Elevated atmospheric CO2 adversely affects freezing tolerance in many evergreens, but the underlying mechanism(s) have been elusive. We compared effects of elevated CO2 with those of daytime warming on acclimation of snow gum (Eucalyptus pauciflora) to freezing temperatures under field conditions. Reduction in stomatal conductance gc under elevated CO2 was shown to cause leaf temperature to increase by up to 3 °C. In this study, this increase in leaf temperature was simulated under ambient CO2 conditions by using a free air temperature increase (FATI) system to warm snow gum leaves during daytime, thereby increasing the diurnal range in temperature without affecting temperature minima. Acclimation to freezing temperatures was assessed using measures of electrolyte leakage and photosynthetic efficiency of leaf discs exposed to different nadir temperatures. Here, we show that both elevated CO2 and daytime warming delayed acclimation to freezing temperatures for 2–3 weeks after which time freeze tolerance of the treated plants in both the FATI and open top chamber (OTC) experiments did not differ from control plants. Our results support the hypothesis that delayed development of freezing tolerance under elevated CO2 is because of higher daytime leaf temperatures under elevated CO2. Thus, potential gains in productivity in response to increasing atmospheric CO2 and prolonging the growing season may be reduced by an increase in freezing stress in frost-prone areas.
There is a pressing need to understand how plants respond to the progressive increase in atmospheric CO2 and associated climate warming. Vegetation at high latitudes or altitudes is particularly vulnerable, with increasing reports of shifting phenological patterns (Walther 2003) such as earlier flowering (Fitter & Fitter 2002), consistent with climate warming. Ironically, such changes in the timing and duration of the growing season can also increase the vulnerability of plants to freezing damage from early or late season frosts, as recently reported in tundra (Molgaard & Christensen 1997) and temperate forests (Norby, Hartz-Rubin & Verbrugge 2003). However, prediction of plant responses to these changes in climate is further complicated by the recent discovery that growth under elevated CO2 can lower freeze tolerance, making even extremely freeze-tolerant species more vulnerable to frost damage at warmer freezing temperatures (Repo, Hänninen & Kellomäki 1996; Lutze et al. 1998; Barker et al. 2005). Such interactive effects of elevated CO2 and temperature have far-reaching consequences for vegetation dynamics. Indeed, Beerling et al. (2001) reported that vegetation at high latitudes is likely to experience increasing sensitivity to frost damage, while Kullman (1998) stated evidence of shifts in the distribution of woody shrubs to lower elevations in Sweden in spite of climate warming. These observations were consistent with enhanced susceptibility to freezing damage under elevated CO2.
How might elevated CO2 affect freezing tolerance? The answer is likely to involve a complex combination of environmental factors and biochemical processes. However, temperature itself is an important cue as exposure to low temperatures is well known to induce freeze tolerance. We have previously shown that leaf temperature of snow gums grown under elevated CO2 is commonly 2–3 °C higher, and can be up to 5 °C higher, than similar leaves grown under ambient CO2 (Barker et al. 2005). This raises the possibility that elevated CO2 could affect freezing tolerance through indirect effects on leaf temperature. Specifically, the sustained reduction in gc that is often seen under elevated CO2 (Morison 1985; Drake, Gonzalez-Meler & Long 1997) can reduce transpiration (Li et al. 2003) leading to increased leaf temperature as a result of reduced evaporative cooling (Siebke et al. 2002). The resultant increase in water use efficiency that occurs from decreased gc has been examined widely in a range of species and environments (Long et al. 2004), yet the impact of the simultaneous increase in leaf temperature on plant function has not received the same level of interest. We hypothesize that higher daytime leaf temperature caused by reduced gC under elevated CO2 could affect acclimation to freezing temperatures, potentially delaying acclimation during autumn and accelerating deacclimation in spring.
We tested this hypothesis with field-based studies on a subalpine evergreen tree species, snow gum (Eucalyptus pauciflora Sieb. ex Spreng), which is one of the most freeze-tolerant type of eucalypts (Sakai, Paton & Wardle 1981). Acclimation of eucalypts to freezing temperatures is believed to occur primarily in response to low minimum temperatures compared with day length (Tibbits & Reid 1987). However, frost damage is greater in snow gum grown under elevated than ambient CO2 even when subject to the same temperature minima under field conditions (Lutze et al. 1998; Barker et al. 2005). Like many species, gC is lower in snow gum grown under elevated than ambient CO2 (Roden, Egerton & Ball 1999), which could increase maximum daytime leaf temperature (Idso, Kimball & Mauney 1987; Jarvis, Mansfield & Davies 1999; Siebke et al. 2002). We used open top chambers (OTC) and infrared lamps for free air temperature increase (FATI) for two concurrent experiments; snow gum seedlings were subject to either elevated/ambient CO2 or elevated/ambient daytime leaf temperatures. The seedlings were planted in late summer and subject to natural vagaries in weather, including the decline in minimum ambient temperature as winter approached. Our study aimed to answer the following question: Are there similarities between effects of elevated CO2 and elevated daytime leaf temperature on the development of freeze tolerance?
MATERIALS AND METHODS
E. pauciflora Sieb. ex Spreng. were grown from seed collected from trees in the Gudgenby Valley, Australian Capital Territory (ACT), at an elevation of 1000 m. The seeds were vernalized under moist conditions at 4 °C for 4 weeks before germinating on soil in a glasshouse. Seedlings of similar size were transferred to individual seedling tubes (70 × 70 × 150 mm) and grown outside under natural conditions in Canberra, ACT, at an elevation of 600 m, from October 2001 to February 2002. Figure 1a shows the daily maximum and minimum temperatures and Fig. 1b shows the daily rainfall data for Canberra during the period when snow gum seedlings were being established. The seedlings were fertilized twice a week with 10% Hoagland solution. A subset of five seedlings was used for initial freeze tolerance experiments before minimum temperature began to decline. These experiments were performed on 20 February 2002. At the 4–6 leaf stage, seedlings were planted in either OTC or in open pasture in front of infrared lamps near Bungendore, New South Wales (NSW) (35°15′S, 149°27′E, at an elevation of 700 m). The seedlings were planted in late summer (28 February 2002). The daily minimum and maximum temperatures observed at the field site for the experimental period are shown in Fig. 1c. A total of 25 mm of rain fell during the experimental period.
Leaf and air temperatures were measured for the OTC and FATI parts of the experiment. In the OTC temperatures on two leaves and that of the adjacent air (less that 5 mm from the measurement leaf) were measured, approximately 50 mm above the ground surface in each treatment of three chamber pairs. However, reliable data were obtained from only one chamber pair, therefore reducing the type of analysis possible. The air temperature 50 mm above the ground outside but close to the chambers was also measured. In the FATI experiment, the temperatures of one leaf and that of the adjacent air were measured, approximately 50 mm above the ground in each treatment in three blocks. Data were only included in the analysis if there were complete and corresponding data for leaf and air thermocouples within each block.
Temperatures were measured with copper–constantan thermocouples (64 µm in diameter) referenced against a platinum resistance thermometer (PT-100). The thermocouples were attached to the leaf lamina on the inward facing surface using a small piece of surgical tape so that they were not exposed to any direct sunlight. The thermocouples were scanned every minute, and a 30 min average was recorded on a data-logger for the FATI and OTC parts of the experiment. An additional measurement for air temperature was also made in a nearby weather screen 1.2 m above the ground. In the screen, a platinum resistance thermometer was scanned every minute and a 30 s average was recorded (DT-100 and DT-500 DataTakers, Data Electronics, Victoria, Australia).
The OTC were arranged in five replicate pairs and flushed with either ambient or CO2-enriched air to approximately twice ambient concentrations. The chambers were essential as described by Ashenden, Baxter & Rafarel (1992) and as modified by Lutze et al. (1998) and Roden et al. (1999). The air was circulated within each pair of chambers by a single fan unit. The CO2 concentration of the air was checked periodically with an infrared gas analyser (Li-6400, Licor, Lincoln, NE, USA), and on average was twice ambient concentration in each of the elevated chambers (see Barker et al. 2005). The CO2 was turned on the day after planting (1 March 2002), and was not injected into the elevated chambers during the night. Each chamber contained 12 seedlings, which were planted in open-ended pots (15 cm wide × 40 cm deep) with soil from the field site. The pots were buried in the ground within the OTC and the soil surface was covered with a layer of straw. A soil profile was simulated within the pots using topsoil and subsoil. The seedlings were watered to field capacity at the time of planting; no further water or fertilizer was applied. A comparison of the minimum and maximum temperatures within and just outside the chambers at plant height are shown in Fig. 1d. Importantly, the minimum temperature within the chambers was very similar to those recorded outside. The chamber mixing fans were turned off during the night to allow chamber temperatures to decline naturally. Maximum temperature, however, was often several degrees higher inside the chamber compared with the outside temperature. This is often observed in experiments using OTC (Norby et al. 1997). The snow gum leaves growing under elevated CO2 in the OTC were commonly 1–3 °C warmer than their ambient grown counterparts (Barker et al. 2005).
Our FATI system was a simplified and modified version of that originally described by Nijs et al. (1996). In this experiment, we did not require the feedback temperature control employed in the Nijs et al. (1996) system, so the output of our lamps was constant while in operation. The degree of warming achieved was controlled by the distance between a lamp and its target seedling. Warmed and control treatments were arranged in five randomized blocks in an open pasture near Bungendore, NSW. Ceramic 150 W Pandorel bulbs and a 30 cm wide aluminium reflector (Vaucluse and Animal Production Services, Adelaide, South Australia) were housed in a weatherproof stainless steel container (35 × 32 × 33 cm). The lamps were positioned 60 cm due south of the seedlings. The lamps produce no visible radiation and, using a net radiometer (Rimco, Middleton, Synchrotac, Mulgrave, Australia) at a distance of 60 cm, they increased radiation input by approximately 79 W m−2. It was aimed to increase daytime leaf temperature by 1–3 °C, a magnitude which we have previously observed in snow gums (Barker et al. 2005). The degree of daytime (0600–1800 h) warming achieved was measured using leaf thermocouples. The difference between leaf and air temperature (ΔT) data are shown in Fig. 2. The ΔT were divided into temperature classes, making it possible to determine the most commonly observed ΔT. There was a clear difference in the distribution of ΔT between control and warmed plants. The control plants most commonly had a leaf temperature very close to air temperature (mean ΔT of 0.11 °C), with only a small proportion of the total observations occurring above the 1 °C temperature class. The leaves of the warmed plants were most commonly 1–1.5 °C warmer (mean ΔT of 1.05 °C) than the air, with the spread of observations to the higher ΔT classes being considerably greater than the control. The FATI treatment was effective in increasing leaf temperature above the control leaves (FATI leaves were significantly warmer than their control counterparts, P = 0.006). There were five replicates of warmed and control treatments. Three seedlings per replicate were planted directly into the ground and watered to field capacity; no further water or fertilizer was applied. The soil around the seedlings was covered with a layer of straw. The heat lamps were turned on at dawn and off at dusk; all plants experienced the same temperature minima at night. The lamps were turned on 6 d after planting the seedlings (6 March 2002).
To determine if the leaf temperatures experienced by plants growing in the FATI experiment were comparable to those growing in the OTC, maximum and minimum leaf temperatures of leaves growing under ambient CO2 were plotted against those of non-warmed control FATI leaves (Fig. 3). Leaf temperatures for the two experiments were significantly correlated (P < 0.01). For the comparison of minimum leaf temperature, there was a cluster around the 1:1 line; however, maximum leaf temperatures were marginally warmer in the FATI plants compared with those measured in the OTC plants.
Freeze tolerance of plants in the OTC and those under FATI treatment was assessed at several intervals over a 4 week period (7 and 28 d after initiation of treatment for FATI, and 7 and 21 d after initiation of treatment for OTC) as a function of change in both the fluorescence parameter (Fv/Fm) and electrolyte leakage from leaf discs frozen to different nadir temperatures. The oldest fully expanded leaves were selected from both OTC and FATI plants. The leaves were collected at dawn, dark-adapted for 30 min at 4 °C and Fv/Fm was measured. For electrolyte leakage measurements 8–10, discs 8 mm in diameter were cut from each leaf. Each disc was randomly allocated to a 10 mL tube and placed in a water bath (Julabo Labortechnik, Seelbach, Germany) at 5 °C for 30 min. The bath was then cooled to −1 °C over 30 min. After the samples were held at −1 °C for 30 min, a small amount of ice was placed in contact with the cut edge of the leaf disc to cause ice nucleation. After a further 30 min at −1 °C, the water bath was cooled at a rate of 1 °C 30 min−1, down to a minimum of −8 and −17 °C for the first and second measurements, respectively, and held at each temperature for 30 min. At each sampling temperature, a subset of the tubes were removed and allowed to thaw on the bench for 10 min prior to the addition of 4 mL of deionized water to each tube. The discs were carefully removed from the solution and dark-adapted at 4 °C for 30 min before measuring Fv/Fm with a plant efficiency analyser (Hansatech, King’s Lynn, UK). The leaf discs were returned to their tubes and stored at 4 °C for 24 h before adding a further 3 mL of deionized water to each tube. The conductivity of the solution was measured using a TPS LC81 conductivity electrode (TPS, Springwood, Australia). The leaf discs were placed in liquid nitrogen for 1 min and then returned to their solution for a further 24 h after which time the conductivity was measured again. Electrolyte leakage at each temperature was expressed as a proportion of total electrolyte leakage (TEL). Second-order polynomial functions were fitted to the electrolyte leakage data to determine the point at which 50% TEL (TEL50) was observed from the leaf disc.
The randomized block design of both the OTC and FATI parts of the experiment was used for statistical analyses. Curves were fitted and data were analysed by one-way analysis of variance and independent t-tests where appropriate using Statistical Package for the Social Sciences software (v12.0.1, SPSS Inc, Sydney, Australia).
Electrolyte leakage, a measure of cell intactness and the change in Fv/Fm, a measure of the functional status of photosystem II, were measured on leaf discs taken from a subset of plants prior to commencement of the experiment for an initial assessment of freeze tolerance (Fig. 4), giving an indication of freeze tolerance in unacclimated seedlings. Both Fv/Fm and electrolyte leakage data show that photosynthetic efficiency and cell intactness began to decline at a nadir temperature of −3 °C and by −6 °C, the Fv/Fm was reduced to 0.1 and close to 100% of the electrolytes had leaked from the cells.
The electrolyte leakage curves for both FATI and OTC parts of the experiment are shown in Fig. 5. Seven days after the commencement of treatments, control plants in both FATI (Fig. 5a) and OTC (Fig. 5c) experiments were more tolerant to lower nadir temperatures than warmed plants or those exposed to elevated CO2. After 28 d of treatment in the FATI experiment (Fig. 5b), acclimation in both control and warmed plants to freezing temperatures had developed considerably, but the latter still suffered greater electrolyte leakage at lower nadir temperatures than the former. Freeze tolerance after 21 d of treatment had also developed for plants grown in the OTC (Fig. 5d); however, the plants growing under elevated CO2 were no longer behind their ambient-grown counterparts with regards to freeze tolerance.
Electrolyte leakage data were further analysed to determine the nadir temperature at which TEL50 was observed from the cells. Table 1 showed that 1 week after treatments commenced, freezing tolerance was lower in seedlings subject to either daytime FATI warming or elevated CO2 than in their counterparts growing under ambient conditions. The TEL50 indicated that plants exposed to either FATI warming or elevated CO2 for 7 d were significantly (P < 0.01) more susceptible to freeze-induced membrane damage than their control counterparts (Table 1). After 21 and 28 d of exposure to elevated CO2 and to FATI warming freezing, respectively, tolerance increased to similar levels in treated and control plants.
Table 1. The temperature at which 50% total electrolyte leakage was observed. Free air temperature increase (FATI) warmed (n = 5) and control plants (n = 3), and plants exposed to elevated (n = 5) or ambient (n = 5) CO2 were studied. The P-values were determined by an independent t-test between treatments within each experiment
OTC, open top chamber; ns, not significant.
−3.84 ± 0.31
−5.78 ± 0.44
−5.89 ± 0.38
−6.58 ± 0.64
−2.93 ± 0.39
−5.00 ± 0.49
−4.73 ± 0.24
−4.64 ± 0.55
The electrolyte leakage results were supported by a change in the Fv/Fm, a measure of the functional status of photosystem II, in thawed, dark-adapted leaves after freezing to different nadir temperatures (Fig. 6). The Fv/Fm values were initially high in all leaves, but declined with decreasing temperature below a threshold of −2 °C. At lower temperatures, Fv/Fm remained significantly higher in control leaves than in those subject to either FATI warming (Fig. 6a, P < 0.001) or elevated CO2 (Fig. 6c, P < 0.01), clearly indicating greater freeze tolerance in the control leaves. Freezing tolerance increased dramatically after 28 and 21 d of FATI warming and exposure to elevated CO2, respectively (Fig. 6b & d); significant differences between treatments and controls were no longer apparent. However, freeze tolerance in plants grown under elevated CO2 appeared to lag behind that of the control ones. These results show that daytime warming and elevated CO2 delayed development of freezing tolerance by 2–3 weeks.
The effect of elevated CO2 on plant freeze tolerance reported in the literature is variable with increased (Betula alleghaniensis; Wayne, Reekie & Bazzaz 1998) to decreased tolerance (Viaccinium myrtillus; Taulavuori et al. 1997) and unchanged levels of freezing tolerance (Picea abies; Wiemken, Kossatz & Ineichen 1996) reported. The lack of consistency among the experiments that examine freezing tolerance under elevated CO2 is likely to be because of species-specific responses in terms of the interaction between elevated CO2, the specific mechanisms employed to develop freeze tolerance and experimental conditions. For example, in agreement with our results Terry, Quick & Beerling (2000) found that between −4 and −6.5 °C, ginkgo (Ginkgo biloba) exposed to elevated CO2 suffered significant membrane damage and a decline in Fv/Fm compared to ambient grown plants. The reduced freeze tolerance in ginkgo was maintained after experimental exposure to freezing temperatures, whereas our results with E. pauciflora show that the freeze tolerance of elevated CO2 or warmed plants was increased to levels similar to those achieved by ambient CO2 or non-warmed plants within 2–3 weeks. Importantly, the ginkgo were grown in glasshouses where the minimum temperature was 3 °C, and so they were not exposed to the natural decline in temperature necessary for acclimation. This highlights the importance of field-based work where plants experience natural seasonal variation in temperature.
The present experiment was conducted in the field, and we did not attempt to control all environmental factors, only influencing temperature and [CO2]. The plants were deep-watered at the time of planting and no further irrigation was supplied. It is therefore possible that the plants experienced water deficit because of the low rainfall, that is common during the part of year when the experiment was conducted (see Materials and Methods section). This does not affect the interpretation of our results; environmental stresses such as low temperature and drought both cause water deficit in plants and therefore, the plant cellular and physiological response is similar (Ingram & Bartels 1996). In fact, similar families of genes and proteins, such as DREB1/CBF genes, are often found to have increased levels of expression when plants are exposed to drought and low temperatures (Hsieh et al. 2002; Seki et al. 2003).
We interpret the similarity in effects of daytime warming and elevated CO2 on acclimation to freezing temperatures as an indication of a common cause, namely an increase in daytime leaf temperature. The data from Fig. 3 show that the leaf temperatures experienced in both the FATI and OTC experiments were very comparable, and therefore we feel confident in interpreting the results together. Both treatments should only affect leaf temperature during daytime, and hence have no effect on minimum leaf temperatures at night. This suggests that the diurnal range in leaf temperature, not just the temperature minima, affects acclimation to freezing temperatures. Nevertheless, cold acclimation is initiated by gradual exposure to decreasing temperatures; the time taken to reach full freeze tolerance is species-dependent but can vary from days to weeks (Glilmour, Hejela & Thomashow 1988; Webb, Uemura & Steponkus 1994). How a plant senses the decreasing temperatures that induce freeze tolerance may be confounded by interactions between elevated CO2, leaf temperature and the signalling pathways responsible for initiating freeze tolerance. Calcium, abscisic acid (ABA) signalling, and protein kinases and phosphatases have been proposed as cold temperature transducers, probably acting in combination rather than in isolation (Xin & Browse 2000). Cytosolic calcium increases transiently when a plant is exposed to cold temperatures (Monroy & Dhindsa 1995; Sheen 1996; Angeli, Malho & Altamura 2003). Elevated leaf temperature can affect membrane fluidity, which is thought to be one of the main triggers for the activity of calcium channels (Monroy & Dhindsa 1995). Firstly reducing calcium influx to the cytosol may interfere with calcium signalling and subsequent cold-regulated gene expression. Secondly, increasing concentrations of the hormone ABA plays a role in the development of freeze tolerance (Chen, Brenner & Li 1983). Reduced gc and changes in xylem sap pH caused by exposure to elevated CO2 could certainly influence the concentration of ABA present and available to act as a signal for the perception of cold temperatures. Finally, changes in xylem sap pH, because of growth under elevated CO2, can reduce NO–3 assimilation resulting in a decline in protein synthesis (Rachmilevitch, Cousins & Bloom 2004), which in turn could affect the rate of acclimation. So there are at least three mechanisms associated with the development of freeze tolerance that may be altered by our experimental conditions.
Under the projected climatic conditions of higher atmospheric CO2 and increased mean temperatures, our results suggest that the success of plant species that are exposed to seasonal frost events may be reduced. The severity of the initial frost will be crucial in determining the degree of photosynthetic depression and of tissue necrosis suffered, and thus how quickly the individual plant can recover. If significant injury occurs early in the growing season, photosynthetic carbon gain may be detrimentally affected. Long-term carbon gain will also be affected by the rate of cold deacclimation, a process which has not been as widely studied as acclimation (Rapacz 2002), yet the speed of deacclimation to cold or freezing temperatures is equally important as acclimation for plant survival (Svenning, Rosnes & Juttila 1997). Adverse effects of elevated CO2 on freezing tolerance were observed in both autumn and spring. As elevated air temperatures can accelerate deacclimation in numerous plant species (Taulavuori et al. 1997; Rapacz 2002), we predict that higher leaf temperatures associated with lower gC under elevated CO2 could increase rates of deacclimation to freezing temperatures in spring. This might explain, for example, the observation that elevated CO2 promoted frost damage in snow gum seedlings, when a severe frost occurred after 2 weeks of mild weather in early spring (Lutze et al. 1998). Apparently, seedlings exposed to elevated CO2 were more deacclimated than their control counterparts. The signalling processes that lead to deacclimation are less understood, but are likely to be similarly affected by leaf temperature and exposure to elevated CO2 as acclimation.
The Intergovernmental Panel on Climate Change presents scenarios that predict a doubling of atmospheric CO2 and an increase in mean global temperature of 1.4–5.8 °C by 2100 (Watson et al. 2001). These two factors would have an additive effect on leaf temperature with far-reaching consequences for plant function. Indeed, our results showed that warmer daytime leaf temperatures under elevated CO2 delay acclimation to freezing temperatures. Paradoxically, this makes plants more susceptible to frost damage as growing seasons lengthen and consequently, the period during which a plant is not acclimated increases. An early-season freezing event during a longer growing season has the potential to have significant long-term impacts on plant growth and survival (Inouye 2000). Thus in a future high-CO2 world, an increase in temperature minima will not necessarily decrease the incidence of frost damage, and may reduce potential gains in productivity from CO2 fertilization as shown in Barker et al. (2005), and may alter species responses to climate warming as shown by Kullman (1998).
The authors would like to thank Mr. Paul Sillis for permission to conduct experiments on his property, Mr. Wayne Pippen for technical assistance, and Professor Martin Canny, Professor Graham Farquhar, Dr. John Evans and Dr. Chris Mitchell for useful comments on drafts of this manuscript.