CO₂ with drought treated at 10 and 15 d of drought stress. After 2 d of rewatering, plants treated with elevated CO₂ under drought recovered more rapidly than those treated with ambient CO₂ under drought. These results suggested that leaves treated with elevated CO₂ had a greater capacity of retaining water or resistance to dehydration during drought stress as well as rapid rehydration or recovery potential on rewatering.

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Cellular hydration status of leaves during drought stress depends on the balance between water retention and water loss, which is governed by stomatal behaviors and osmotic adjustment among various other physiological factors (Farooq et al., 2009; Kramer and Boyer, 1995). Osmotic adjustment helps to maintain the cell water balance with active accumulation of solutes in the cytoplasm, thereby minimizing the harmful effects of drought (Clifford et al., 1998). Elevated CO₂ could alleviate leaf dehydration through osmotic adjustment under drought stress. In this study, elevated CO₂ treatment promoted osmotic adjustment, as shown by the decreases in the level of ψ_S under drought stress conditions (Fig. 2). Osmotic adjustment is associated with the accumulation of some osmoregulation substances such as water-soluble carbohydrates (Geissler et al., 2009; Pérez-López et al., 2010; Yoshiba et al., 1997). Soluble sugars are the predominant forms of osmoregulants in C₃ perennial grasses during the early phase or a moderate level of drought stress (DaCosta and Huang, 2006) and decreases in ψ_S during drought stress are accompanied by increases in sucrose content in tall fescue (Fu et al., 2010). In the present study, soluble sugar content increased along with lowering ψ_S under elevated CO₂ concentration, particularly under drought conditions (Fig. 3). These results suggested that elevated CO₂ could facilitate osmotic adjustment in association with soluble sugar accumulation and consequently retained more water in leaves of tall fescue. Other solutes such as proline and glycine betaine also play roles in osmotic adjustment. How elevated CO₂ may affect osmotic adjustment in relation to the accumulation of those solutes is yet to be determined. Several previous studies also reported enhanced osmotic adjustment by elevated CO₂ in different plant species (Ferris and Taylor, 1994; Pérez-López et al., 2010). Some other studies have demonstrated that the elevated CO₂ concentration had minimal effect on ψ_S during drought stress (Aranda et al., 2008; Polley et al., 1999; Robredo et al., 2007), and a few studies even showed decreased osmotic adjustment in response to drought under elevated CO₂ concentration (Tschaplinski, et al., 1993, 1995). The variable results of elevated CO₂ effects on osmotic adjustment under drought stress from different studies may be the result of different plant species, growth stages, drought severity levels or duration as well as CO₂ concentrations examined in different experiments. Nevertheless, our results suggested that the enhanced osmotic adjustment in association with sugar accumulation by elevated CO₂ concentration could play roles in maintaining greater RWC in tall fescue leaves under drought stress in this study.

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Elevated CO₂ concentration could also alleviate leaf dehydration through lowering the rate of water loss. In this study, T_r (Fig. 4A) was suppressed by elevated CO₂ under both well-watered conditions and drought stress. Drought stress led to a rapid decline in T_r under both CO₂ regimes. Plants under the elevated CO₂ treatment had significantly lower T_r than those under the ambient CO₂ treatment during the first 10 d of drought stress and by 15 d of drought stress, T_r declined to the lowest level and there were no significant differences between the two CO₂ treatments. The reduction in T_r by elevated CO₂ concentration also has been reported by others (Assmann, 1993; Morison and Gifford, 1983; Tyree and Alexander, 1993). Along with T_r decline, g_S decreased in plants exposed to both drought and elevated CO₂ but increased during rewatering (Fig. 4B).

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The partial closure of stomata induced by elevated CO₂ may contribute to the increases in leaf instantaneous WUE in tall fescue. In this study, WUE of tall fescue increased with drought stress under both CO₂ treatments, and it was significantly enhanced by elevated CO₂ under drought stress (Fig. 4C). The WUE in plants treated with the elevated CO₂ concentration was 1.54-, 2.09-, and 1.85-fold greater than those plants under the ambient CO₂ treatment at 5, 10, and 15 d of drought stress, respectively. Under rewatering conditions, no differences in WUE were detected in the two CO₂ treatments. Improved WUE by elevated CO₂ concentration has also been reported in various plant species exposed to drought stress (Centritto et al., 2002; Chun et al., 2011; Ghannoum et al., 2002; Laila and Adel, 2002; Retuerto and Woodward, 1993; Tschaplinski et al., 1995). WUE has been suggested as an effective criterion to select for superior drought-performing turfgrasses (Ebdon and Kopp, 2004). The improvement in WUE with elevated CO₂ has a great potential for water conservation and reducing irrigation in turfgrass management.

Stomata remained open in well watered and excessive CO₂ conditions (Fig. 5A) but closed under drought stress with ambient CO₂ (Fig. 5B) or under elevated CO₂ with adequate water (Fig. 5C) or under drought stress with elevated CO₂ (Fig. 5D). On rewatering, stomata of drought-stressed leaves reopened under ambient CO₂ (Fig. 5E) but remained at least partially closed under elevated CO₂ (Fig. 5F). These results suggested that elevated CO₂ concentration could induce stomata closure at least partially, leading to the reduction in the rate of water loss through transpiration either under well-watered conditions or drought stress. However, CO₂-induced stomatal closure may inhibit photosynthetic capacity under drought stress and photosynthetic recovery after rewatering, as discussed subsequently.

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Effects of elevated CO₂ concentration on photosynthetic rate and carboxylation enzyme activity under drought stress and rewatering.

Leaf P_n of tall fescue was enhanced by elevated CO₂ under both well-watered conditions and drought stress (Fig. 6A). Leaf P_n under both CO₂ regimes declined during 15-d drought stress, but the decline was less pronounced (36% reduction from 8.21 to 5.23 μmol·m⁻²·s⁻¹) under the elevated CO₂ treatment than that under the ambient CO₂ treatment (58% reduction from 7.42 to 3.12 μmol·m⁻²·s⁻¹). Elevated CO₂-enhanced P_n has been reported under non-stress and stress conditions in other plant species (Albert et al., 2011; Campbell et al., 1988; Sage et al., 1989; Vu et al., 1997), but few reported effects of elevated CO₂ on recovery in photosynthesis in response to rewatering (Widodo et al., 2003). In this study, on rewatering, leaf P_n with the elevated CO₂ treatment recovered more rapidly than that did with the ambient CO₂ treatment, and at 2 d of rewatering, P_n of elevated CO₂-treated plants was ≈37% greater than that under ambient CO₂ treatment. However, leaves under elevated CO₂ still maintained lower g_S compared with those exposed to ambient CO₂ after rewatering, which could have limited the recovery potential in P_n (Fig. 6A) even in fully rehydrated leaves (Fig. 1) during rewatering. Factors regulating the recovery in P_n under elevated CO₂ concentration on rewatering after prolonged periods of drought stress were not well understood. The results in this study indicated that greater P_n during rewatering in CO₂-enriched plants relative to that under ambient CO₂ was not the result of the elimination in stomatal limitation, but could be the result of other factors such as metabolic activity of carboxylation.

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Rubisco is the key enzyme for carbon fixation of photosynthesis, and its activity is controlled by another enzyme, Rubisco activase. Rubisco activity showed a significant decline compared with the well-watered control at 10 and 15 d of drought stress under both CO₂ treatments (Fig. 6B). No significant differences were observed in Rubisco activity between ambient CO₂ and elevated CO₂ treatments under well-watered conditions, but Rubisco activity was significantly higher in CO₂-enriched plants than those with ambient CO₂ at 10 and 15 d of drought stress (Fig. 6B). Rubisco activation state decreased to a significantly lower level at 10 and 15 d of drought stress than that of the well-watered control plants under both ambient and elevated CO₂ treatments (Fig. 6C). The drought-induced decline in Rubisco activation state was to a lesser extent under the elevated CO₂ (reduction by 19%) than that under ambient CO₂ treatment (reduction by 35%). Drought-induced decline in Rubisco activity and activation state has been reported in other plant species (Erice et al., 2006; Vu et al., 1998). At 10 and 15 d of drought stress, Rubisco activation state was significantly greater under the elevated CO₂ treatment than that under the ambient CO₂ treatment, suggesting that increasing CO₂ concentration could enhance the photosynthetic capacity of tall fescue by stimulating Rubisco carboxylation activities under drought stress. Previous studies reported enhancement in leaf photosynthesis by elevated CO₂ under heat stress was associated with the up-regulation of Rubisco (Prasad et al., 2009). After 2 d of rewatering, both Rubisco activity and activation state increased to a significantly higher level than that at 15 d of drought but did not fully recover to the well-watered control level under either ambient or elevated CO₂ treatment. Both Rubisco activity and activation state were significantly greater in plants exposed to elevated CO₂ than those under ambient CO₂ concentration at 2 d of rewatering. These results indicated that the resumption of Rubisco activity and activation state could contribute to the greater recovery of P_n on rewatering after drought stress, particularly under elevated CO₂.

In summary, elevated CO₂ improved water status in tall fescue during drought stress, which could be the result of CO₂-induced stomatal closure with limited transpirational water loss and enhanced osmotic adjustment in association with soluble sugar accumulation. Elevated CO₂ enhanced net photosynthetic rate in tall fescue leaves even with lower g_S but enhanced Rubisco and Rubisco activase activity during both drought and rewatering. The mitigating effects of elevated CO₂ on drought inhibition of photosynthesis and the enhanced recovery in photosynthesis on rewatering were mainly the result of the elimination of metabolic limitation from drought damages associated with increased enzyme activities for carboxylation.