The effect of induced heat waves on Pinus taeda and Quercus rubra seedlings in ambient and elevated CO2 atmospheres

Authors


Summary

  • Here, we investigated the effect of different heat-wave intensities applied at two atmospheric CO2 concentrations ([CO2]) on seedlings of two tree species, loblolly pine (Pinus taeda) and northern red oak (Quercus rubra).
  • Seedlings were assigned to treatment combinations of two levels of [CO2] (380 or 700 μmol mol−1) and four levels of air temperature (ambient, ambient +3°C, or 7-d heat waves consisting of a biweekly +6°C heat wave, or a monthly +12°C heat wave). Treatments were maintained throughout the growing season, thus receiving equal heat sums. We measured gas exchange and fluorescence parameters before, during and after a mid-summer heat wave.
  • The +12°C heat wave, significantly reduced net photosynthesis (Anet) in both species and [CO2] treatments but this effect was diminished in elevated [CO2]. The decrease in Anet was accompanied by a decrease in Fv′/Fm′ in P. taeda and ΦPSII in Q. rubra.
  • Our findings suggest that, if soil moisture is adequate, trees will experience negative effects in photosynthetic performance only with the occurrence of extreme heat waves. As elevated [CO2] diminished these negative effects, the future climate may not be as detrimental to plant communities as previously assumed.

Introduction

Atmospheric carbon dioxide concentration ([CO2]) is rapidly increasing because of anthropogenic contributions. According to the fourth assessment report (AR4) of the IPCC, [CO2] is expected to reach 700 μmol mol−1 by the year 2100 (Meehl et al., 2006; IPCC, 2007). Models show that, because of the rise in [CO2] and other glasshouse gasses, global air temperature is expected to rise by between 1.7°C and 4.4°C by the end of the 21st century (SRES: A1B) (IPCC, 2007). In addition to the rise in air temperature, plants are likely to face an increase in frequency and severity of weather extremes, such as heat waves (Meehl & Tebaldi, 2004; Tebaldi et al., 2006; IPCC, 2007; Ballester et al., 2010). Although there is no generally accepted definition for a heat wave, heat waves have been defined using temperature–mortality criteria (Montero et al., 2010) or, more commonly, statistical–meteorological criteria (Frich et al., 2002; Meehl & Tebaldi, 2004; Meehl et al., 2006; IPCC, 2007; Ballester et al., 2010). A heat wave was defined by Frich et al. (2002) and Tebaldi et al. (2006) as at least five consecutive days with maximum temperatures at least 5°C higher than the climatological norm of the same calendar days. This definition was adopted by the IPCC (2007) and will be used here.

The individual effects of elevated [CO2] (Ceulemans & Mousseau, 1994; Long et al., 2004; Ainsworth & Rogers, 2007) or elevated temperature (Saxe et al., 2001; Sage & Kubien, 2007) on plant performance have been studied intensively. A general conclusion from this research was that a rise in [CO2] or temperature had beneficial effects on both photosynthesis and biomass production. For example, when [CO2] was doubled, increases in net photosynthesis were reported ranging from 43% to 192% in Pinus taeda (Teskey, 1997; Tissue et al., 1997; Ellsworth, 1999; Wertin et al., 2010; Frenck et al., 2011) and from 30% to 256% in Quercus rubra (Kubiske & Pregitzer, 1996; Anderson & Tomlinson, 1998; Cavender-Bares et al., 2000).

Generally, an increase in air temperature also has a positive effect on net photosynthesis and growth (Sage & Kubien, 2007; Way & Oren, 2010). However, most elevated temperature studies have applied a constant increase in air temperature, while models predict an increase in extreme heat events, that is, heat waves (Ballester et al., 2010). Plant responses to heat waves have received little study. De Boeck et al. (2011) reported that the maximum rate of net photosynthesis was diminished by summer and autumn heat waves in a well-watered experimental plant community containing three perennial herbaceous species (Plantago lanceolata, Rumex acetosella and Trifolium repens). The community was able to minimize heat stress through transpirational cooling. When combined with drought stress, heat waves exacerbated the negative effect of drought stress. Hamerlynck et al. (2000) observed that in ambient [CO2] a heat wave greatly decreased net photosynthesis of Larrea tridentata, a desert perennial shrub. However, Dreesen et al. (2012) observed that heat waves did not diminish plant photosynthesis in an experimental group of annual and biannual plants under well-watered conditions. They suggested that annual and biannual species may be less sensitive to heat stress than perennial species. Hamilton et al. (2008) and Wang et al. (2008) investigated the interactive effect of elevated [CO2] and short-term heat stress on photosynthesis in C3 and C4 species by imposing a +15°C heat treatment for 4 h in either 370 or 700 μmol mol−1 [CO2]. They showed an increase in thermotolerance under elevated [CO2] in C3 species, but a decrease in thermotolerance in C4 species. Elevated [CO2] mitigated the effect of heat stress in L. tridentata during a 9-d heat wave (Hamerlynck et al., 2000). In that experiment plants grown at high [CO2] (700 μmol mol−1) showed less negative effects of heat stress during the heat wave, and a faster recovery, than plants grown at low [CO2] (360 μmol mol−1). By contrast, elevated [CO2] (700 μmol mol−1) did not compensate for a decrease in net photosynthesis in Brassica napus plants that were subjected to a +5°C heat treatment (Frenck et al., 2011).

These discrepancies in literature illustrate that more research is needed on the interaction between [CO2] and heat-wave stress in plants, in particular for seedlings as the regeneration of species is dependent on seedling survival and tree seedlings tend to be very sensitive to environmental stresses (Gazol & Ibanez, 2010; Becerra & Bustamante, 2011).The objective of this study was to determine the effect of heat waves on Anet in ambient and elevated atmospheric [CO2] in moist soil conditions on seedlings of two co-occurring forest tree species, P. taeda and Q. rubra. We hypothesized that: heat waves would be more detrimental to Anet than a constant elevation of air temperature; elevated [CO2] would diminish the negative effects of heat stress induced by heat waves; and a deciduous broadleaf tree species would be more susceptible to heat stress than an evergreen needle-leaf tree species.

Materials and Methods

Study site

The study site was located at Whitehall Forest of the University of Georgia in Athens, USA (33°57′N, 83°19′W, altitude 230 m). Eight treatment chambers, measuring 3.62 m long by 3.62 m wide by 2.31 m high, were constructed at the site. The treatment chambers were constructed with lumber bases and PVC pipe frames bent to form a half-cylinder shape that supported a cover of 6 mm clear polyethylene film (GT Performance Film; Green-Tek Inc., Edgerton, WI, USA) (Boyette & Bilderback (1996). The chambers were placed in an open field, spaced 3.7 m apart to minimize shading, and oriented facing south to maximize daily sun exposure.

Plant material

Two species were examined in this study: an evergreen conifer, loblolly pine (P. taeda L.) and a broadleaf deciduous species, northern red oak (Q. rubra L.). Seeds of both species were planted on 1 December, 2009 in 0.5 l pots in a glasshouse. After the seeds germinated, seedlings were transplanted to 7.6 l pots and transported to the treatment chambers. The P. taeda seeds were from a single family in Morgan County, GA (Family 15042; Plum Creek Timber Company, Athens, GA, USA). Quercus rubra seeds were obtained from a wild collection in Tennessee (Louisiana Forest Seed Company, Lecompte, LA, USA).

Thirty seedlings of P. taeda (P) and Q. rubra (Q) were placed in each treatment chamber. The P. taeda and Q. rubra seedlings were placed on separate sides of the chambers. Pots were evenly spaced within the chamber.

Soil was kept moist by watering the pots via drip irrigation four times a day to field capacity. In April, May, June and July each pot was fertilized with 30 g of 15-9-12 extended release fertilizer (OsmocotePlus #903286; Scotts-Sierra Horticultural Products, Marysville, OH, USA) and 0.2 g chelated iron was supplied to P. taeda in June and July (Sprint 138, Becker Underwood, Ames, IA, USA). In May, 0.04 ml of Imidacloprid was applied topically to the soil in each pot to prevent pests (Advanced 12 Month Tree and Shrub Insect Control; Bayer,Monheim am Rhein, Germany). A topical fungicide was sprayed on the seedlings in June.

Experimental design and setup

Each treatment chamber was assigned to one of eight treatment combinations: ambient [CO2] (A) (380 μmol mol−1) or elevated [CO2] (E) (700 μmol mol−1) in combination with one of four air temperature treatments: ambient (Tamb), elevated +3°C (Tamb + 3), 6°C heat wave (HW6), or 12°C heat wave (HW12). Heat waves were imposed for a period of 7 d. The HW6 treatment was imposed every other week and the HW12 treatment was imposed every fourth week. During the non-heat wave intervals the temperature was ambient. Seedlings in the different treatments are identified in the following manner: Q for Quercus, P for Pinus, followed by A for ambient [CO2] and E for elevated [CO2], the last character represents the temperature treatment (Tamb, Tamb+ 3, HW6 and HW12).

Temperature and [CO2] treatments were initiated on 2 May 2010. Each month, treatment combinations among the chambers and seedling positions within the chambers were rotated to minimize any potential chamber effects. The Tamb treatment was used as a control to represent the current ambient temperature. The Tamb + 3 treatment was used as a second control to ascertain the effect of a constant increase in air temperature that produced the same average monthly temperature and heat sum as the HW6 and HW12 treatments.

To maintain the target [CO2], a solid-state nondispersive infrared CO2sensor (Model GMT222; Vaisala Inc., Woburn, MA, USA) was suspended in each treatment chamber. The sensor continuously measured [CO2] and directly controlled a solenoid valve that released CO2 into the chambers from a cylinder of industrial grade compressed CO2 (Airgas National Welders, Toccoa, GA, USA). An oscillating fan was installed in each chamber to disperse the CO2 evenly throughout.

The temperature in each chamber and outside, 1.45 m south of the chambers, was measured with matched type T thermocouples every 3 min and averaged and recorded every 15 min using a data logger (Campbell 23X, Logan, UT, USA). Each thermocouple was housed in a ventilated radiation shield (Model SRS100; Ambient Weather, Chandler, AZ, USA) mounted on a pole 1 m above ground level. The data logger continuously compared air temperature in the chambers with the outside temperature and controlled an air conditioner (Model FAM186R2A, Frigidaire, Augusta, GA, USA) and heaters (Model 3VU33A, Dayton Electric, Niles, IL, USA) in each chamber to maintain the treatment air temperatures. Instrumentation was located at the same place in each chamber in order to minimize any potential chamber effect.

Measurements

Foliar gas exchange and fluorescence parameters were measured during the sixth +6°C and third +12°C heat wave from 20 July to 26 July. During that heat wave, mean daily temperatures in the treatment chambers ranged from 30°C (Tamb) to 40°C (HW12) (Table 1).

Table 1. Mean (SE) daily air temperature (Tavg) in treatment chambers under [CO2] and temperature treatment combinations and outside before (Pre), during (Dur), and after (Aft) a heat wave treatment
[CO2] TreatmentTemperature treatmentHeat-wave treatment period
PreDurAft
Tavg (°C)
  1. Tamb, ambient temperature; Tamb + 3, ambient temperature elevated +3°C; HW6, 6°C heat wave; HW12, 12°C heat wave.

AmbientTamb28.76 (0.14)30.63 (0.16)30.61 (0.16)
Tamb + 330.92 (0.14)33.54 (0.18)33.14 (0.17)
HW628.43 (0.13)34.85 (0.16)30.38 (0.15)
HW1228.86 (0.15)39.69 (0.2)30.62 (0.17)
ElevatedTamb27.98 (0.12)30.13 (0.15)30.03 (0.15)
Tamb + 331.18 (0.15)32.88 (0.17)33.02 (0.18)
HW628.75 (0.14)35.93 (0.17)30.66 (0.17)
HW1229.23 (0.16)40.17 (0.19)30.51 (0.16)
Ambient (outside) temperature27.97 (0.12)30.04 (0.15)30.49 (0.21)

Measurement protocol

Four seedlings of each species in each [CO2] and temperature treatment combination were selected for measurement. Gas exchange measurements were made on sunny or mostly sunny days before (13, 16 and 19 July), during (20, 22, 24 and 26 July) and after the heat wave (27 and 29 July and 1 August) beginning at 12:30 h. All measurements were conducted on representative foliage of the most recent fully developed flush.

Gas exchange and fluorescence measurements

Gas exchange and fluorescence parameters were measured concurrently with a portable photosynthesis system (LI-6400; LiCor Biosciences, Lincoln, NE, USA) fitted with a fluorescence head (6400-40 LCF; LiCor Biosciences) and a CO2-mixer (6400-01; LiCor Biosciences). Fluorescence parameters – quantum yield of photo-system II (PSII) (ΦPSII) and maximum quantum yield of light saturated PSII (Fv’/Fm’) – were calculated as described in Maxwell & Johnson (2000). We determined the optimum intensity and time for saturating and desaturating flashes on representative foliage before the measurements. Net photosynthesis (Anet), stomatal conductance (gs), leaf transpiration (E) and fluorescence parameters were measured on the four selected seedlings throughout the experiment. The temperature of the leaf cuvette was set to match the temperature in each treatment chamber at the start of the measurements. The light source of the fluorescence head was maintained at 1500 μmol photons m−2s−1. [CO2] in the cuvette was maintained at 380 or 700 μmol CO2 mol−1 for ambient or elevated [CO2] treatments, respectively. Total leaf area for P. taeda was determined using the method described in Fites & Teskey (1988).

Acclimatization experiment

To determine if either P. taeda or Q. rubra seedlings were acclimatizing to repetitive exposure to + 6°C heat waves, three individuals from the Tamb treatment were placed in the +6°C treatment chamber during a heat wave (20–27 August). Measurements were conducted as described earlier on these seedlings and on three seedlings that had been grown in the HW6 treatment. This experiment allowed us to investigate whether seedlings with no previous exposure to a +6°C heat wave had similar performance during a heat wave to those that had previous exposure.

Statistical analysis

The average of the three measurements before the heat wave (Pre) served as a baseline to compare with measurements during (Dur) and after (Aft) the heat wave. Measured and calculated parameters were analysed using multifactorial repeated measures anova with species (Q, P), [CO2] treatment (A, E), temperature treatment (Tamb, Tamb + 3, HW6, HW12) and time (Pre, Dur1, Dur2, Dur3, Dur4, Aft1, Aft2) as fixed factors. Student's t-tests were used to compare between-treatment combinations. Measurements from the acclimatization experiment were compared using paired t-tests. Statistical tests, correlation and regression analysis were performed using proc mixed, proc corr and proc reg, respectively, in SAS 9.0 (SAS Institute Inc., Cary, NC, USA).

Results

Maximum daily temperatures in the HW6 and HW12 treatments during the heat wave events were > 5°C higher than the climatological norm (Fig. 1), indicating that both treatments successfully simulated a heat wave (Frich et al., 2002; Tebaldi et al., 2006).

Figure 1.

Maximum daily temperature in the +6°C heat-wave treatment (HW6) (open circles) and +12°C heat-wave treatment (HW12) (triangles) compared with average maximum temperature, increased by 5°C, of the same days over a 30-yr period (closed circles). The grey area represents the period during which a heat wave was applied.

Gas exchange

Net photosynthesis differed significantly between species and [CO2] treatments and among temperature treatments (Table 2). Averaged across all treatments Anet was 13% greater in Q. rubra than in P. taeda (< 0.05). Average Anet was 78% higher in elevated [CO2] compared with ambient [CO2] in Q. rubra seedlings and 79% higher in P. taeda seedlings.

Table 2. Summary (P-values) of the multivariate repeated measures anova of the effects of species, [CO2] treatment, temperature treatment (Temp) and time on net photosynthesis (Anet), stomatal conductance (gs), transpiration (E), maximum quantum yield of light-saturated PSII (Fv′/Fm′) and quantum yield of light-saturated PSII (ΦPSII)
Effect A net g s E Fv’/FmΦPSII
Species< 0.001   0.14   0.0053< 0.0001< 0.0001
CO2< 0.0001< 0.001< 0.001   0.031   0.11
Species × CO2   0.34   0.52   0.27   0.001   0.42
Temp< 0.0001   0.27   0.0021< 0.001< 0.0001
Species × Temp   0.62   0.89   0.74< 0.001   0.92
CO2 × Temp   0.23   0.67   0.89< 0.001   0.081
Species × CO2 × Temp   0.31   0.63   0.55   0.67   0.088
Time< 0.0001< 0.0001< 0.0001< 0.001< 0.0001
Species × time< 0.0001< 0.0001< 0.0001< 0.0001< 0.0001
CO2 × time< 0.0001   0.56   0.79   0.52< 0.0001
Species × CO2 × time   0.0030   0.16   0.082   0.0084   0.85
Temp × time< 0.0001   0.0076< 0.0001< 0.0001< 0.001
Species × Temp × time< 0.0001   0.0026   0.0082   0.10   0.13
Temp × CO2 × time   0.093   0.14   0.21   0.87   0.54
Species × Temp × CO2 × time   0.0012   0.050   0.023   0.45   0.16

Net photosynthesis also differed with time, and there were several significant interactions with time, species and [CO2] and temperature treatments (Table 2). Averaged across the three pretreatment measurements, Anet was similar in all temperature treatments within a species and [CO2] treatment combination (Table 3, Fig. 2). In both species, at the start of the heat wave there was an immediate decline in Anet in the HW12 treatment.Net photosynthesis remained suppressed in P. taeda through the duration of the heat wave and was significantly lower (< 0.05) than the control treatments (Tamb, Tamb+ 3), with the exception of PEHW12 on Dur1 when compared to Tamb. In P. taeda in the elevated [CO2] treatment, the decline was not significant on the first day of the heat wave, but by the third day Anet had declined by 69% (< 0.001), compared with the pre-heat-wave period, and it remained significantly lower (< 0.05) than the control treatments for the rest of the heat-wave period. On the first and third day of the heat wave, Anet of Q. rubra seedlings in the HW12 treatment was significantly lower (< 0.05) than the control treatments. On the fifth day, no significant differences with both control treatments were found, except for QEHW12, compared with Tamb+ 3(< 0.05). Anet was significantly different from Tamb and Tamb+ 3 on the last day of the heat wave, but this was not the case for QEHW12. Anet remained positive throughout the heat wave in both species in the HW6 and HW12 treatments with the exception of PAHW12, where mean Anet was − 0.57 μmol m−2 s−1 on the last day of the heat treatment (Fig. 2). Coinciding with slightly cooler temperatures on the fifth day of the heat wave (Day 85, Fig. 1), a transient increase in Anet was observed in PAHW12, PEHW12 and QAHW12 that reversed on the last day (Day 87). This result indicates that Anet was responding to daily temperatures rather than to an accumulated stress caused by the duration of the heat wave.

Figure 2.

Mean net photosynthesis (Anet, left panels) and transpiration (E, right panels)of Pinus taeda and Quercus rubra seedlings grown under [CO2] and temperature treatment combinations. Seedlings grown at ambient temperature (Tamb) are indicated by green squares and ambient temperature +3°C (Tamb + 3) by yellow triangles. Seedlings subjected to +6°C heat waves every other week (HW6) are indicated by orange circles; seedlings subjected to + 12°C heat waves every fourth week (HW12) are indicated by red diamonds. The grey area represents the period during which a heat wave was applied to the HW6 and HW12 treatments. Error bars represent ± 1 SE.

Table 3. Mean (SE) net photosynthesis (Anet) of Pinus taeda and Quercus rubra seedlings grown under [CO2] and temperature treatment combinations before (Pre) a heat wave treatment and percent difference from that value on measurement days during (Dur 1–7) and after the heat wave (Aft 1 and 3)
Treatment combinationAnet (μmol m−2 s−1)Difference (%)
PreDur1Dur3Dur5Dur7Aft1Aft3
  1. Tamb, ambient temperature; Tamb + 3, ambient temperature elevated +3°C; HW6, 6°C heat wave; HW12, 12°C heat wave. Significant differences between Dur and Aft measurements and their respective Pre measurement depicted with: *, < 0.05; ***, < 0.001.

Pinus taeda
Ambient [CO2]
Tamb7.99 (0.72)− 12.74− 15.21     7.26       21.03  − 2.68− 22.00
Tamb + 36.80 (0.47)     4.23− 17.53− 18.47   − 29.83    16.16− 21.59
HW67.60 (0.53)− 25.28− 24.63− 24.39   − 10.66  − 9.49− 9.47
HW126.99 (0.48)− 60.12***− 92.34***− 69.93***   − 108***− 22.28− 29.03
Elevated [CO2]
Tamb10.24 (0.41)    19.45− 13.20    11.61        5.50    13.28    30.33*
Tamb + 311.90 (0.66)    12.91      8.67− 14.92   − 20.89*      0.25      7.22
HW613.07 (1.03)   − 8.39− 34.96***− 14.78   − 27.06*   − 1.72       2.60
HW1211.26 (0.67)− 11.69− 69.15***− 50.23***   − 60.3*** − 34.22***    − 2.22
Quercus rubra
Ambient [CO2]
Tamb7.90 (0.49)     0.10− 12.22− 23.27   − 33.96*− 12.27  − 8.57
Tamb + 38.75 (0.51)  − 2.71− 23.52− 33.26*   − 36.86*− 16.21− 18.63
HW68.16 (0.49)  − 2.78  − 6.65− 24.00   − 39.76*   − 6.04− 15.95
HW128.19 (0.58)− 42.04*− 83.52***− 47.23***   − 73.17***− 49.02***− 55.75***
Elevated [CO2]
Tamb11.76 (0.84)    14.88     4.13− 19.54*   − 21.17*     7.19      3.88
Tamb + 312.99 (0.46)   − 6.45   − 9.28− 22.28*   − 32.68***   − 6.42− 11.12
HW614.48 (0.55)      4.20− 26.08***− 52.03***   − 34.01***    11.92    23.76*
HW1213.47 (0.83)− 54.39***− 42.77***− 48.62***   − 25.21*− 5.24− 41.39***

Compared with the pre-heat-wave period, a decline in Anet in Q. rubra seedlings in the Tamb and Tamb+ 3 treatments occurred during the heat-wave period (Fig. 2). In P. taeda no significant changes were observed over time in the Tamb and Tamb+ 3 treatments, except in PE Tamb+ 3 on the last day of the heat wave (Table 3). No significant differences were found in Anet of the HW6 seedlings compared with either control treatment (Tamb or Tamb+ 3) and Anet followed a similar pattern in these three treatments before, during and after the heat wave (Fig. 2).

Net photosynthesis was negatively related to leaf temperature (TL) in both species (Fig. 3, Table 4). A similar decrease in Anet in response to TL was observed in both ambient and elevated [CO2] treatments, but Anet was consistently higher in elevated [CO2] at any measured TL in both species. The effect of TL on Anet was consistent in many of the individual temperature treatments but because the different temperature treatments encompass different ranges of TL, differences in the slopes of the regression analyses should be interpreted with caution (Fig. 3, Table 4).

Figure 3.

Net photosynthesis (Anet), stomatal conductance (gs),maximum quantum yield of light saturated PSII (Fv′/Fm′) and quantum yield of light-saturated PSII (ΦPSII) of Pinus taeda and Quercus rubra seedlings grown under [CO2] and temperature treatment combinations as a function of leaf temperature. Seedlings grown at ambient or elevated [CO2] are indicated by circles or squares, respectively. Seedlings grown at ambient temperature (Tamb) are indicated by green symbols and ambient temperature +3°C (Tamb + 3) by yellow symbols. Seedlings subjected to +6°C heat waves every other week (HW6) are indicated by orange symbols; seedlings subjected to +12°C heat waves every fourth week (HW12) are indicated by red symbols. Lines represent linear regressions across all temperature treatments at ambient [CO2] (solid) and elevated [CO2] (dotted).

Table 4. Linear regression analysis of net photosynthesis (Anet), stomatal conductance (gs), maximum quantum yield of light-saturated PSII (Fv′/Fm′) and quantum yield of light-saturated PSII (ΦPSII) on leaf temperature
Treatment combination A net P g s P E P Fv/Fm P ΦPSII P
  1. Tamb, ambient temperature; Tamb + 3, ambient temperature elevated +3°C; HW6, 6°C heat wave; HW12, 12°C heat wave. Values in bold represent slopes and P-values across temperature treatments for each species × [CO2] combination. When slopes are significant across temperature treatments, slopes and P-values are shown for individual temperature treatments. Significant P-values are depicted with: *, < 0.05; **, < 0.01; ***, < 0.0001.

Pinus taeda – ambient [CO2] − 0.55 *** − 3.54E-03 **    0.15 *** − 1.02E-02 ***   
Tamb  − 1.15E-02 *− 0.39 *    
Tamb + 3− 0.62 ** − 1.09E-02 **− 0.43**    
HW6       0.33 **    
HW12− 0.56 ***     0.16 *− 9.01E-03 ***  
Pinus taeda – elevated [CO2]− 0.64 ***− 2.81E-03**    0.16 *** − 9.31E-03 ***   
Tamb  − 1.01E-02 * − 0.34 *     
Tamb + 3− 0.88 ** − 8.92E-03 ** − 0.33 **     
HW6− 0.82 *** − 4.49E-03 *   − 1.02E-02 ***   
HW12− 0.46 ***      0.22 ** − 7.23E-03 **   
Quercus rubra – ambient [CO2]− 0.40 ***       0.40 *** − 4.93E-03 * − 2.25E-03 **
Tamb          
Tamb + 3       0.38 ** 1.76E-02 *   
HW6− 0.35 **      0.53 ***     
HW12− 0.28 **      0.43 ***     
Quercus rubra – elevated [CO2]− 0.56 ***       0.35 ***   − 2.06E-03 **
Tamb− 0.75 *      0.28 *     
Tamb + 3          
HW6− 0.84 ***      0.46 ***     
HW12− 0.42 **       − 1.84E-03 *

In ambient [CO2], stomatal conductance (gs), averaged across all measurements (before, during and after the heat wave) was 0.11 mol H2O m−2 s−1in both P. taeda and Q. rubra (Fig. 4). It was reduced in elevated [CO2] compared with ambient [CO2] by 21% (= 0.0016) in Q. rubra and 14% (= 0.036) in P. taeda (Table 2). In Q. rubra there was no effect of TL on gs (Table 4). In P. taeda there was a small negative effect of increased TL on gs (Fig. 3) that was significant in all treatment combinations except PAHW12, PEHW12 and PAHW6 (Table 4). For gs there were few significant interactions among main treatment factors and no significant interactions between time and CO2 or temperature treatments and no higher-order interactions with the exception of a species × temperature treatment × time interaction (Table 2). There was no significant relationship between gs and VPD (Table 5).

Figure 4.

Mean stomatal conductance (gs) of Pinus taeda and Quercus rubra seedlings grown under [CO2] and temperature treatment combinations and vapor pressure deficit (VPD) in the heat wave treatment chambers. Seedlings grown at ambient temperature (Tamb) are indicated by green squares and ambient temperature +3°C (Tamb + 3) by yellow triangles. Seedlings subjected to +6°C heat waves every other week (HW6) are indicated by orange circles; seedlings subjected to + 12°C heat waves every fourth week (HW12) are indicated by red diamonds. The VPD in the HW6 and HW12 treatments is indicated by semi-filled circles and diamonds, respectively. The grey area represents the period during which a heat wave was applied to the HW6 and HW12 treatments. Error bars represent ± 1 SE.

Table 5. Linear regression analysis of stomatal conductance (gs) and transpiration (E) on vapor pressure deficit (VPD) and net photosynthesis (Anet) on stomatal conductance (gs), maximum quantum yield of light-saturated PSII (Fv′/Fm′), and quantum yield of light saturated PSII (ΦPSII)
Treatment combinationIndependent variable
VPD g s Fv′/FmΦPSII
Dependent variable
g s P E P A net P A net P A net P
  1. Tamb, ambient temperature; Tamb + 3, ambient temperature elevated +3°C; HW6, 6°C heat wave; HW12, 12°C heat wave. Values in bold type represent slopes and P-values across temperature treatments for each species × [CO2] combination. When slopes are significant across temperature treatments, slopes and P-values are shown for individual temperature treatments. Significant P-values are depicted with: *, < 0.05; **, < 0.01; ***, < 0.0001.

Pinus taeda – ambient [CO2]   0.67 *** 41.80 *** 23.34 ***   
Tamb  1.45 *** 41.02 ***     
Tamb + 3  0.74 * 35.14 ***     
HW6  1.15 *** 38.03 ***     
HW12  0.71 *** 38.51 ** 37.02 ***   
Pinus taeda- elevated [CO2]   0.77 *** 71.14 *** 30.14 ***   
Tamb  1.41 *** 47.06 ***     
Tamb + 3  1.60 *** 76.18 ***     
HW6  1.19 *** 43.89 *** 29.65 **   
HW12  0.77 *** 73.96 *** 24.25 *   
Quercus rubra- ambient [CO2]   0.92 *** 41.90 ***    7.92 ** 67.54 ***
Tamb  1.14 *** 36.85 ***   52.35 ***
Tamb + 3  1.35 *** 45.11 **   60.32 ***
HW6  1.16 ***     57.68 ***
HW12  0.91 *** 47.69 ***   60.86 ***
Quercus rubra – elevated [CO2]   0.75 *** 45.34 ***    78.93 ***
Tamb  1.05 *** 44.92 *   91.02 ***
Tamb + 3  0.45 * 111.08 ***     
HW6  1.23 ***     97.77 ***
HW12  0.69 *** 51.85 *   82.73 **

Corresponding with higher gs, transpiration was significantly higher in ambient compared with elevated [CO2] (P < 0.05) in both species (Fig. 2). There was a substantial rise in E during the heat wave in the HW6 and HW12 treatments (Fig. 2) and E was positively related to VPD in all treatment combinations (Table 5).There was also a positive relationship between E and TL (Table 4) but the effects of VPD and TL on E cannot be separated in this experiment. A positive relationship was found between Anet and gs (Fig. 5) in both species and all treatment combinations except QAHW6 and QEHW6 (Table 5). A steeper slope in the relationship between Anet and gs was found under elevated [CO2] compared with ambient [CO2].

Figure 5.

Net photosynthesis (Anet) of Pinus taeda and Quercus rubra seedlings grown under [CO2] and temperature treatment combinations as a function of stomatal conductance (gs). Seedlings grown at ambient or elevated [CO2] are indicated by circles or squares, respectively. Seedlings grown at ambient temperature (Tamb) are indicated by green symbols and ambient temperature +3°C (Tamb + 3) by yellow symbols. Seedlings subjected to +6°C heat waves every other week (HW6) are indicated by orange symbols; seedlings subjected to +12°C heat waves every fourth week (HW12) are indicated by red symbols. Lines represent linear regressions across all temperature treatments at ambient [CO2] (solid) and elevated [CO2] (dotted).

Quercus rubra seedlings that had not been exposed to previous heat waves had statistically similar Anet during a heat wave as seedlings in the HW6 treatment, which had been previously subjected to five heat waves (data not shown), indicating that there had been no photosynthetic acclimatization after repeated exposure to biweekly heat waves. The same result occurred in P. taeda seedlings, but there was an nonsignificant trend: Anet of P. taeda seedlings that had not been previously exposed to +6°C heat waves had 20–31% lower Anet compared to seedlings in the HW6 treatment (= 0.058 and = 0.15 in ambient and elevated [CO2], respectively).

In both P. taeda and Q. rubra, reductions in Anet in seedlings grown in elevated [CO2] were comparable to, or less than, reductions in Anet in seedlings grown in ambient [CO2], indicating that elevated [CO2] diminished the effect of heat stress on Anet (Table 3). Averaged across the heat-wave period in the HW12 treatment, Anet under ambient [CO2] decreased 20% more in Q. rubra and 35% more in P. taeda, compared with a decrease under elevated [CO2]. The mitigating effect of elevated [CO2] on the response of Anet during the heat wave was greatest in QEHW12 (Fig. 2). In these seedlings, Anet at the beginning of the heat wave (Day 81) was substantially lower than Anet of seedlings in the control treatments, but by the end of the heat wave (Day 87) Anet was similar among these treatments, indicating recovery during the heat wave.

Fluorescence parameters

Before the heat wave there were no significant differences in Fv′/Fm′ or ΦPSII between species or among treatment combinations (Fig. 6). In Q. rubra seedlings, averaged across temperature treatments, Fv′/Fm′ was significantly higher in elevated than in ambient [CO2] (< 0.001).This effect was not observed in P. taeda seedlings. With the exception of QAHW12, PAHW12 and PEHW12, Fv′/Fm′ was similar before and during the heat wave in most treatment combinations. In the last two treatments a significant reduction (< 0.01) was observed throughout the heat wave. In the QAHW12 treatment, Fv′/Fm′ decreased initially during the heat wave but by day 5 had returned to values not statistically different from those of seedlings in the control treatments. A positive correlation was found between Fv′/Fm′ and Anet in the PAHW12 and PEHW12 treatments (r = 0.74 and r = 0.62, respectively). In P. taeda seedlings, Fv′/Fm′, but not ΦPSII, was negatively related to TL in both ambient and elevated [CO2] (Fig. 3, Table 4). In Q. rubra seedlings, ΦPSII was negatively related to TL in both ambient and elevated [CO2], but a negative relationship between Fv′/Fm′ and TL was only evident in ambient [CO2]. A stronger correlation between ΦPSII and Anet for HW12 Q. rubra seedlings in ambient [CO2] (r = 0.51) was found, when compared with elevated [CO2] (r = 0.39). Consistent with the response of Anet we found a mitigating effect of elevated [CO2] on reductions in Fv′/Fm′ and ΦPSII during the heat-wave period. There was a more severe reduction in Fv′/Fm′ under ambient [CO2] (−42%, < 0.01) than under elevated [CO2] (− 34%, < 0.01) in P. taeda seedlings in the HW12 treatment. In addition, a less negative slope in the relationship between Fv′/Fm′ and TL was found in PEHW12, compared with PAHW12 (Table 4). This effect of elevated [CO2] was also present for the reduction in ΦPSII in QAHW12 (−21%, = 0.04) and QEHW12 (−16%, = 0.16). After the heat wave we observed a continuation of the inhibition in ΦPSII values for QAHW12, which was consistent with the reduction in Anet during this period.

Figure 6.

Maximum quantum yield of light-saturated PSII (Fv′/Fm′) and quantum yield of light-saturated PSII (ΦPSII) of Pinus taeda and Quercus rubra seedlings grown under [CO2] and temperature treatment combinations. Seedlings grown at ambient temperature (Tamb) are indicated by green squares and ambient temperature +3°C (Tamb + 3) by yellow triangles. Seedlings subjected to +6°C heat waves every other week (HW6) are indicated by orange circles; seedlings subjected to +12°C heat waves every fourth week (HW12) are indicated by red diamonds. Error bars represent ± 1 SE.

Discussion

The effect of a heat wave on plant performance

An immediate and significant decline in Anet was observed in seedlings that were subjected to a +12°C heat wave, but not in seedlings subjected to a +6°C heat wave. Our first hypothesis, that heat waves would be more detrimental to Anet than a constant elevation of air temperature, was confirmed, but it depended on the severity of the heat wave. Our results were similar to results of studies that imposed short term temperature increases on other plant species, for example, Phaseolus vulgaris (Hüve et al., 2011), Quercus pubescens (Haldimann & Feller, 2004), Pisum sativum (Haldimann & Feller, 2005) and Vitis amurensis (Luo et al., 2011). These studies applied the temperature treatment for a few hours, while we imposed the temperature treatments for a week. After the third day of the +12°C heat wave, Anet values stabilized at positive values and did not show signs of further reduction, indicating that the photosynthetic apparatus did not accrue additional stress or damage as the heat wave continued. During the heat-wave period, we observed a slow decline in Anet of the Tamb and Tamb + 3 seedlings of Q. rubra at ambient [CO2]. As the ambient temperature was higher during this period when compared with the period before the heat wave (Table 1), air temperature might have been supra-optimal for photosynthesis (Gunderson et al., 2010).

Decreases in photosynthetic performance in response to heat stress have generally been attributed to stomatal limitations, increased respiration, photorespiration and/or heat damage to different parts of the photosynthetic apparatus (Saxe et al., 2001). As gs was not reduced during the heat wave in either P. taeda or Q. rubra, reductions in Anet cannot be explained by stomatal limitation. We found that gs was negatively related to leaf temperature in P. taeda but not in Q. rubra. Reports of the response of gs to heat stress have been highly variable. For example, a decrease in gs with rising temperature was reported in two oak species (Quercus macrocarpa and Quercus muehlenbergii) (Hamerlynck & Knapp, 1996), an increase was reported in gs in wheat (Triticum aestivum) and barley (Hordeum vulgare) (Bunce, 2000) and soybean (Glycine max) (Wilson & Bunce, 1997), and no change in gs was reported in two eucalyptus species (Eucalyptus saligna and Eucalyptus sideroxylon)(Ghannoum et al., 2010). We also found no significant relationships between VPD and gs, perhaps because of the high temperatures and VPDs in which the experiment was conducted. Similar to findings in P. taeda (Bongarten & Teskey, 1986; Teskey et al., 1986), gs exhibited diminished responses to changes in VPD at high values of VPD. It was noted by Salvucci & Crafts-Brandner (2004a) that plants under heat stress, in the presence of adequate water supply, keep their stomata open to evaporatively reduce their leaf temperature. We observed no stomatal closure and high transpiration rates during the heat wave, suggesting that seedlings of Ptaeda and Q. rubra employed transpirational cooling to cope with heat stress. This result raises potential concerns about depletion of soil water if heat waves persist in time or increase in severity (Heath, 1998). In the absence of an adequate water supply it can be expected that leaf temperature will rise substantially, leading to more severe reductions in photosynthetic performance, and possibly increased tree mortality (Allen et al., 2010; Albert et al., 2011).

As the +12°C heat wave caused no apparent stomatal limitations to the diffusion of [CO2], inhibition of the photosynthetic apparatus must have contributed to the reduction in Anet. It has been shown that PSII is a thermolabile component of the photosynthetic apparatus. For example, thermal deactivation of oxygen evolution in PSII was observed in spinach (Spinacia oleracea)(Pueyo et al., 2002). In addition to reduction in oxygen evolution, heat stress has been shown to cause increased proton permeability of the thylakoid membranes (Bukhov et al., 1999), limitation of electron transport (Wise et al., 2004), cellular lesions (Hüve et al., 2011) and reversible deactivation of Rubisco activase (Haldimann & Feller, 2004; Salvucci & Crafts-Brandner, 2004b; Sharkey, 2005; Allen et al., 2010), all of which result in decreased photosynthetic activity.

InPAHW12 and PEHW12 we found a decrease in F0′ in response to the heat wave (data not shown). This result is consistent with the findings of Haldimann & Feller (2004), and suggests that the thylakoid membrane was not significantly damaged and was stable during heat stress. In contrast to P. taeda, an increase in F0′ was found inQAHW12, which indicates increased thylakoid proton permeability. This finding is in contrast to Ghouil et al. (2003), who concluded that through acclimatization, the thermal stability of the thylakoid membrane was enhanced in Q. suber seedlings exposed to temperatures up to 50°C. A similar acclimatization process may have taken place with repeated exposure to heat waves in P. taeda, but not in Q. rubra. Our analysis showed a similar relationship between Anet and ΦPSII values in QAHW12 and QEHW12. This relationship indicates that electron transport was reduced by heat stress in Q. rubra, resulting in a reduction in the production of ATP and NADPH, which would eventually cause reduced Anet. Values of ΦPSII remained constant for PAHW12 and PEHW12, which indicates maintenance of electron transport during the heat wave. As electron transport remained constant but a decrease in Anet was observed in both PAHW12 and PEHW12, electrons must have been transferred to alternative sinks such as photorespiration. Because increased leaf temperature results in reduced CO2 solubility relative to O2, it is likely that photorespiration increased during the heat wave (Wang et al., 2012). Photorespiration provides a mechanism that prevents over-reduction of the electron transport chain, thus protecting the photosynthetic apparatus from heat stress (Wingler et al., 2000; Leakey et al., 2003). In addition, a significant reduction in Fv′/Fm′ was found for PAHW12 and PEHW12, indicating a reduction in the efficiency of excitation energy capture and an increase in the thermal dissipation of excitation energy (Maxwell & Johnson, 2000; Rohacek, 2002).

We observed recovery of Anet during the 3 d after the heat wave in PAHW12 but not QAHW12, which indicates that the photosynthetic apparatus sustained damage, and lends support to our third hypothesis that a deciduous broadleaf tree species would be more susceptible to heat waves than an evergreen needle-leaf tree species. In a previous study of Q. rubra at the same site in which ambient temperature was raised by a constant 3°C or 6°C, no evidence of damage to photosynthetic apparatus was observed (Wertin et al. 2011). This result, combined with the lack of reduction to Anet in the +6°C heat wave in this study, suggests that the daily maximum temperatures in the +12°C heat wave, which exceeded 45°C, may have been the cause of the damage. These results also indicate that a mean increase in air temperature of +3°C caused by a series of heat waves may have a more negative effect on photosynthesis than a constant elevation of temperature. However, we observed partial recovery of Anet indicating that inhibition of photosynthesis during the heat wave was to some extent reversible. As damage to PSII is only slowly, if at all, reversible (Sinsawat et al., 2004), recovery of PSII cannot explain the reversible inhibition of Anet. However, a reversible reduction in Rubisco activation state through thermal deactivation of Rubisco activase has been reported (Haldimann & Feller, 2004, 2005; Sharkey, 2005). These studies showed that after alleviation of heat stress, Anet quickly returned to pre-stress values. In addition, leaf temperature decreased after the heat wave, thus reducing photorespiration (Sage & Kubien, 2007),suggesting another explanation for the partial recovery of Anet.

No evidence of thermal acclimatization of Anet to higher temperatures

Through thermal acclimatization of photosynthesis, plants can increase their optimum temperature for Anet (Hikosaka et al., 2006). This phenomenon has been reported for some conifer and deciduous tree species (Medlyn et al., 2002; Hikosaka et al., 2006; Ghannoum et al., 2010; Gunderson et al., 2010), but most studies have shown no acclimatization of photosynthesis to temperature (e.g. Ow et al., 2008a,b). Our observations indicated that a constant temperature increase of 3°C did not have a significant effect on Anet in P. taeda or Q. rubra, compared with Anet at ambient temperature. Wertin et al. (2010) also found that a temperature increase of 2.3°C did not result in thermal acclimatization of Anet in P. taeda. They attributed the lack of response to the broad temperature optimum for Anet in P. taeda (Teskey et al., 1987). In addition, seedlings exposed repeatedly to the +6°C heat-wave treatment did not acclimatize, although there was some inconclusive evidence of possible acclimatization in P. taeda.

Less heat stress under elevated [CO2]

The higher Anet we observed under elevated [CO2] was consistent with previous results in P. taeda (Teskey, 1997; Tissue et al., 1997) and Q. rubra (Kubiske & Pregitzer, 1996; Anderson & Tomlinson, 1998; Cavender-Bares et al., 2000), as well as many other species. Our data also revealed that elevated [CO2] compensated for the negative effect of heat stress on Anet, which is consistent with other studies (Teskey et al., 1987; Faria et al., 1996, 1999; Taub et al., 2000; Hamilton et al., 2008; Wang et al., 2008)and supported our second hypothesis that elevated [CO2] would mitigate heat stress induced by heat waves. This result suggests that under predicted future atmospheric [CO2] conditions, well-watered seedlings will be better able to cope with heat waves than in current [CO2] conditions. However, this finding must be interpreted with caution. As photosynthesis can acclimatize by downregulation to higher [CO2] (Wang et al., 1996; Long et al., 2004; Ainsworth & Rogers, 2007), the potential for future long-term mitigating effects of elevated [CO2] on heat stress might be less than the effect seen in our study. If this is the case, the future impact of heat waves on Anet may be similar to that at the present time. It is also important to consider that drought stress may strongly exacerbate heat stress regardless of [CO2].

Our results showed that Anet was significantly reduced in seedlings that were subjected to a +12°C heat wave, but that Anet remained positive throughout most or all of the heat wave and the overall level of stress was less than expected, especially under elevated [CO2]. The decline in Anet during the heat wave was similar in P. taeda and Q. rubra, but the underlying mechanism was different; in P. taeda, reduced Anet was linked to lower Fv′/Fm′, while in Q. rubra it was linked to lower ΦPSII.

We conclude that even under well-watered conditions, a future increase in the frequency or severity of heat waves will have detrimental effects on photosynthesis of P. taeda and Q. rubra. We observed a mitigating effect of elevated [CO2] on the thermal stress imposed by a severe heat wave. However, photosynthetic downregulation caused by long-term exposure to elevated [CO2] could negate this mitigating effect and lower available soil water could exacerbate the stress caused by heat waves.

Acknowledgements

We thank M. Jacobson and P. Belonger of Plum Creek Timber Company for their generous donation of loblolly pine seed. We also thank the CWO, Faculty of Bioscience Engineering, Belgium for the travel grant to M.A. and I.B., allowing the research stay at the University of Georgia. The project was supported by a grant from the United States Department of Energy NICCR Program (07-SC-NICCR-1060).

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