CO elevation improves photosynthetic performance in 2 progressive warming environment in white birch seedlings

White birch (Betula paperifera Mash) seedlings were exposed to progressively warming in greenhouses under ambient and elevated CO concentrations for 2 5 months to explore boreal tree species’ potential capacity to acclimate to global climate warming and CO elevation. In foliar gas exchange, in vivo 2 situ carboxylation characteristics and chlorophyll fluorescence were measured at temperatures of 26 C and 37 C. Elevated CO significantly increased net o o 2 photosynthetic rate (Pn) at both measurement temperatures, and Pn at 37 C o was higher than that at 26 C under elevated CO . Stomatal conductance (gs) o 2 was lower at 37 C than at 26 C, while transpiration rate (E) was higher at 37 o o o C than that at 26 C. Elevated CO significantly increased instantaneous o 2 water-use efficiency (WUE) at both 26 C and 37 C, but WUE was markedly o o enhanced at 37 C under elevated CO . The effect of temperature on maximal o 2 carboxylation rate (Vcmax), PAR-saturated electron transport rate (Jmax) and triose phosphate utilization (TPU) varied with CO and the Vcmax and Jmax 2, were significantly higher at 37 C than at 26 C under elevated CO . However, o o 2 there were no significant interactive effects of CO and temperature on TPU. 2 The actual photochemical efficiency of PSII (∆F/ Fm’), total photosynthetic linear electron transport rate through PSII (JT) and the partitioning of JT to carboxylation (Jc) were higher at 37oC than at 26oC under elevated CO2. Elevated CO2 significantly suppressed the partitioning of JT to oxygenation (Jo/JT). The data suggest that the CO2 elevation and progressive warming greatly enhanced photosynthesis in white birch seedlings in an interactive fashion. Referees

months to explore boreal tree species' potential capacity to acclimate to global climate warming and CO elevation. In foliar gas exchange, in vivo there were no significant interactive effects of CO and temperature on TPU. 2 The actual photochemical efficiency of PSII (DF/ Fm'), total photosynthetic linear electron transport rate through PSII (JT) and the partitioning of JT to carboxylation (Jc) were higher at 37 C than at 26 C under elevated CO . Global climate warming and increases in atmospheric CO 2 concentration are currently key topics for scientists, politicians and the general public alike 1 . Such changes have been observed in the past 150 years and supported by modeling results for the longer term, e.g., warming ocean water, shrinking mountain glaciers, retreating snow cover, and CO 2 concentration dynamics in Arctic/Antarctic ice cores 2-6 . It is projected that the global temperatures will increase by an average of 3°C with a range of 2 to 4.5°C under the scenario of doubling atmospheric CO 2 concentration by the end of the century 2 . Global climate warming will likely have profound and diverse impacts on biological systems 2, 4-13 .
Increases in CO 2 and temperature to a certain extent should have positive impact on photosynthesis and growth, as the current atmospheric CO 2 concentration is below the saturation point for RuBisCO (Ribulose-1,5-bisphosphate carboxylase oxygenase) 14 . Furthermore, higher CO 2 concentrations suppress photorespiration and increase the partitioning of photosynthetic electron transport to carboxylation 14 . However, the situation will become complicated if the temperature goes beyond plants' ability to acclimate, or when the rate of temperature increase exceeds the pace of acclimation. In such cases, temperature and CO 2 will have opposite effects on photosynthesis, i.e., the higher temperature induced increase in photorespiration may exceed the beneficial effect of CO 2 elevation, resulting in a decline in net photosynthesis. Consequently, the direction and magnitude of change in net photosynthesis will be determined by the relative magnitudes of the two opposite effects 15 . Kirschbaum 16 has conducted a theoretical analysis on the dependence of photosynthesis on temperature and CO 2 concentration for C3 plants and found that at 35°C, photosynthesis at the ambient CO 2 concentration reaches only 50% of the rate at saturating CO 2 concentration, whereas the corresponding value at 5°C is 77%. Therefore, there is greater potential photosynthetic enhancement by CO 2 elevations at higher temperatures. This theory has been supported by the results of a number of studies 15, 17-19 . Long 20 has suggested that the increase in atmospheric CO 2 will not only increase photosynthetic rate, but also alter the photosynthetic response to temperature. Mooney et al. 21 indicate that the photosynthetic acclimation to elevated temperature and CO 2 mainly involves changes in the heat stability of the thylakoids and RuBisCO activity. Hence, high temperature and CO 2 elevations may have synergistic effect on photosynthesis and CO 2 elevations may lead to improved acclimation to high temperatures. However, such interactions may vary with plant species 22,23 and other environmental conditions. Variations in acclimation ability can change the interactions within and between species and the composition and functioning of plant communities under future climatic conditions. Furthermore, in most past studies, high temperature treatments are achieved in one step, which is in contrast with the gradual, progressive increases in temperature occurring in global climate changes.
The boreal forest is an important terrestrial ecosystem with a high carbon sequestration potential 24 . As the global climate change accelerates, the boreal forest has been experiencing progressive increases in temperatures and CO 2 . The response of the boreal forests could have great impact on the global carbon balance 25 .
White birch is one of the most widely distributed tree species in the boreal forest. The growth conditions of white birch in northwest Ontario are characterized by a long cold winter and short summer. For example, the annual mean temperature in Thunder Bay region is 2.4°C while the January and July average temperatures are -14.9°C and 17.6°C (based on Environment Canada's online weather records for the time period of January 1943 to December 2003). Nevertheless, based on our past experience in growing white birch seedlings in greenhouses, it appears that the species is capable of acclimating to continuous warming to more than 40°C in the early afternoon on sunny summer days (see Figure 1). In this current study, we test the hypothesis that CO 2 elevation will enhance the photosynthetic performance of white birch seedlings growing in a progressively warming environment.

Plant materials
White birch (Betula papyrifera Mash.) seedlings were grown from seeds in the greenhouses at the Thunder Bay campus of Lakehead University. The growing medium was a mixture of peat moss and vermiculite (1:1 (v/v)).

Experiment design
The seedlings were subject to a progressive warming in the greenhouses as the season progressed from March to August ( Figure 1). The temperatures in all the greenhouses were monitored and recorded using a computerized environment control system (Argus, Vancouver, Canada). The highest recorded temperature in the greenhouse was 44.8°C in the later stages of the experiment ( Figure 1). The seedlings were grown under two CO 2 concentrations (i.e., the ambient (360 µmol mol -1 ) and elevated (650 µmol mol -1 )). The two CO 2 treatments were conducted simultaneously in separate greenhouses with identical design and dimensions. The CO 2 elevation was achieved using Argus CO 2 generators (Argus, Vancouver, Canada). A photoperiod of 16-hour was maintained (the natural light was supplemented by high-pressure sodium lamps on cloudy days, early mornings and late evenings).
The moisture content of the growing medium was maintained at around 50%, as measured using a HH2 Moisture Meter (DELTA-T DEVICES, Cambridge, UK). The seedlings were watered up to twice a day during the summer to maintain the soil moisture condition. The seedlings were fertilized weekly with a solution of 100 µmol mol -1 N, 35 µmol mol -1 P and 66 µmol mol -1 K.

Simultaneous measurements of in situ gas exchange and chlorophyll fluorescence
The foliage gas exchange was measured using a PP-Systems CIRAS-1 open gas exchange system (Hitchin, Hertfordshire, UK). The environmental conditions in the broad-leaf chamber were controlled automatically. The environmental conditions for measuring the Pn-C i (C i = intercellular CO 2 concentration) curve were as follows: 26°C and 37°C air temperature, which were close to the highest temperatures in the early and late period of the experiment, 800 µmol m -2 s -1 PAR (PAR = photosynthetically-active radiation) and 50% relative humidity. The in vivo maximal carboxylation rate (V cmax ), PAR-saturated electron transport rate (J max ), triose phosphate utilization (TPU) and other relevant parameters were calculated from the Pn-C i curves according to Farquhar et al. 26 , van Caemmerer and Farquhar 27 , Sharkey 28 , Harley and Sharkey 29 and Harley et al. 30 . The Pn-C i curves were fit using the Photosyn Assistant software (Dundee Scientific, Scotland, UK) to estimate V cmax , J max and TPU. The parameters for the kinetics of RuBiscCO, i.e., Kc, Ko and t, and their temperature dependencies were adopted from Harley et al. 30 and Wullschleger 31 .
Three seedlings were selected randomly from each treatment combination for the measurement. The measurement was taken on the top 5 th mature leaf. All the in situ measurements were made between 9:00 and 11:30 AM with the seedlings in their original positions and conditions of the treatments.
The chlorophyll fluorescence was measured using a FMS-2 portable pulse-modulated fluorometer (Hansatech Instruments Ltd. Norfolk, UK). The probe was integrated in the leaf chambers of the gas exchange system and the control software for the two systems was also integrated to allow the simultaneous measurement of gas exchange and chlorophyll fluorescence. The following variables were obtained: fluorescence intensity at any time, F; the maximal fluorescence in light, F m '; the actual photochemical efficiency of PSII in light, (F m '-F)/F m ' or DF/F m ', which is the efficiency under the actual degree of reaction centre closure 32 . F m ' was obtained by illuminating the foliage with a pulse of strong light (around 14000 µmol photons m -2 s -1 ) for 800 ms. The DF/ F m ' was measured simultaneously with each gas exchange measurement. Both gas exchange and chlorophyll fluorescence were measured after 5 months of the treatments.
The apparent rate of total electron transport (J T ) and its partitioning between carboxylation (J c ) and oxygenation (J o ) were calculated based on the methods of Farquhar et al. 26 , Genty et al. 33 and Epron et al. 34 .

Statistical analysis
All the data were examined graphically for the normality of distribution (probability plots for residual analysis) and the homogeneity of variance (scatter plots) using the Data Desk (version 6.01, Data Description, Inc. 1996) 35 before the Analysis of Variance (ANOVA) was carried out. Some of the data were log-transformed to meet the two assumptions for ANOVA. The data were analyzed using the two-way ANOVA procedure of the Data Desk. When the interaction between temperature and CO 2 was significant, Scheffe's F test for post hoc pairwise comparisons was conducted.

Results
In situ photosynthetic gas exchange There was a significant (P<0.01) interactive effect of temperature and CO 2 on Pn (Figure 2). Pn was higher (P<0.01) at 37°C than at 26°C under elevated CO 2 (Figure 2), but there was no significant (P>0.05) temperature effect on Pn under ambient CO 2 . CO 2 elevation significantly increased Pn at both temperatures (P<0.05, P<0.001 at 26°C and 37°C, respectively). g s significantly (P<0.05) decreased at 37°C under both ambient and elevated CO 2 (Figure 2), and there was no significant (P>0.05) CO 2 effect on g s . Meanwhile high temperature significantly (P<0.05) stimulated E under both ambient and elevated CO 2 (Figure 2). Water-use efficiency (WUE) was significantly (P<0.05) higher at 26°C than that at 37°C under both CO 2 regimes. CO 2 elevation greatly (P<0.001) increased WUE at both temperatures.
High temperature significantly reduced C i under both ambient and elevated CO 2 (P<0.05, P<0.01, respectively) and, also, elevated CO 2 significantly (P<0.001) increased C i at both temperatures.
In vivo RuBisCO activity V cmax , J max and TPU at 37°C were significantly (P<0.001) higher than those at 26°C (Figure 3). The temperature dependencies of V cmax and J max were changed by CO 2 , and those values at 37°C enhanced much more (P<0.05) under elevated CO 2 than under ambient CO 2 .
Photosystem II efficiency and electron transport partitioning to carboxylation and oxygenation There was a significant (P<0.001) interactive effect of CO 2 and temperature on (F m '-F)/F m ' and J T (Figure 4). (F m '-F)/F m ' and J T greatly increased at 37°C as compared to at 26°C under elevated CO 2 , and there was no significant temperature effect on (F m '-F)/F m ' and J T under ambient CO 2 . ± SD, n=3-4) for current year white birch seedlings after they were exposed to continuous warming under ambient CO 2 and elevated CO 2 concentrations for 5 months. The in situ measurements were taken at 26°C and 37°C under ambient CO 2 and elevated CO 2 . The significance levels (*** = P<0.001, ** = P<0.01, * = P<0.05). If the interaction between measurement temperature and CO 2 was significant for a given parameter, Scheffe's F test for post hoc pairwise comparisons was conducted. Means sharing the same letter or letters are not significantly different.

Figure 2. Pn, g s , E and WUE (mean
The pattern of CO 2 and temperature effects on J c was almost the same as (F m '-F)/F m ' and J T (Figure 4), and J c was greater (P<0.001) at 37°C than that at 26°C under elevated CO 2 , and there was no significant temperature effect on J c under ambient CO 2 . Elevated CO 2 greatly suppressed J o /J T , and there was no significant (P > 0.05) effect of temperature on J o /J T (Figure 4).

Discussion
Our results suggest that the photosynthetic mechanisms of white birch seedlings have high capacity to acclimate to a progressively warming environment, particularly under elevated CO 2 . This result is in contrast to the results of most studies with a single step warming treatment. Larcher 36 has suggested that plants' optimal temperature is closely related to the climate in which they grow. The measurement temperatures of 26°C and 37°C used in this study are believed to be the normal (or optimal) and stressful temperature, respectively, for most boreal forest tree species growing at their natural environments. Zhang et al. 37 have found that the Pn of mature oak even in warm-temperate zones decline greatly at temperatures over 30°C, as compared to measurements at temperatures of 20-30°C, which occurs naturally north of temperate zones or even warm-temperate zones. However, in this experiment the Pn of white birch didn't decline at 37°C under ambient CO 2 , as compared to that at 26°C; furthermore, Pn increased substantially at 37°C under elevated CO 2 . These results indicate that the photosynthetic mechanisms of white birch acclimated to the progressive warming environment, and this high temperature acclimation was greatly strengthened by elevated CO 2 . Long 20 argued that CO 2 elevation could change the photosynthesis dependence of temperature.
The activity of RuBisCO is highly temperature-dependent. According to Jordan and Ogren 38 , the Rubisco's specificity for CO 2 /O 2 decreases as increasing temperatures over the optimal range, but the   increase in RuBisCO oxygenation will exceed that of carboxylation because the solubility of CO 2 declines faster than that of O 2 at even higher temperatures, resulting in a decline in net photosynthetic rate. White birch's acclimation to warming was also evidenced by V cmax measured at the two different temperatures and two CO 2 regimes. V cmax at 37°C was much higher than at 26°C under both ambient and elevated CO 2 , indicating a shift in the temperature dependency of RuBisCO. Furthermore, the partitioning of total electron transport to oxygenation was not significantly different between the two temperatures under either ambient CO 2 or elevated CO 2 , suggesting that the higher temperature did not change the RUBisCO specificity for CO 2 /O 2 which could be a contributing factor for the enhanced acclimation of photosynthesis to the progressive warming. Overdieck et al. 15 have also found that both the temperature treatment alone and the combination of elevated CO 2 and temperature depressed V cmax in Scots pine at temperatures below the optimum range, but increased V cmax when the temperature was above the optimum. Additionally, the magnitude of the change in V cmax increased as temperature increased.
The decrease in C i at the high temperature could be attributable to either enhanced RuBisCO activity or declines in stomatal conductance or both. Not only V cmax , but J max and TPU were also higher at 37°C than at 26°C, suggesting that the CO 2 assimilation process, including carboxylation, electron transport for RuBP regeneration, ATP supply and the translocation of the primary photosynthates, all maintained at high levels in the warm environment. In this study, there was no down-regulation of RuBisCO activity in association with the CO 2 elevation, to the contrary, CO 2 elevation greatly increased V cmax and J max at 37°C, as well as Pn at both 26°C and 37°C.
While high temperature enhanced V cmax under both ambient and elevated CO 2 , the increases in actual PSII efficiency (DF'/F m ') and J c associated with the high temperature only occurred under elevated CO 2 , suggesting that the high temperature did not significantly affect the total electron transport, and its partitioning to carboxylation, t under the ambient CO 2 . Conversely, the partitioning of total electron flow to oxygenation increased more than 40% in response to the high temperature under elevated CO 2 . The reduced electron transport partitioning to carboxylation and low C i might explain why Pn was relatively low at 37°C under the ambient CO 2 , even though the corresponding V cmax was quite high, implying that the slow electron transport to carboxylation and CO 2 supply at high temperature under ambient CO 2 didn't match the high activity of RuBisCO. This again confirms Kirschbaum's theoretical analysis that photosynthesis has a higher potential to be stimulated by CO 2 elevation at high temperatures than at low temperatures 16 .
Author contributions QLD and SZ conceived and designed the experiment. SZ conducted the measurements and analyzed the data. SZ and QLD wrote and revised the manuscript.

Competing interests
No competing interests were disclosed.