Effects of extreme high temperature, drought and elevated CO2 on ...

Plant Ecology 148: 183–193, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.

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Effects of extreme high temperature, drought and elevated CO2 on photosynthesis of the Mojave Desert evergreen shrub, Larrea tridentata Erik P. Hamerlynck1,2 , Travis E. Huxman1, Michael E. Loik3 & Stanley D. Smith1 1 Department

of Biological Sciences, University of Nevada, Las Vegas, NV 89154, USA (e-mail: [email protected]); 2 Department of Biological Sciences, Rutgers University, Newark, NJ 07012, USA; 3 Department of Environmental Studies, University of California, Santa Cruz, CA 95064, USA Received 13 October 1999; accepted in revised form 14 December 1999

Key words: Chlorophyll fluorescence, Drought, Elevated CO2 , High temperature, Larrea tridenata, Photosynthesis

Abstract The interaction of extreme temperature events with future atmospheric CO2 concentrations may have strong impacts on physiological performance of desert shrub seedlings, which during the critical establishment phase often endure temperature extremes in conjunction with pronounced drought. To evaluate the interaction of drought and CO2 on photosynthesis during heat stress, one-year-old Larrea tridentata [DC] Cov. seedlings were exposed to nine days of heat with midday air temperature maxima reaching 53 ◦ C under three atmospheric CO2 concentrations (360, 550 and 700 µmol mol−1 ) and two water regimes (well-watered and droughted). Photosynthetic gas exchange, chlorophyll fluorescence and water potential responses were measured prior to, during and one week following the high temperature stress event. Heat stress markedly decreased net photosynthetic rate (Anet), stomatal conductance (gs ), and the photochemical efficiency of photosystem II (Fv /Fm ) in all plants except for well-watered L. tridentata grown in 700 µmol mol−1 CO2 . Anet and gs remained similar to pre-stress levels in these plants. In droughted L. tridentata, Anet was ca. 2× (in 550 µmol mol−1 CO2 ) to 3× (in 700 µmol mol−1 CO2 ) higher than in ambient-CO2-grown plants, while gs and Fv /Fm were similar and low in all CO2 treatments. Following heat stress, gs in all well-watered plants rose dramatically, exceeding pre-stress levels by up to 100%. In droughted plants, gs and Anet rose only in plants grown at elevated CO2 following release from heat. This recovery response was strongest at 700 µmol mol−1 CO2 , which returned to Anet and gs values similar to pre-heat following several days of recovery. Extreme heat diminished the photosynthetic down-regulation response to growth at elevated CO2 under well-watered conditions, similar to the action of drought. Ambient-CO2-grown L. tridentata did not show significant recovery of photosynthetic capacity (Amax and CE) after alleviation of temperature stress, especially when exposed to drought, while plants exposed to elevated CO2 appeared to be unaffected. These findings suggest that elevated CO2 could promote photosynthetic activity during critical periods of seedling establishment, and enhance the potential for L. tridentata to survive extreme high temperature events.

Introduction Of all terrestrial ecosystems, arid lands are predicted to be the most responsive to increasing atmospheric CO2 due to the impacts of increased water-use efficiency on productivity (Melillo et al. 1993), and the general alleviation of stress on seedling establishment and survival (Smith et al. 1997). Concurrent with increased CO2 levels, annual mean temperatures are expected to increase by 1.5 to 4 ◦ C (Watson et al. 1990),

potentially causing shifts in the distribution and abundance of key species (Peters 1992). Detailed modeling efforts have shown that minor increases in global mean temperature could markedly increase the severity and frequency of regional temperature extremes (Wagner 1996). Abiotic extremes have strong impacts on seedling establishment in aridland systems, and the interaction of temperature extremes and unpredictable, protracted drought induce episodic seedling mortality that can be a strong determinant of community struc-

184 ture and function (Jordan & Nobel 1979; Rundel & Gibson 1996; Pockman & Sperry 1997). This is especially true for long-lived desert species, which are highly susceptible to physical stresses during seedling establishment (Smith et al. 1997). How future elevated CO2 levels may interact with extreme abiotic events to affect seedling establishment of long-lived desert species is unknown. While much research has shown that elevated CO2 and increased elevated mean growth temperature can increase productivity in many plants (Chen et al. 1994; Morgan et al. 1994; Hunt et al. 1996; Read & Morgan 1996), studies addressing the interactive effects of temperature extremes and elevated CO2 have yielded contrasting results (Coleman et al. 1991; Long 1991; Bassow et al. 1994; Roden & Ball 1996a,b). Long (1991) postulated that elevated CO2 would result in increased assimilation rates and carbon pools that would provide a dissipation pathway for excess light energy, thereby reducing photoinhibitory effects and increasing productivity under high temperature. This was supported by Bassow et al. (1994), who showed that elevated CO2 proportionally stimulated more biomass production in tree seedlings exposed to a 45 ◦ C temperature transient compared to control plants, despite a 60% reduction of stomatal conductance. However, Roden & Ball (1996a,b) found that Eucalyptus species showing pronounced photosynthetic down-regulation under elevated CO2 experienced a stronger midday depression of photosynthesis, but this was reduced under water-limited conditions. Consistent with Roden & Ball (1996a,b), application of a heat shock (up to 49 ◦ C) did not result in disproportionate changes in biomass accumulation for Abutilon theophrasti, Sinapsis alba, or Amaranthus retroflexus with respect to growth differences between ambient and elevated CO2 under normal temperature regimes (Coleman et al. 1991). These studies have assessed the responses of broad-leaved, relatively mesophytic species from fairly resource-rich habitats, making extrapolation to xeromorphic desert species from relatively resourcepoor environments tenuous (Chapin et al. 1993). In this regard, the response of small-leaved desert species to these extreme abiotic events under future elevated CO2 conditions may provide additional information into the mechanistic responses of plants to environmental stress. Here we present a study assessing the response of photosynthesis and water relations to a nine-day high temperature (3–4 h at 53 ◦ C daily maximum) treatment of the dominant evergreen shrub of the Mo-

jave Desert, Larrea tridentata [DC] Cov, exposed to chronic drought and 3 levels of atmospheric CO2 . As elevated CO2 usually improves the water status of plants, a result of lower stomatal conductance and transpiration (Field et al. 1995), elevated CO2 was expected to improve plant performance during and following exposure to extreme high temperature as compared to an ambient CO2 growth environment. During drought, this may mean L. tridentata could maintain physiological activity under high temperatures that would otherwise induce strong stomatal and nonstomatal limitations to photosynthesis (Mooney et al. 1978; Long 1991; Giardi et al. 1996; Hamerlynck & Knapp 1996). In addition, L. tridentata has strong seasonal acclimatory responses to high temperature and water deficit, resulting in a remarkable tolerance to temperature extremes (Mooney et al. 1977, 1978; Seeman et al. 1984; Smith et al. 1997). Given that drought diminishes photosynthetic down-regulation in L. tridentata under elevated CO2 (Huxman et al. 1998a), understanding the interaction of non-stomatal and stomatal responses to varying watering regimes and temperature extremes may have important implications for this important component of North American warm deserts under future atmospheric conditions.

Materials and methods Growth facility and conditions One-month-old seedlings of Larrea tridentata were transplanted into large pots (0.15 m dia. by 1 m tall), and exposed to ambient, 550 and 700 µmol mol−1 atmospheric CO2 in a climate-controlled glasshouse facility at the University of Nevada, Las Vegas for one year prior to experimentation. Climate control towers maintained temperature in the 550 and 700 µmol mol−1 glasshouse bays within ±5% of those in the ambient CO2 bay. Temperature and humidity was recorded every minute with a Campbell CR-21X datalogger (Campbell Instruments, Logan, UT, USA) using a shaded type-T (copper/constantan) thermocouple and a Vaisala Humicap resistance hygrometer suspended 2 m above the floor. Humidity was not controlled, and ranged from ca. 60% to 10% from early morning to late afternoon, respectively, and varied ±2% between each bay. Temperature differences between the bays never exceeded 1 ◦ C. The glasshouse utilized natural light (maximum photosynthetic photon flux densities (PPFD) of

185 1600 µmol m−2 s−1 ), and followed external temperature conditions, with maximum and minimum temperature cut-offs of 45◦ and 0 ◦ C, respectively, to reduce transplant mortality. CO2 set-point was maintained within ±5% by measuring CO2 every minute with an infrared gas analyzer (Model 6252; LiCOR Inc., Lincoln, NE, USA) under computer control, and injecting CO2 into the air-circulation system as needed. Single bays were used to insure that identical environmental conditions existed between CO2 treatments. Soil consisted of an 80:20 mix of commercially obtained sand and silt. Volumetric soil moisture (θ ) was measured weekly at 10 and 50 cm depths using horizontally set TDR probes. There were no detectable differences in θ between CO2 treatments. Prior to imposing drought, plants were supplied every 2 weeks with 400 ml of 10% strength Hoagland’s solution. Three months before sampling, watering for plants marked for drought treatment was completely curtailed. θ ranged between 0.05 and 0.07 in well-watered soils, but was often below the resolution capability of the TDR system (minimum θ ca. 0.02) in drought treatments, making reliable estimation of θ difficult. All plants were exposed to a nine-day high temperature treatment in September 1997. Varying accessory evaporative cooler input over the course of the day increased midday maximum temperature, while circulation was maintained by running all fans. Reducing the relative cooler inputs starting at 1000 h PDT led to a 2 ◦ C d−1 increase for four days from a maximum of 45◦ to a high of 53 ◦ C, which was achieved over five days. The temperature in the glasshouse followed a normal increase from overnight lows of 28 ◦ C, reaching and maintaining the maximum temperature from 1200 to 1600 h, after which the air-cooling system was reactivated, and the glasshouse was allowed to cool to the overnight low. VPD was allowed to increase with the higher ambient temperatures. After the ninth day, the pre-heat maximum temperature of 45◦ was reestablished. There were no detectable changes in soil moisture at the depths measured during the heat stress event in either watering treatment. Midday leaf ψ, gas exchange, and Fv /Fm Since we were interested in the performance of L. tridentata under maximally stressful conditions, midday water potential (ψ) was estimated using a Scholander type pressure chamber (Soil Moisture Stress, Inc., Santa Barbara, CA, USA). Stem ψ from four L. triden-

tata from each CO2 × drought treatment combination were taken 3 days prior to temperature stress (‘Prestress’), at the last day of the temperature stress period (‘Peak-stress’), and 11 days following cessation of the high temperature treatment (‘Post-stress’). Three days before heat stress, midday (ca. 1130 to 1430 h) measurements of net CO2 assimilation rate (Anet ) and stomatal conductance to water vapor (gs ) were made on four plants per CO2 × drought treatment using an open-flow portable photosynthesis system (Li-6400, LiCOR Inc., Lincoln, NE, USA). All measurements were made under saturating light (1500 µmol m−2 s−1 photosynthetic photon flux density, PPFD) and treatment CO2 concentrations, which were achieved by mixing CO2 from an internal source with ambient air. Due to the small size of individual leaves, two to three leaflets were used to ensure a strong gas exchange signal. For pre- and post-stress measurements, the block temperature of a Peltierthermoelectric block attached to the cuvette was set to 30 ◦ C and chamber humidity from 25 to 38%. This resulted in leaf temperatures close to external conditions, and leaf-to-air vapor pressure deficits (VPDl ) of 2.6 to 2.9 kPa. During the heat stress period, the cuvette temperature was set to 45 ◦ C, with relative humidity ranging between 19 and 25%, resulting in leaf temperatures ca 1–2 ◦ C cooler than ambient and VPDl of 3.3 to 3.8 kPa. This temperature was used to avoid unstable and excessive leaf temperatures resulting from the introduction of hot external air used to set cuvette [CO2 ] and heat generated by the LED light source apparent at higher cuvette temperatures (data not shown). In-situ chlorophyll fluorescence was measured in three L. tridentata per treatment combination in conjunction with gas exchange measurements. Entire seedlings were dark adapted for one hour by an aluminum foil cover. Fluorescence was determined using a pulse-amplitude-modulated (PAM) fluorimeter (Model MFMS/2S, Hansatech Instruments, UK) under PC control (Minirec v 2.1, Hansatech Instruments, Ltd.). F0 was established with a low light source at 583 nm light at 2 µmol m−2 s−1 PPFD, with a demodulator gain of 80 and a response time of 0.1 s, and was averaged over 30 s. Maximal fluorescence (Fm ) was determined by using 0.5 s pulses of saturating (8500 µmol m−2 s−1 PPFD) pulse-modulated light at a frequency of 1 Hz supplied by fiber optic cable carrying light from a Hansatech FLS1 light source. The photochemical efficiency of photosystem II (Fv /Fm ) was calculated as: Fv /Fm = (Fm − F0 )/Fm . All water

186 relations, photosynthetic gas exchange, and chlorophyll fluorescence measurements were made on shoots produced prior to the heat treatment. Photosynthetic down-regulation Two weeks prior to temperature treatment, response curves of net photosynthesis to internal CO2 concentration (A/Ci ) were made on well-watered and droughted L. tridentata from the three CO2 treatments. To assess longer-term impacts of heat and drought stress, four weeks after the relaxation of high temperature and three days following a 1-l watering, A/Ci measurements were repeated on well-watered and drought-treated plants. This watering did not produce new growth in any of the CO2 /watering treatments, with the exception of ambient CO2 /droughted plants, which displayed considerable leaf shedding and production. Three plants in each CO2 /drought treatment were sampled using the Li 6400 photosynthesis system. Cuvette temperature set to 30 ◦ C, resulting in leaf temperatures of 29–32 ◦ C and VPDl of 2.8–3.0 kPa, under saturating PPFD (1500 µmol m−2 s−1 ). Two to three leaflets were enclosed in the cuvette for each curve. Changes in Ci were achieved by changing cuvette [CO2 ] by mixing CO2 from an internal source with ambient air. A nonlinear least-squares regression (Sigmaplot v4.0, SPSS), using the exponential model of Jacob et al. (1995) was used to estimate Ci saturated rate of photosynthesis (Amax ) and the initial linear slope of the curve, an indicator of the carboxylation efficiency (CE). Statistical analyses A split-plot, repeated measures three-way ANOVA (Statistix v 4.0, Analytical Software, St. Paul, MN, USA) was used to test for significant interactive effects of CO2 , drought and high temperature treatment on Anet , gs , and Fv /Fm through the time course of the high temperature period. This model was used because the experimental units differed between CO2 , watering, and temperature treatments. CO2 and day of sampling were the whole plot factors (3 − 1 = 2 df and 7 − 1 = 6 df, respectively), with the CO2 × sampling day interaction as the whole plot error term (12 error df). The sub-plot factors were watering treatment and all remaining two-way and three-way interactions. For ψ data, the sub-plot error structure differed in that time was broken down into three distinct periods (pre-heat, peak-heat and post-heat). Of specific interest was the three-way interaction of CO2 × drought

× time, which would show how the interactive effects of CO2 concentration and drought behaved differently through a time period before, during and after the high temperature event. The sub-plot error term was the CO2 × drought × time × replicate plant interaction. Post-hoc general linear contrasts (Scheffe’s F ) were used to test the significance of contrasts that might contribute to any significant interaction effects. An α of 0.05 was considered significant in all analyses. All fluorescence data were arcsine transformed to meet ANOVA data distribution assumptions (Zar 1974). Three-way ANOVA was used to test for significance of CO2 concentration, drought, and pre- and post-high-temperature exposure effects on the A/Ci derived parameters of Amax and CE, with an α of 0.05 considered significant. Means separation was made using Scheffe’s pairwise tests. Post-hoc general linear contrasts (Scheffe’s F ) were made on specific contrasts that might contribute to any significant interaction effects. Of specific interest were the two-way (CO2 × drought, CO2 × pre/post temperature, and drought × pre/post temperature exposure) and the three-way (CO2 × drought × pre/post temperature exposure) interactions. Throughout, data are presented as means ± one standard error.

Results Midday leaf ψ, gas exchange and Fv /Fm Midday leaf water potential (ψ) was similar between all CO2 treatments in well-watered plants (ca. −2.7 MPa), while ψ in droughted plants was lowest in ambient CO2 (−5.9 MPa) compared to elevated CO2 treatments (−4.6 and −3.4 MPa for 550 and 700 µmol mol−1 CO2 , respectively; Figure 1). There were no significant differences in plant ψ between well-watered and droughted L. tridentata at the highest CO2 treatment, regardless of time period (Figure 1), resulting in a significant two-way interaction between CO2 and watering treatments (F = 6.74; P ≤ 0.05; df = 2, 45). Comparing pre- and post-heat periods, only L. tridentata growing in ambient CO2 showed any significant reduction in ψ at the end of the heat treatment (Figure 1), giving a CO2 by time interaction (F = 3.16; P ≤ 0.05; df = 4, 45). A significant watering × time interaction (F = 4.25; P ≤ 0.05; df = 2, 45) was due to ψ in well-watered plants being similar between pre-heat and peak-heat periods (−3.2 and −2.8 MPa, respectively), followed by an

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Figure 1. Midday water potential (ψ) of Larrea tridentata growing under two watering regimes, three atmospheric CO2 concentrations, prior to, during and following a nine day high temperature transient of 53 ◦ C. Each bar is the mean of four measurements, with ± one S.E. of the mean. General linear contrasts contributing to two-way interactions are: ∗ differences between well-watered and droughted plants within a CO2 treatment and time period; lower case letters indicate differences between CO2 treatments within a watering regime and time period; superscripted lower case letters indicate differences between time periods within a CO2 /watering regime. Mean separation main effects are indicated by subscripts following panel or axis labels.

increase after alleviation of heat stress (−1.8 MPa). Droughted L. tridentata on the other hand had similar pre- and post-heat ψ (−4.4 and −4.2 MPa, respectively), which were both higher than ψ measured on the last day of heat treatment (−5.3 MPa; Figure 1). Net photosynthesis (Anet) increased significantly with growth CO2 concentration in L. tridentata (F = 43.0; P < 0.05; df = 2, 12). Pooled across the experiment, Anet in L. tridentata grown at 700 µmol mol−1 CO2 (14.1 µmol m−2 s−1 ) was significantly higher than 550 µmol mol−1 (9.7 µmol m−2 s−1 ), which in

turn was significantly higher than for plants exposed to ambient CO2 (7.4 µmol m−2 s−1 ). Drought reduced Anet significantly throughout the experiment (F = 197.6; P ≤ 0.05; df = 1, 126), declining to only 36% (5.5 µmol m−2 s−1 ) of the value observed in wellwatered plants (15.3 µmol m−2 s−1 ). As expected, a significant three-way interaction of CO2 level, watering, and time occurred (F = 2.3; P ≤ 0.05; df = 12, 126), reflecting the treatment-specific effect of the imposition and release from the high temperature treatment (Figure 2). This interaction probably occurred through the maintenance of Anet throughout the extreme high temperature event or recovery of Anet upon release from heat stress in L. tridentata at the highest CO2 concentration (Figure 2). A three-way interaction between CO2 , drought and time also significantly influenced stomatal conductance (gs ) in L. tridentata (F = 2.6; P ≤ 0.05; df = 12, 126). However, unlike Anet responses, gs in all CO2 /watering combinations initially increased from high-temperature to post-temperature recovery (Figure 2). During the high-temperature transient, gs in well-watered L. tridentata grown at 700 µmol mol−1 did not decline from pre-heat levels, as compared to ambient-grown and 550 µmol mol−1 counterparts. Under droughted conditions, gs in L. tridentata grown at 700 µmol mol−1 was higher, and displayed a more pronounced mid-recovery increase, than for droughted plants in the other two CO2 treatments (Figure 2). The photochemical efficiency of PS II (Fv /Fm ) did not respond significantly to the interactive effects of CO2 , drought and temperature, but rather showed a significant drought effect (F = 29.1; P ≤ 0.05; df = 1, 84) and two-way watering × time interaction (F = 5.4; P ≤ 0.05; df = 6, 84). Fv /Fm declined from 0.66 under well-watered to 0.57 under drought (Figure 2). The two-way interaction was likely due to the stronger positive post-temperature recovery apparent in well-watered L. tridentata, which exceeded pre-stress Fv /Fm by as much as 31% (Figure 2). In droughted conditions, post-heat, only L. tridentata exposed to 700 µmol mol−1 CO2 showed a recovery of Fv /Fm to pre-heat treatment levels, while plants exposed to 360 and 550 µmol mol−1 had up to 20% reductions from pre-heat levels. Photosynthetic down-regulation Elevated CO2 did not significantly alter Ci -saturated photosynthesis (Amax ) pooled across all watering/heat treatments, with no significant two-way interactions

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Figure 2. Time course of net photosynthetic rate (Anet ), stomatal conductance (gs ), and quantum efficiency of PS II (Fv /Fm ) of Larrea tridentata prior to, during, and following a nine day high temperature treatment, under well-watered and droughted conditions and three CO2 concentrations. Each point is the mean of four (gas exchange) and three (fluorescence) measurements; bars indicate ± one S.E. Solid bar indicates length of high temperature transient period. 9 indicate days when midday water potential measurements were made.

between CO2 concentration and drought or pre/posthigh temperature recovery. Amax was significantly reduced under drought (−34% compared to wellwatered plants; F = 13.4; P ≤ 0.05; df = 1, 24) and was 32% lower following high temperature treatment (F = 9.5; P ≤ 0.05; df = 1, 24; Figure 3). A significant two-way interaction in Amax between drought and pre- and post-high temperature periods (F = 30.3; P ≤ 0.05; df = 1, 24) was due to lower Amax in droughted plants compared to well-watered plants prior to heat stress (Figure 3; F = 14.1, 4.3 and 4.4, for 360, 550 and 700 µmol mol−1 CO2 treatments, respectively; df = 1, 24). Upon release from heat stress, there were no significant differences in Amax between well-watered and droughted plants (Figure 3). However, within well-watered L. tridentata, Amax declined significantly after recovery from heat stress across all

CO2 treatments (F = 8.7; P ≤ 0.05; df = 2, 24), but was similar in all droughted plants (Figure 3). Carboxylation efficiency (CE) reduced significantly in response to elevated CO2 (F = 5.4; P ≤ 0.05; df = 2, 24), drought (F = 6.6; P ≤ 0.05; df = 1, 24), and heat (F = 8.7; P ≤ 0.05; df = 1, 24; Figure 3). However, these responses were complicated by significant two-way interactions between drought and recovery period (F = 26.3; P ≤ 0.05; df = 1, 24) and a three-way interaction between CO2 , drought and pre/post heat stress (F = 10.8; P ≤ 0.05; df = 2, 24). The two-way interaction was likely due to well-watered L. tridentata having lower CE following temperature recovery (F = 32.64; P ≤ 0.05; df = 1, 24), while CE in droughted plants was constant across all CO2 treatments (Figure 3). Three factors could account for the significant three-

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Figure 3. Maximum photosynthetic capacity (Amax ) and carboxylation efficiency (CE) of Larrea tridentata prior to and following a nine day high temperature treatment, under three CO2 concentrations, and two watering treatments. Each bar is the mean of three measurements, error bars indicate ± one S.E. Significance of post-hoc general linear contrasts contributing to significant interactions are: ∗ difference between watering treatments within a CO2 treatment and temperature period, different lower case letters indicate CO2 effects within a watering treatment prior to and following temperature stress, capital letters over a bracket indicate CO2 effects pooled across watering treatments within a temperature period.

way interaction. First, prior to the heat treatment, well-watered L. tridentata had significantly lower CE under elevated CO2 compared to ambient-CO2-grown plants (F = 4.27; P ≤ 0.05; df = 1, 24), while droughted plants did not. After recovery from high temperature and drought, this pattern was reversed, with drought-treated L. tridentata showing a reduction in CE in response to CO2 , while well-watered plants did not. Second, when pooled across watering treatments, there was no significant effect of CO2 on CE in L. tridentata, but there was after release from drought and high-temperature treatments. Finally, differences in watering treatments were reversed within ambient-CO2-grown L. tridentata (Figure 3).

Discussion Elevated CO2 largely offset the interactive effects of drought and high temperature on water relations and photosynthesis in seedlings of Larrea tridentata, an evergreen shrub that is well adapted to water and heat stress (Mooney et al. 1977, 1978; Smith et al. 1997). Larrea tridentata under elevated CO2 did not show any change in midday ψ during heat stress, while ambient CO2 grown counterparts ψ were low (Figure 1). There is evidence that L. tridentata increases fine root mass under elevated CO2 (BassiriRad et al. 1997), which could elevate ψ, but this does not always occur. Indeed, Huxman et al. (1999) found that L. tridentata did not increase total root mass, root/shoot ratio, or

190 root hydraulic conductivity. Thus, the improved water status under elevated CO2 was likely due to increased soil water availability resulting from lower gs and transpiration (Field et al. 1995). The lack of differences in θ in this study and Huxman et al. (1999) suggest that ψ in coarse-grained soils could be highly sensitive to minor changes in θ (Nobel 1991), but without predawn water potential data this remains unresolved. Heat stress reduced subsequent realized photosynthetic rates (Anet) in L. tridentata following the return to lower temperature. How elevated CO2 altered these reductions in Anet depended on watering treatment (Figure 2). The reduced Anet realized 14 d after heat stress in droughted plants under elevated CO2 were not likely the result of thermal damage, since Amax and CE were already low and did not further decrease after heat stress (Figure 3). Thus, the similar Anet apparent in plants in both elevated CO2 treatments under drought was likely due to stronger stomatal limitations in response to water deficit. In contrast, reduced Anet in well-watered plants could well have been an increase in combined stomatal (Figure 2) and non-stomatal limitations (Figure 3). The heat stress imposed in this experiment may have caused some degree of thermal acclimation (Mooney et al. 1978; Seemann et al. 1984), as evidenced by the slight recovery of Anet apparent on the last day of heat stress in most CO2 /watering treatments (Figure 2). Indeed, the Anet rates in ambient CO2 were comparable to those reported for plants acclimated to summertime conditions (Mooney et al. 1978). The reductions in gs during heat stress in most treatments were likely a result of increasing VPDl , not direct effects of high temperature per se (Mooney et al. 1978; Meinzer et al. 1990). It is interesting to note that gs in well-watered L. tridentata under 700 µmol mol−1 CO2 was not reduced during heat stress, even though VPDl did increase. Given that photosynthetic capacity was reduced by heat stress (Figure 3), it might be that a combination of favorable water status and high CO2 availability did result in L. tridentata eliciting a stomatal stress response (Chapin et al. 1993). Indeed, under drought conditions, gs and Anet in L. tridentata grown at 700 µmol mol−1 CO2 followed trends similar to those in well-watered 550 µmol mol−1 and ambient-CO2grown plants (Figure 2). The striking rise in stomatal conductance in L. tridentata upon release from heat stress is unique and, to our knowledge, unreported. Past research has shown that many woody plants reduce stomatal conductance

during, and for long periods following temperature extremes (Bassow et al. 1994; Kozlowski et al. 1994; Hamerlynck & Knapp 1996; Roden & Ball 1996b). However, these studies have examined broad-leaved species, which often rely on evaporative cooling to maintain leaf temperatures near air temperature (Kozlowski et al. 1994). With such leaf morphology, long-term reductions in gs could result in leaf temperatures more frequently exceeding air temperatures (Nobel 1991). This, coupled to any alterations in carbon metabolism, might reduce the ability of broad-leaved evergreens to dissipate excess light energy under elevated CO2 (Roden and Ball 1996a,b; Hymus et al. 1999). We found little evidence that elevated CO2 exacerbated the effects of heat related photo-oxidative stress in the microphyllous L. tridentata (Figure 2). Fv /Fm more closely followed changes in water availability and temperature, a common response in some plants (Giardi et al. 1996; Loik & Harte 1996), and was only slightly influenced by elevated CO2 in L. tridentata. Indeed, in drought-stressed L. tridentata, Fv /Fm was markedly higher under elevated CO2 , and 8 d after the release from heat stress, were highest in plants growing in twice-ambient CO2 concentrations (Figure 2). These findings support assertions that elevated CO2 can result in changes that enhance the dissipation of excess light energy (Long 1991; Huxman et al. 1998b; Hymus et al. 1999). The pronounced and rapid opening of stomata in L. tridentata may be a stress recovery mechanism in aridland microphylls. Microphyllous leaves maintain leaf temperature near air temperature primarily via convection, not transpiration (Gibson 1996). By reducing any stomatal limitations to photosynthesis upon release from stress, L. tridentata may facilitate rapid recovery from high-temperature stress. This contrasts with findings in woody broadleaved species, which commonly have prolonged periods (>7 d) of stomatal closure immediately following heat stress (Bassow et al. 1994; Hamerlynck & Knapp 1996). However, the broadleaf species in these studies likely had lower temperature tolerances compared to L. tridentata, and the prolonged reduction in gs in these were a consequence of impaired photosynthetic capacity (Wong et al. 1979; Seemann et al. 1984; Hamerlynck & Knapp 1996). The transient increase in gs following release from heat stress likely fit into the optimization of the water/carbon use strategy of L. tridentata. This is supported by the differing responses between wellwatered and droughted plants (Figure 2). Under

191 well-watered conditions, pronounced transient gs increase was accompanied by higher Anet under ambient CO2 concentrations. Indeed, under 550 and 700 µmol mol−1 CO2 , Anet did not respond to increased gs . In contrast, under water-limiting conditions, the increased gs and Anet transient was most pronounced under the highest CO2 concentration (Figure 2). Thus, any carbon-gain benefit during recovery from heat stress this response may impart may be tightly coupled to both carbon availability and cost of water resources expended to maintain it. This behavior is remarkably similar to transient stomatal responses to short-term fluctuations in light level, which are thought to optimize water-use efficiency and productivity in plants from systems characterized by highly variable water and light resources (Knapp & Smith 1989, 1991; Knapp 1992). Indeed, transient stomatal responses to light variability are strongly reduced under water limitation (Knapp & Smith 1989, 1991; Knapp 1992), in leaves with low photosynthetic capacity (Hamerlynck & Knapp 1996), and in elevated CO2 (Knapp et al. 1994) in a manner similar to the transient gs increases apparent in our study (Figure 2). The increase in gs following release from heat stress during drought may have implications for future conditions in North American deserts. While exposed to drought conditions, only at 700 µmol mol−1 CO2 did gs in L. tridentata markedly increase after alleviation from temperature stress (Figure 2). Midday ψ for these plants suggests that more water was available to them than in ambient-CO2-grown plants (Figure 1). This may mean that elevated CO2 could increase productivity in L. tridentata following unfavorable temperature episodes, but that total water use could also increase due to greater transpirational losses accompanying increased stomatal opening. However, given that ambient-CO2-grown L. tridentata shed drought/temperature stressed leaves, the savings incurred by retaining leaves during and following drought and temperature stress may further increase whole plant water-use efficiency of this shrub, a response not readily observable on a scale of leaf-level gas exchange (Morgan et al. 1994; Read & Morgan 1996). Photosynthetic down-regulation at elevated CO2 is thought to be an important response since it reflects changes in resource allocation that might affect higher order processes (Griffin & Seemann 1996). It could be that shifting resources such as nitrogen away from C-fixation enzymes, especially Rubisco, might improve performance and perhaps fitness of

plants at elevated CO2 (Sharkey 1985; Sage 1996). Down-regulation has been found to be a function of seasonal temperature patterns (Lewis et al. 1996; Stirling et al. 1997), water-status (Oechel et al. 1995; Huxman et al. 1998a), and species-specific growth patterns (Stirling et al. 1997). Generally, warmer temperatures promote down-regulation (Lewis et al. 1996), while water limitations reduce it by decreasing carbohydrate accumulation that may feedback on down-regulation (Oechel et al. 1995; Huxman et al. 1998a). Our findings for L. tridentata suggest that heat stress under both well-watered conditions and drought reduced maximum photosynthetic capacity and mesophyll carboxylation efficiency (Figure 3). The increase in CE apparent in ambient CO2 /droughted plants was a result of new tissue production, not improved enzyme kinetics in previously droughted tissue. However, L. tridentata in both elevated CO2 treatments retained their original leaves, and Amax and CE did not change after alleviation of both heat and drought stress (Figure 3). Our findings suggest that even a single, rather short-duration high temperature event can impact regulatory responses in L. tridentata under moisture conditions that otherwise would be conducive to down-regulation. These findings support the Lewis et al. (1996) view that predictive modeling efforts utilizing mechanistic sub-routines to characterize ecosystem-level responses to elevated CO2 should account for distinct photosynthetic regulatory responses across a range of environmental conditions and time scales. In summary, this study shows that elevated CO2 reduced the impact of drought and high temperature stress on Larrea tridentata, potentially reducing the effects of extremely high temperatures and dry soils that currently limit seedling establishment and survival of this desert xerophyte (Jordan & Nobel 1979; Smith & Nowak 1990). Increasing productivity of this evergreen dominant of North American deserts could also be enhanced by extending activity into dry seasons or by enchancing photosynthesis after small rainfall events in the hot summer months. However, our findings do show that water status has a greater proportional impact on physiological processes in L. tridentata than does CO2 . This suggests that altered regional climate patterns in the deserts of North America could have a more pronounced impact than future CO2 concentrations (Smith et al. 1999). However, if elevated CO2 does indeed result in periods of higher stomatal conductance following high temperature periods, total

192 water use and seasonal water balance may change in the future (Smith et al. 1995, 1998).

Acknowledgements This research was supported by a NSF-EPSCoR grant to the State of Nevada, a grant from the NSF/DOE/NASA/USDA TECO program (NSF IBN9524036) to SDS, and a DOE/EPSCoR Traineeship to TEH.

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