Effects of Elevated CO on Physiological Responses of

RESEARCH

Effects of Elevated CO2 on Physiological Responses of Tall Fescue to Elevated Temperature, Drought Stress, and the Combined Stresses Jingjin Yu, Lihua Chen, Ming Xu,* and Bingru Huang* ABSTRACT Drought and elevated temperature often occur alone or in combination in many areas, limiting cool-season grass growth. Rising atmospheric CO2 concentration may affect plant adaptation to drought and high temperature. The objective of this study was to investigate the effectiveness of elevated CO2 in mitigating the negative effects of drought or elevated temperature alone or a combination of these stresses on physiological processes in a perennial grass species. The effects of these treatments on water relations, photosynthesis, and respiration were determined in tall fescue (Festuca arundinacea Schreb. cultivar Rembrandt). Grass plants were subjected to the following treatments in growth chambers: heat stress (30°C or 5°C above the optimal level of 25°C), drought stress by maintaining soil water content at 50% of field capacity, or the combined two stresses for 28 d. Stressed and unstressed control plants were exposed to a constant level of either ambient CO2 (400 μL L−1) or elevated CO2 (800 μL L−1). At ambient CO2 concentration, drought and the combined stress for 28 d caused significant decline in leaf relative water content (RWC), photochemical efficiency (ratio of variable to maximum fluorescence [Fv:Fm]), net photosynthetic rate (A), stomatal conductance (gs), maximal ribulose1,5-bisphosphate carboxylase oxygenase (Rubisco)limited rate of photosynthesis (Vcmax), and maximal electron transport-limited rate of photosynthesis (Jmax) but increased membrane electrolyte leakage (EL) and dark respiration rate (Rd). Elevated temperature to 5°C above the optimal level resulted in the increases in gs, EL, and Rd but had no significant effects on the other physiological parameters. Drought stress for 28 d was more detrimental than increasing temperature by 5°C for tall fescue and the combined stress was more detrimental than either stress alone. Elevated CO2 mitigated the degree of change in all physiological factors under drought or heat stress and resulted in increases in A (162%) and RWC (19%) and a reduction in EL (21%) under the combined stress. These results suggest that elevated CO2 could improve tall fescue tolerance to drought and elevated temperature by enhancing plant water status, cellular membrane stability, and photosynthesis capacity and by suppressing gs for water loss and C consumption through lowering respiration rate.

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J. Yu and L. Chen, School of Soil and Water Conservation, Beijing Forestry Univ., Beijing, People’s Republic of China 100083; J. Yu and M. Xu, Dep. of Ecology, Evolution and Natural Resources, Rutgers Univ., New Brunswick, NJ 08901; M. Xu, Key Lab of Ecosystem Network Observation and Modeling, Institute of Geographic Sciences and Natural Resources, Chinese Academy of Sciences, Beijing, People’s Republic of China 100101; B. Huang, Dep. of Plant Biology and Pathology, Rutgers Univ., New Brunswick, NJ 08901. Received 16 Jan. 2012. *Corresponding authors ([email protected]; mingxu@ igsnrr.ac.cn). Abbreviations: A, photosynthetic rate; ATP, adenosine triphosphate; Cinitial, conductivity of the incubation solution; Cmax, conductivity of incubation solution with killed tissues; DW, dry weight; EL, electrolyte leakage; Fv:Fm, ratio of variable to maximum fluorescence; FW, fresh weight; g s, stomatal conductance; Jmax, maximal electron transportlimited rate of photosynthesis; Rd, dark respiration rate; Rubisco, ribulose-1,5-bisphosphate carboxylase oxygenase; RuBP, ribulose-1,5bisphosphate; RWC, leaf relative water content; SWC, soil volumetric water content; TW, turgid weight; Vcmax, maximal Rubisco-limited rate of photosynthesis.

T

emperature is a primary factor limiting growth of cool-season plant species, as it is often increased to above the optimal temperatures for plant growth during summer months in many areas. Drought stress caused by a decline in precipitation and fresh water availability for irrigation is also a major factor limiting plant growth. Simultaneous drought and elevated temperature stress often occurs in the summer in many areas, which is even more detrimental to plant growth than either stress alone (Jiang and Huang, 2000, 2001b). These two abiotic stresses are becoming increasing concerns due to the threats of global warming and predictions of increased Published in Crop Sci. 52:1848–1858 (2012). doi: 10.2135/cropsci2012.01.0030 © Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.

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average temperatures, which are predicted to rise by 2 to 6°C in the 21st century (IPCC, 2007). In addition, global precipitation may decline and become unevenly distributed both spatially and temporally (Sankaranarayanan et al., 2010; Chun et al., 2011). Drought and elevated temperature alone or in combination affects various cellular and physiological processes, including cellular membrane integrity, water relations, photosynthesis, and respiration (Rachmilevitch et al., 2006). Improving plant tolerance to either stress alone or in combination is of great significance for maintaining plant productivity with changing climatic conditions. Along with climate changes, the atmospheric CO2 concentration has increased more than 100 μL L−1 since the beginning of the industrialization era with the concentration rising at a rate of approximately 2 μL L−1 per year currently (IPCC, 2007). Increased CO2 concentration may promote plant growth and improve plant adaptation to climate changes, particularly for C3 species (Kirkham, 2011). Various physiological processes are sensitive to changes in temperature, drought stress, and atmospheric CO2. Both leaf photosynthesis and respiration are highly temperature dependent (Faria et al., 1996; Qaderi et al., 2006). Elevated temperature often leads to decreases in net photosynthesis and increases in dark respiration, limiting plant growth (Salvucci and Crafts-Brandner, 2004; Fry and Huang, 2004). Leaf photosynthesis is also regulated by plant water status and soil water availability. Drought stress leads to significant reductions in photosynthesis because water is pivotal to various biophysical and biochemical processes therein (Hsiao and Acevedo, 1974; Hu et al., 2010). Drought effects on photosynthesis are coupled with stomatal conductance, which is regulated by guard cell water potential and hormone balance (Hsiao and Acevedo, 1974; DaCosta and Huang, 2007). Previous work has shown that elevated CO2 may mitigate the negative effects of drought and heat stress through the regulation of photosynthesis (Hamerlynck et al., 2000; Qaderi et al., 2006; Robredo et al., 2007). The positive effects of elevated CO2 on photosynthesis have been associated with reduced photorespiration and increased ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) carboxylation (Leakey et al., 2006; Reddy et al., 2010). Many previous studies have examined the mitigating effects of elevated CO2 on drought or heat stress alone; however, effects of elevated CO2 on physiological responses of plants to combined drought and heat stress are not well documented and limited data were reported in this aspect (Forseth and Ehleringer, 1982; Aranjuelo et al., 2005; Qaderi et al., 2006). Furthermore, most of the work has focused on annual crops (Aranjuelo et al., 2005; Sankaranarayanan et al., 2010; Wang et al., 2008), vegetables (Hamilton et al., 2008; Wang et al., 2008), and woody species (Faria et al., 1996; Reddy et al., 2010). Despite their important use as turf and forage grasses, few studies have investigated the potential beneficial CROP SCIENCE, VOL. 52, JULY– AUGUST 2012

effects of elevated CO2 on the alleviation of physiological damage in perennial grasses exposed to drought or elevated temperature acting alone or in combination. Tall fescue is a widely used cool-season perennial grass species in both forage and turf scenarios (Fry and Huang, 2004). This species exhibits good drought avoidance traits such as deep rooting characteristics but has a limited ability to tolerate drought and high temperatures (Fry and Huang, 2004). Understanding physiological effects of elevated CO2 on tall fescue responding to drought and/or heat stress alone or simultaneously has a great potential to develop stress-tolerant germplasm and novel practices such as CO2 fertilization for turf and forage grass management. With this, the objective of this study was to investigate whether elevated CO2 can mitigate the negative effects of drought or elevated temperature alone or combined for tall fescue by monitoring physiological processes associated with water relations, photosynthesis, and respiration. To our knowledge, this is the first report investigating elevated CO2 interaction with drought and temperature stress in a perennial turfgrass system.

MATERIALS AND METHODS Plant Materials and Growing Conditions Tall fescue (‘Rembrandt’) plants were collected from field plots managed as turfgrass in the research farm at Rutgers University in Adelphia, NJ, and transplanted into pots (10 cm diameter by 60 cm long) fi lled with a mixture of fi ne sand and soil (1:1 v/v). Plants were maintained in a greenhouse with an average temperature regime of 21/16°C (day/night) and 810 μmol m−2 s−1 photosynthetic active radiation in natural sun light, and 65% relative humidity for 70 d (May–June 2011) to establish canopy and roots. During this establishment period, plants were watered every other day and fertilized once weekly with half-strength Hoagland’s solution (Hoagland and Arnon, 1950). Plants were cut once a week to maintain a canopy height of 5 to 6 cm. Plants were moved to a growth chamber with the temperature set at 25/18°C (day/night), 70% relative humidity, 650 μmol m−2 s−1, and a 12-h photoperiod for 2 wk before treatment. After moving to growth chambers, plants were not cut during the entire experimental period.

Treatments and Experimental Design The experiment consisted of three factors (water, temperature, and CO2) and was arranged in a split plot design with CO2 as the main plot and temperature and drought stress as subplots with four replicates for each treatment and eight pots in each growth chamber. The CO2 treatments included ambient CO2 (400 ± 10 μmol mol−1) and elevated CO2 (800 ± 10 μmol mol−1). Temperature was controlled at two levels: 25/20°C (day/night, optimal temperature control) and 30/25°C (day/ night, elevated temperature). Plants were subjected to two irrigation treatments: well-watered control plants were irrigated three times weekly with 400 mL of water at each time of irrigation to maintain soil water content at the field capacity (through

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irrigating plants until drainage ceased); drought stress was irrigated three times per week with 200 mL of water to maintain soil water content at 50% of the field capacity. Plants were acclimated to two CO2 levels in the growth chambers for 4 wk before imposition of drought and/or temperature treatments. Multiple growth chambers (Environmental Growth Chamber) were used to control CO2 and temperature. Four chambers were maintained at the ambient CO2 level with two of them set to elevated temperature (30/25°C) and the other two at the optimal temperatures (25/20°C). The same temperatures were used for the elevated CO2 chambers, with two at elevated temperature and two at the optimal temperature. All plants in the wellwatered control and drought treatments were randomly placed inside the growth chambers with elevated or ambient CO2 at the normal temperature or elevated temperature. Plants were relocated among the different chambers once per week to minimize confounding effects of environmental variation between different chambers. 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, Inc.). The CO2 levels were continuously monitored through an infrared gas analyzer (Li-820, LICOR, Inc.) 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 μL L−1 of the target levels (400 and 800 μmol mol−1).

and 1500 μmol mol−1) with a 3-min period for stabilization between switching to a different concentration. The response of A to chloroplastic CO2 concentration was analyzed by nonlinear regression (Miao et al., 2008) to obtain maximal Rubiscolimited rate of photosynthesis (Vcmax) and maximal electron transport-limited rate of photosynthesis ( Jmax). Dark respiration rate was measured using the Li-6400 infrared gas analyzer with leaves enclosed in the chamber without light supply.

Soil and Leaf Water Status

Statistical Analyses

Soil volumetric water content (SWC) and leaf relative water content (RWC) are the most commonly used parameters to indicate soil water availability and leaf hydration status, respectively (Flexas and Medrano, 2002). The SWC in a 0 to 20 cm soil layer of each pot was measured during 0800 to 0830 h in the morning using time domain reflectometry (TDR) (Soil Moisture Equipment Corp.). Leaf relative water content of fully expanded leaves was determined based on fresh weight (FW), turgid weight (TW), and dry weight (DW) using the following formula: RWC (%) = [(FW – DW)/(TW – DW)] × 100. Fully expanded leaves were immediately weighed for FW after being excised from the plants and then placed into tubes fi lled with deionized water for 12 h in dark at 4°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 at least 72 h and again weighed for DW (Barrs and Weatherley, 1962).

Leaf Photosynthesis, Stomatal Conductance, and Dark Respiration Leaf net photosynthetic rate (A), stomatal conductance (g s), and dark respiration rate (Rd) were taken using six individual leaves (second fully expanded from the top) from each pot using a 6 cm 2 cuvette with a portable infrared gas analyzer (Li-6400, LICOR, Inc.). Leaves were placed in a leaf chamber with a built-in red and blue light source of the Li-6400 and A and g s were determined at the light level of 800 μmol photon m−2 s−1, which was the light saturation point for tall fescue leaves. The response curves of A to intercellular CO2 concentration were generated by varying the ambient CO2 concentration in 12 steps (400, 200, 150, 120, 80, 50, 400, 600, 800, 1000, 1300,

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Photochemical Efficiency and Cell Membrane Stability Leaf photochemical efficiency was determined by measuring chlorophyll fluorescence (the ratio of variable to maximum fluorescence [Fv:Fm]) with a fluorescence induction monitor (Bioscientific Ltd.) following 30 min dark acclimation. Cellular membrane stability was estimated by measuring electrolyte leakage (EL) (Blum and Ebercon, 1981). For EL analysis, fresh leaves (0.2 g) were placed in test tube containing 20 mL deionized water and shaken on a conical shaker for 24 h under room temperature to measure the initial conductivity of the incubation solution (Cinitial) with a conductivity meter (YSI Instrument). Leaves were then killed by autoclaving at 140°C for about 20 min and the conductivity of incubation solution with killed tissues (Cmax) was measured after samples being shaken for another 24 h. The relative EL was calculated as Cinitial/Cmax × 100 (Blum and Ebercon, 1981).

Data were analyzed using statistics software (SAS 9.0; SAS Institute, 1994). The ANOVA with a fixed model was used to determine differences among treatments in SWC, RWC, Fv:Fm, EL, Vcmax, Jmax, A, gs, and Rd. When a particular F test was significant, means were tested with LSD at a confidence level of 0.05.

RESULTS Soil Water Status under Elevated CO2, Temperature, and Drought Stress Soil volumetric water content was maintained at the field capacity (11%) at 25°C under well-watered conditions. The SWC decreased significantly during drought stress under both CO2 concentrations, but pots exposed to elevated CO2 concentration had higher SWC than those at ambient CO2 under drought stress (Fig. 1A). The SWC was maintained at the well-watered level under elevated temperature and elevated CO2 treatment (Fig. 1B). The combination of drought and elevated temperature caused rapid decline in SWC at both ambient and elevated CO2 concentrations (Fig. 1C).

Physiological Responses (RWC, EL, and Fv:Fm) to CO2, Temperature, and Drought Stress

Drought stress alone caused significant reduction in RWC, but plants exposed to elevated CO2 had significantly higher RWC than that at ambient CO2 (Fig. 2A). Elevated temperature alone had no significant effect on RWC regardless of CO2 concentrations, and elevated CO2 did not affect RWC at either ambient or elevated

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temperature (Fig. 2B). Elevated temperature in combination with drought stress caused a more severe decline in RWC than drought or elevated temperature alone under either ambient or elevated CO2 treatment (Fig. 2C). Plants treated with elevated CO2 had significantly higher RWC than those at ambient CO2 under the combined treatment of drought and elevated temperature (Fig. 2C). Drought stress alone for 7 d had no effect on EL, but prolonged stress (28 d) caused significant increases in EL regardless of CO2 treatments (Table 1). Elevated temperature alone had no significant effect on EL at either ambient or elevated CO2 at 7 d, but at 28 d it caused a significant increase in EL at ambient CO2 level (Table 1). The combination of drought and elevated temperature resulted in more significant increases in EL compared to either drought or elevated temperature alone. Elevated CO2 suppressed the increase in EL induced by drought alone or by combined drought and elevated temperature at 28 d (Table 1). For Fv:Fm, no treatment effects of drought stress alone were detected at 7 d at both CO2 levels. Drought stress alone for 28 d caused a significant decline in Fv:Fm under ambient CO2, but elevated CO2 suppressed the overall decrease (Table 1). Elevated temperature alone did not impose negative effects on Fv:Fm at either CO2 concentration at both 7 and 28 d. The combination of elevated temperature and drought stress led to a significant decrease in Fv:Fm at both ambient and elevated CO2 treatment at 28 d (Table 1).

Photosynthetic Rate, Carboxylation Capacity (Vcmax and Jmax), and Stomatal Conductance as Affected by CO2, Temperature, and Drought

Figure 1. Soil volumetric water content (SWC) under drought stress (A), elevated temperature (B), and combined drought and heat stress (C) at two CO2 levels (400 and 800 μmol mol−1). The treatment symbols are 25-800-W for optimal temperature, wellwatered, and elevated CO2, 25-400-W for optimal temperature, well-watered, and ambient CO2, 25-800-D for optimal temperature, drought stress, and elevated CO2, 25-400-D for optimal temperature, drought stress, and ambient CO2, 30-800W for elevated temperature, well-watered, and elevated CO2, 30400-W for elevated temperature, well-watered, and ambient CO2, 30-800-D for elevated temperature, drought stress, and elevated CO2, and 30-400-D for elevated temperature, drought stress, and ambient CO2. Vertical bars indicate LSD values (P ≤ 0.05) for the comparison of treatments.


Leaf net A decreased significantly during drought stress at ambient CO2 treatment but not at elevated CO2 concentration (Fig. 3A). Elevated CO2 caused an increase in A under both well-watered and drought conditions at 25°C over the entire treatment period, except at 21 d (Fig. 3A). Elevated temperature had no significant effects on A regardless of CO2 concentrations over the treatment period (Fig. 3B). Elevated CO2 also did not cause changes in A under either ambient or elevated temperature excluding those at 7 and 28 d (Fig. 3B). Leaf A declined rapidly during the plants’ exposure to the combined drought and elevated temperature after 7 d at ambient CO2 but not until 14 d at elevated CO2 concentration (Fig. 3C). Additionally, elevated CO2 led to significantly greater A during the treatment periods under the combination of drought and elevated temperature conditions (Fig. 3C). Drought stress alone caused an increase in Vcmax at ambient CO2 at 7 d but did not cause changes in Jmax. Both parameters were significantly decreased by drought stress for 28 d at ambient CO2 but not at elevated CO2 level. Elevated temperature alone did not have significant effects

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on Vcmax and Jmax at 7 or 28 d of treatment regardless of CO2 concentrations. The combined drought and heat stresses had no effects on either parameter at 7 d but resulted in significantly lower Vcmax and Jmax at both CO2 levels at 28 d. Elevated CO2 induced significantly lower Vcmax and Jmax under combined stresses (Table 1). Stomatal conductance declined rapidly under drought stress alone at both ambient and elevated CO2 (Fig. 4A). Elevated CO2 led to an increase in g s under drought stress at 21 and 28 d of treatment but resulted in significantly lower g s at 25°C under well-watered conditions (Fig. 4A). Elevated temperature alone caused significant increase in g s compared to plants exposed the optimal temperature at ambient CO2 at 7 and 14 d and under elevated CO2 concentration at 7, 14, and 21 d (Fig. 4B). Elevated CO2 did not have significant effects on g s under elevated temperature alone during most of the experimental periods except at 7 d (Fig. 4B). Under the combined drought and elevated temperature conditions, gs declined rapidly with duration of the treatments (Fig. 4C). Elevated CO2 had no significant effects on g s under these conditions except at 7 d (Fig. 4C).

Leaf Dark Respiration Rate as Affected by CO2, Temperature, and Soil Water Status

Leaf Rd increased significantly at ambient CO2 concentration at 14 and 28 d but did not change at elevated CO2 concentration during drought stress (Fig. 5A). Elevated CO2 caused significant decline in Rd after 14 d under drought stress but had no effect on Rd under well-watered conditions at 25°C (Fig. 5A). Elevated temperature alone caused increases in Rd compared to the optimal temperature control at ambient CO2 over the treatment period, but Rd was not affected at elevated CO2 concentration except at 28 d (Fig. 5B). The combination of drought and elevated temperature increased Rd significantly at ambient CO2 concentration over the treatment period but did not significantly affect Rd at 7 and 21 d at elevated CO2 concentration (Fig. 5C).

DISCUSSION Significant interactive effects of temperature, CO2, and drought stress on leaf physiological activities were detected in tall fescue managed as turfgrass in our study. Overall, prolonged periods (28 d) of elevated temperature to 30°C (5°C above the optimal level of 25°C) had limited extent of adverse effects on tall fescue while drought stress alone for 28 d caused remarkable physiological damages. The combined drought and elevated temperature stress was even more detrimental than either stress alone, shown by the decline in photosynthetic activities (Fv:Fm and A) (Table 1; Fig. 3), water status (RWC) (Fig. 2), and cell membrane stability (increases in EL). Elevated CO2 mitigated these adverse effects, particularly for drought stress alone or 1852

Figure 2. Leaf relative water content (RWC) of tall fescue under drought stress (A), elevated temperature (B), and combined drought and heat stress (C) at two CO2 levels (400 and 800 μmol mol−1). The treatment symbols are 25-800-W for optimal temperature, well-watered, and elevated CO2, 25-400-W for optimal temperature, well-watered, and ambient CO2, 25-800-D for optimal temperature, drought stress, and elevated CO2, 25400-D for optimal temperature, drought stress, and ambient CO2, 30-800-W for elevated temperature, well-watered, and elevated CO2, 30-400-W for elevated temperature, well-watered, and ambient CO2, 30-800-D for elevated temperature, drought stress, and elevated CO2, and 30-400-D for elevated temperature, drought stress, and ambient CO2. Vertical bars indicate LSD values (P ≤ 0.05) for the comparison of treatments.

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Table 1. Effects of two temperature regimes (25 and 30°C), two CO2 levels (400 and 800 μmol mol−1), and water availability (wellwatered and drought-stressed plants) on electrolyte leakage (EL), photochemical efficiency of photosystem II (ratio of variable to maximum fluorescence [Fv:Fm]), maximum carboxylation rate (maximal ribulose-1,5-bisphosphate carboxylase oxygenase [Rubisco]-limited rate of photosynthesis [Vcmax]), and maximal electron transport-limited rate of photosynthesis (Jmax) at 7 and 28 d of treatments. Treatments Days 7

Water well-watered

Temperature °C 25 30

drought

25 30

28

well-watered

25 30

drought

25 30

Physiological parameters EL

CO2 −1

μmol mol 400 800 400 800 400 800 400 800 400 800 400 800 400 800 400 800

Fv:Fm

% 8.27 ± 0.7 aA† 10.04 ± 0.9 aA 8.69 ± 0.9 aA 10.15 ± 0.4 aA 9.21 ± 0.4 bA 11.27 ± 0.5 aA 9.94 ± 0.7 abA 11.54 ± 0.4 aA 8.34 ± 0.2 bB 12.71 ± 0.9 aB 11.41 ± 0.6 aB 12.46 ± 0.8 aB 29.84 ± 1.2 bA 18.25 ± 1.1 cA 39.83 ± 1.6 aA 31.360.8 bA

Vcmax

Jmax −2

0.80 aA 0.80 aA 0.79 aA 0.79 aA 0.80 aA 0.78 aA 0.79 aA 0.79 aA 0.80 aA 0.78 aA 0.79 aA 0.79 aA 0.63 ± 0.1 bB 0.78 aA 0.60 ± 0.1 bB 0.40 cB

–––––––––––μmol m 36.79 ± 0.6 aB 30.99 ± 3.5 aA 34.95 ± 4 aA 32.53 ± 1.8 aA 39.72 aA 32.11 ± 5.3 aA 29.54 ± 2.1 aA 32.97 ± 1.6 aA 42.43 aA 42.25 ± 10.7 aA 34.09 ± 2.1 aA 39.08 ± 3.1 aA 25.62 bB 27.49 aA 15.92 cB 14.66 dB

−1

s ––––––––––– 83.27 ± 1.6 aA 81.07 ± 11 aA 86.07 ± 8.6 aA 71.97 ± 3.2 aA 84.76 aA 78.1 ± 12.8 aA 71.64 ± 6.3 aA 72.18 ± 0.4 aA 91.97 aA 83.89 ± 8.2 aA 82.16 ± 1.1 aA 89.64 ± 5.4 aA 63.80 aB 61.41 bA 46.52 cB 40.55 dB

†

The different small letters indicate significant differences (P < 0.05) between treatments as determined by LSD at 7 and 28 d. The capital letters mean significant differences (P < 0.05) between well-watered and drought treatments at a given level of temperature and CO2.

elevated temperature alone, relative to plants grown under the ambient CO2 level. The CO2–induced alleviation of injury from drought alone or elevated temperature alone and the combined stresses are discussed below.

Physiological Effects and CO2 Mitigation of Elevated Temperature Stress Elevated temperature alone (30°C) had no significant effects on RWC, Fv:Fm, A, Vcmax, and Jmax, but prolonged periods of exposure (28 d) to high temperature resulted in increases in EL (37%) after 28 d of treatment at ambient CO2 level and Rd (47%) during the entire treatment period as well as a transient rise in gs at 7 and 14 d of treatment. The differential responses of physiological parameters to elevated temperature alone suggested that this level of temperature (5°C above the optimal temperature) was more detrimental to EL, gs, and Rd than to water status and photosynthesis in tall fescue. Previous studies in other plant species have also reported that EL, gs, and Rd were sensitive to increasing temperatures. Du et al. (2009) reported that EL was increased 10 times that of the control level following exposure to 34°C in tall fescue, suggesting that high temperature may cause significant decline in cellular membrane stability. The transient increase in gs under elevated temperatures could be reflective of rapid stomatal response to changing temperatures but was not enough to cause changes to leaf photosynthesis. Respiration is a major avenue of C loss for plants and increased respiration rate can cause carbohydrate CROP SCIENCE, VOL. 52, JULY– AUGUST 2012

depletion especially with increasing temperatures (Lambers et al., 1999). Increases of Rd in response to elevated temperature have been reported in various plant species (Cowling and Sage, 1998; Rodríguez-Calcerrada et al., 2010), including tall fescue (Volenec et al., 1984). Tall fescue genotypes with higher Rd had lower carbohydrate accumulation under elevated temperatures (Volenec et al., 1984). The suppressive effects of elevated CO2 on Rd under elevated temperature conditions may therefore reduce consumption of carbohydrates, which may be used as C reserves to support plant growth in a short term. Elevated CO2 offset the physiological inhibition induced by elevated temperatures in tall fescue in this study by reducing g s (20%), Rd (7%), and EL. Similar results have been reported in other plant species. Faria et al. (1996) observed that elevated CO2 led to the reduction in g s under elevated temperature by 5°C above 25°C in cork oak (Quercus suber L.) seedlings. Elevated CO2 suppressed the increase of Rd in various plant species (Idso and Kimball, 1992; Drake et al., 1997). The specific mechanisms CO2 may reduce EL on elevated temperature stress are not clear, but the protective effect of elevated CO2 in membrane stability under salinity stress has been attributed to enhanced antioxidant capacity (Pérez-López et al., 2009). Elevated CO2 may mitigate the negative impacts of elevated temperature in tall fescue through the suppression of water loss, change in C balance, and enhanced membrane stability.

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Figure 3. Leaf net photosynthetic rate (A) of tall fescue under drought stress (A), elevated temperature (B), and combined drought and heat stress (C) at two CO2 levels (400 and 800 μmol mol−1). The treatment symbols are 25-800-W for optimal temperature, well-watered, and elevated CO2, 25-400-W for optimal temperature, well-watered, and ambient CO2, 25-800D for optimal temperature, drought stress, and elevated CO2, 25-400-D for optimal temperature, drought stress, and ambient CO2, 30-800-W for elevated temperature, well-watered, and elevated CO2, 30-400-W for elevated temperature, well-watered, and ambient CO2, 30-800-D for elevated temperature, drought stress, and elevated CO2, and 30-400-D for elevated temperature, drought stress, and ambient CO2. Vertical bars indicate LSD values (P ≤ 0.05) for the comparison of treatments.

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Figure 4. Leaf stomatal conductance (gs) of tall fescue under drought stress (A), elevated temperature (B), and combined drought and heat stress (C) at two CO2 levels (400 and 800 μmol mol−1). The treatment symbols are 25-800-W for optimal temperature, well-watered, and elevated CO2, 25-400-W for optimal temperature, well-watered, and ambient CO2, 25-800D for optimal temperature, drought stress, and elevated CO2, 25-400-D for optimal temperature, drought stress, and ambient CO2, 30-800-W for elevated temperature, well-watered, and elevated CO2, 30-400-W for elevated temperature, well-watered, and ambient CO2, 30-800-D for elevated temperature, drought stress, and elevated CO2, and 30-400-D for elevated temperature, drought stress, and ambient CO2. Vertical bars indicate LSD values (P ≤ 0.05) for the comparison of treatments.

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Physiological Effects and CO2 Mitigation of Drought Stress

Figure 5. Leaf dark respiration rate (Rd) of tall fescue under drought stress (A), elevated temperature (B), and combined drought and heat stress (C) at two CO2 levels (400 and 800 μmol mol−1). The treatment symbols are 25-800-W for optimal temperature, wellwatered, and elevated CO2, 25-400-W for optimal temperature, well-watered, and ambient CO2, 25-800-D for optimal temperature, drought stress, and elevated CO2, 25-400-D for optimal temperature, drought stress, and ambient CO2, 30-800W for elevated temperature, well-watered, and elevated CO2, 30400-W for elevated temperature, well-watered, and ambient CO2, 30-800-D for elevated temperature, drought stress, and elevated CO2, and 30-400-D for elevated temperature, drought stress, and ambient CO2. Vertical bars indicate LSD values (P ≤ 0.05) for the comparison of treatments.


The decreases in photosynthesis under drought stress may be attributed to both stomatal and nonstomatal (metabolic) limitation (Long et al., 2004; Hu et al., 2010). The former is usually induced by stomatal closure (Escalona et al., 1999) and the latter is caused by metabolic impairment, such as reduced photochemical and carboxylation efficiency (Flexas and Medrano, 2002; Long et al., 2004). In our study, drought stress caused gs to decrease on average 38% compared to that under well-watered conditions, which was associated with the decrease in A (34%) observed. The decrease in A was also due to metabolic factors, including the reduction in Vcmax (37%), Jmax (29%), and Fv:Fm (11%) and the increase of EL (128%). Reduction in Fv:Fm and the increased EL induced by drought stress have been widely reported in various turfgrass species (Merewitz et al., 2010; Chai et al., 2010). Significantly lower Vcmax at ambient CO2 concentration during drought stress compared to wellwatered conditions suggests that carboxylation efficiency might be severely limited by drought treatment, as reported in other plant species (Flexas et al., 2004; Hu et al., 2010). Reduced Jmax has been associated with a decline in ribulose1,5-bisphosphate (RuBP) regeneration capacity, which may be caused by lack of adenosine triphosphate (ATP) or nicotinamide adenine dinucleotide phosphate supply as well as lower activity of enzymes involved in RuBP regeneration, such as fructose-2,6-bisphosphatase, 3-phosphoglycerate kinase, and glyceraldehyde-3-phosphate dehydrogenase (Maroco et al., 2002; Flexas et al., 2004; Hu et al., 2010). Although not measured in this study, it is reasonable to assume that changes in these parameters could be related to drought-induced decline in A in tall fescue. In this study, elevated CO2 mitigated the negative influence of drought on photosynthetic capacity by promoting both gs and metabolic activities. Under elevated CO2 concentration at 28 d, Vcmax (10%) and gs (238%) of drought-stressed plants at 25°C was maintained at a higher level compared to plants under ambient CO2. In addition, Fv:Fm at elevated CO2 at 28 d was significantly higher (24%) than at ambient CO2 under drought conditions at 25°C, indicating that elevated CO2 could enhance photochemical efficiency of the photosystem II (Hamerlynck et al., 2000; Aranjuelo et al., 2005). Carbon dioxide mitigation of drought-induced decline in A (on average 307%) has been reported in various other plant species such as myrtle oak (Quercus myrtifolia Willd.) in open-top chambers (Li et al., 2007) and rice (Oryza sativa L.) in a controlledenvironment chamber experiment (Widodo et al., 2003). Drought-stressed plants of tall fescue under elevated CO2 also maintained higher RWC (21%) and lower EL (30%). The maintenance of higher RWC in plants exposed to elevated CO2 has been associated with enhanced osmotic adjustment and slower water depletion from the soil in other

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plant species (Clifford et al., 2000; Hamerlynck et al., 2000; Qaderi et al., 2006; Kirkham, 2011). Results showing that elevated CO2 inhibits water loss from plants and increases water use efficiency have also been reported by many studies (Chaudhuri et al., 1990; Clifford et al., 2000; Qaderi et al., 2006; Robredo et al., 2007). Although it is not clear how tall fescue under elevated CO2 may maintain higher RWC, deep rooting is an important trait controlling water uptake and use. Therefore, it would be interesting to determine whether improved leaf water status is associated with better root growth in future studies. Drought stress increased Rd (24%) in tall fescue under ambient CO2, which may cause increased carbohydrate consumption and eventually a depletion of carbohydrates, particularly when photosynthesis was greatly inhibited. The increase in Rd under drought treatment may be governed by numerous factors, such as the increased ATP demand associated with protein turnover and ion transport, repairing cell damage, and increased wastage respiration (Stocker, 1961; Mooney, 1969; Zagdańska, 1995; Slot et al., 2008; Metcalfe et al., 2010). It has been previously reported that Rd decreases about 20% by doubling ambient CO2 concentration and plants tend to become more efficient in C usage (Drake et al., 1997). In this study, elevated CO2 suppressed the increase in Rd (27%) under drought stress, which could contribute to better drought tolerance as it relates to carbohydrate availability.

Physiological Effects and CO2 Mitigation of the Combined Stress of Drought and Elevated Temperature The combined drought and elevated temperature had more detrimental effects than either stress alone under ambient CO2 conditions, which is in agreement with previous studies in tall fescue and other grass species such as perennial ryegrass (Lolium perenne L.) and Kentucky bluegrass (Poa pratensis L.) (Jiang and Huang, 2001a, 2001b). Elevated CO2 enhanced RWC (19%) and A (162%) and reduced EL (21%), providing evidence for the mitigating effects on photosynthesis, plant water retention, and membrane stability, particularly following prolonged periods of treatment. However, Fv:Fm (33%), Vcmax (8%), and Jmax (13%) all declined further under the combination of elevated CO2, drought and elevated temperature, indicating that long-term simultaneous drought and elevated temperature together causes irreversible physiological damage in tall fescue and elevated CO2 may have acted as an additional stress in this case. Hamerlynck et al. (2000) also found that elevated CO2 of 550 μmol mol−1 induced lower Fv:Fm than a control (360 μmol mol−1) under the combined drought and heat conditions in an evergreen shrub [Larrea tridentata (DC.) Coville]. The reduction in Vcmax under elevated CO2 at elevated temperature and drought stress combined reported by Aranjuelo et al. (2005) in nodulated alfalfa (Medicago sativa L.) was attributed to a decline in carboxylation 1856

efficiency due to inhibition of Rubisco activity. The specific mechanisms of how elevated CO2 in combination with drought and elevated temperature affect photochemical efficiency and carboxylation capacity in perennial grasses deserve further investigation. Limited information is available as to the biochemical factors regulated by the three-way interaction of CO2, temperature, and drought stress. Such information is particularly important for improving plant performance under the environment with all three factors occurring simultaneously due to continued global warming and climate changes. Acknowledgments The authors wish to thank the Chinese Scholarship Council, Rutgers Center of Turfgrass Science, and the National Special Research Program on Public Welfare in Forestry (grant number 200804022) for funding support. Thanks also go to Emily Merewitz, Patrick Burgess, and David Jespersen for critical review of the manuscript.

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