Abstract
Warming shifts the thermal optimum of net photosynthesis (ToptA) to higher temperatures. However, our knowledge of this shift is mainly derived from seedlings grown in greenhouses under ambient atmospheric carbon dioxide (CO2) conditions. It is unclear whether shifts in ToptA of field-grown trees will keep pace with the temperatures predicted for the 21st century under elevated atmospheric CO2 concentrations. Here, using a whole-ecosystem warming controlled experiment under either ambient or elevated CO2 levels, we show that ToptA of mature boreal conifers increased with warming. However, shifts in ToptA did not keep pace with warming as ToptA only increased by 0.26–0.35 °C per 1 °C of warming. Net photosynthetic rates estimated at the mean growth temperature increased with warming in elevated CO2 spruce, while remaining constant in ambient CO2 spruce and in both ambient CO2 and elevated CO2 tamarack with warming. Although shifts in ToptA of these two species are insufficient to keep pace with warming, these boreal conifers can thermally acclimate photosynthesis to maintain carbon uptake in future air temperatures.
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Introduction
Photosynthesis is the largest annual carbon flux between the atmosphere and the biosphere1, taking up ~123 gigatons of carbon per year from the atmosphere2. Terrestrial photosynthesis is ~11 times higher than annual anthropogenic CO2 emissions1, offsetting a significant fraction of anthropogenic CO2 emissions3,4. Thus, relatively small changes in terrestrial photosynthesis due to global change drivers, such as warming and drought, could increase the rate of atmospheric CO2 accumulation and associated climate warming predicted by Terrestrial Biosphere Models (TBMs)5 that are a key component of global climate models.
To improve predictions of CO2 exchange between terrestrial vegetation and the atmosphere in the warmer, elevated CO2 climates of the future, it is critical to account for acclimation of photosynthesis to both warming and elevated CO2 within TBMs6,7. The photosynthetic temperature sensitivity functions currently employed within TBMs were developed using data largely derived from young trees grown in greenhouse warming experiments under ambient atmospheric CO2 conditions6,8. Thus, it is unclear whether these thermal responses accurately represent mature trees growing in natural conditions in the field and whether they hold under elevated atmospheric CO2 conditions.
Photosynthesis is regulated by several types of processes (biochemical, biomechanical and diffusional) which are all temperature dependent9,10,11. In the short-term (minutes to hours), photosynthesis responds non-linearly to temperature, increasing up to a thermal optimum (ToptA) and decreasing at supra-optimal temperatures. The decrease of photosynthesis at supra-optimal temperatures is caused by various processes including increased membrane fluidity12,13, impaired redox reactions between protein complexes and electron carriers14, reduced intracellular CO2 availability due to stomatal closure15, deactivation of the key photosynthetic enzyme Rubisco (ribulose-1,5-biphosphate carboxylase/oxygenase)16, and the release of previously-fixed CO2 through high respiration and photorespiration rates5,9,10,11. When exposed to long-term warming (days to years), plants generally acclimate photosynthesis by increasing the ToptA8,11,17,18,19,20,21,22,23, thereby increasing net carbon uptake at the new warmer temperature. This acclimation to high temperatures can involve decreased thylakoid membrane fluidity24, expression of a more heat-stable Rubisco25 and Rubisco activase11, expression of heat shock proteins11, and decreases in respiration26,27,28. However plants differ greatly in their ability to thermally acclimate ToptA, with reported values in the literature ranging from increases in the ToptA of 0.16–0.78 °C per 1 °C of warming8,11,19,22,29,30,31. Among the conifers that dominate the boreal forest, some species have shown the ability to acclimate ToptA30,32,33 to warming, while others have not34. Whether such stark differences in acclimation capacity are truly representative (i.e., do some species acclimate while others do not) or result from modest sampling intensity is as of yet unclear. Moreover, these studies on boreal conifers have been conducted on seedlings in growth chambers and greenhouses, and it is unclear whether these photosynthetic acclimation responses translate to mature trees growing in the variable air temperatures found in the forest. Furthermore, these studies rarely investigate whether increases in ToptA match increases in growth temperature. In a three-year field warming study on broad-leaved boreal and temperate seedlings, shifts in ToptA occurred but were much smaller than increases in growth temperatures19. However, no study to date has explored whether mature field-grown conifers, the trees that represent the majority of the boreal forest, can adjust ToptA to compensate for the increasing air temperatures expected over the next few decades.
Photosynthesis and ToptA are also affected by elevated CO2. Elevated CO2 concentrations stimulate photosynthesis because CO2 is the substrate for Rubisco35,36,37, the carboxylating enzyme in C3 photosynthesis. In the long term, this initial stimulation of photosynthesis often (but not always38) diminishes39 due to acclimation of the photosynthetic biochemistry to elevated CO2 concentrations and plant sink limitations36,40,41. In some instances, the initial stimulation of photosynthesis by high CO2 completely disappears, mainly due to nitrogen limitation42. By increasing the concentration of CO2 around Rubisco, growth in elevated CO2 concentrations also suppresses photorespiration43, a process that releases previously fixed CO2. Given that high temperatures stimulate photorespiration5,9,44, plants grown and measured under elevated CO2 have a higher ToptA than those grown and measured at current CO2 levels9,18,23,30, reflecting the suppression of photorespiration at high temperatures by elevated CO29,30,45.
Studies of the thermal sensitivity of photosynthesis have focused on ambient CO2-grown plants6,8,11,17,20,46, and less on how elevated CO2 may alter temperature acclimation5,47. Because of this, the temperature sensitivity functions currently employed in TBMs are derived from ambient CO2-grown plants6,48. To date, only a handful of studies have assessed the effect of elevated CO2 on thermal acclimation of photosynthesis23,30, and only one has investigated the effect of elevated [CO2] on the temperature sensitivity parameters of net photosynthesis and its underlying biochemical processes (maximum Rubisco carboxylation rate—Vcmax, and maximum electron transport rates—Jmax)30. This latter study, conducted on boreal conifer seedlings grown in pots for six months, reported that elevated CO2 had little effect on thermal acclimation of the temperature sensitivity parameters of Vcmax and Jmax (i.e., their thermal optima and activation energies)30. In the same study, warming increased ToptA by 0.36–0.65 °C per 1 °C warming regardless of CO2 treatments. But elevated CO2-grown seedlings had a ToptA that was generally 3.6–4 °C higher than their ambient CO2-grown counterparts when measured at prevailing growth CO2, likely due to direct suppression of photorespiration by elevated CO2.
The key photosynthetic temperature sensitivity parameters employed in TBMs include ToptA, as well as the thermal optima (ToptV and ToptJ) and activation energies (EaV and EaJ) of Vcmax and Jmax6,7. The responses of these parameters to long-term changes in temperature, either due to experimental warming or natural seasonal variation, are primarily driven by thermal acclimation and less influenced by adaptation to different thermal environments8,21. This implies that results generated in this study, using boreal tree species, could have implications for plants grown in natural conditions from different thermal environments.
In this study, we assessed the thermal acclimation of photosynthesis and its underlying biochemical processes (i.e., Vcmax and Jmax) in mature trees (~45 years) of tamarack (also known as larch), a deciduous conifer, and black spruce, an evergreen conifer, exposed to either ambient (hereafter aCO2) or elevated CO2 (≈+460 ppm above ambient; hereafter eCO2) combined with a warming of up to +9 °C above ambient temperatures in a regression-based design with five temperature treatments (ambient +0, +2.25, +4.5, +6.75, and +9). The data presented were collected after 2 years of warming combined with one year of CO2 treatment at the Oak Ridge National Laboratory’s SPRUCE (Spruce and Peatland Responses Under Changing Environments; https://mnspruce.ornl.gov) project site at the U.S. Forest Service’s Marcell Experimental Forest, in Minnesota, USA (47°30.476’ N; 93°27.162’ W).
Here we show that ToptA of mature boreal conifers increased with warming, and this warming-induced increases in ToptA were correlated with simultaneous increases of the thermal optima of underlying photosynthetic biochemical processes (Vcmax and Jmax). However, shifts in ToptA did not keep pace with warming as ToptA only increased by 0.26–0.35 °C per 1 °C of warming. But when estimated at the mean growth temperature, net photosynthetic rates increased with warming in eCO2 spruce, while remaining constant in aCO2 spruce and in both aCO2 and eCO2 tamarack with warming. Our overall finding is that, although shifts in ToptA of these two species are insufficient to keep pace with warming, these boreal conifers can thermally acclimate photosynthesis to maintain carbon uptake in future air temperatures.
Results
Shifts in thermal optimum of net photosynthesis (T optA)
The ToptA increased by 0.26 and 0.35 °C per 1 °C warming in tamarack and black spruce, respectively, and this shift was similar for both aCO2- and eCO2-grown trees (Fig. 1, Supplementary Figs. 1 and 2, and Supplementary Table 1). In addition, ToptA was 3 °C higher in eCO2-grown than ambient-grown tamarack, while CO2 had no effect on ToptA in black spruce (Fig. 1 and Supplementary Table 1). Warming-induced increases in ToptA were correlated with increases of the thermal optima of photosynthetic biochemical processes, ToptV (0.35 and 0.44 °C per 1 °C warming for tamarack and black spruce, respectively) and ToptJ (0.26 and 0.55 °C per 1 °C warming for tamarack and black spruce, respectively) (Fig. 2, Supplementary Figs. 3–7, and Supplementary Tables 1 and 2). There was no evidence of acclimation of the activation energy for Vcmax in either species (Supplementary Fig. 3e, f). However, in black spruce the activation energy of Jmax declined non-linearly with warming in eCO2-grown trees but not in aCO2-grown counterparts, while in tamarack it was unaffected by warming (Supplementary Figs. 3g, h and Supplementary Tables 1 and 2). Furthermore, neither stomatal conductance nor respiration were correlated with the shifts in ToptA seen in either species (Supplementary Table 3a, b).
Exceedance of T optA by mean growth temperature
Photosynthesis typically acclimates to prolonged exposure to warming within 10 days19,20,21. Therefore, we assessed to what extent warming-induced shifts in ToptA matched the increases in growth temperature (expressed as the difference between mean air temperature for the 10 days preceding each measurement and the respective ToptA; ΔMeanTg). This approach assumes that leaf and air temperatures are similar, a reasonable assumption considering the tight coupling between leaf and air temperature in small leaves49, such as conifer needles. In aCO2-grown tamarack and black spruce, mean daytime growth temperature exceeded ToptA (ΔMeanTg > 2 °C) across all warming treatments (+2.25 to +9 °C) (Fig. 3 and Supplementary Table 4). However, eCO2 reduced the ΔMeanTg for tamarack in the +2.25 °C treatment, while for black spruce, eCO2 had weak or no effect on ΔMeanTg across all warming treatments (Fig. 3b, d and Supplementary Table 4).
Elevated CO2 impacts on thermal sensitivity of net photosynthesis
We also examined the impact of the treatments on the model parameter representing the spread of the instantaneous temperature response of net photosynthesis (b in Eq. 2, see “Methods”). A high b value represents a narrower temperature response curve of photosynthesis and thus higher sensitivity to short-term temperature fluctuations21. In both species, b was unaffected by warming in aCO2-grown trees. However, the impact of eCO2 differed between the two species. In tamarack, b was constant in eCO2-grown trees across the warming treatments, but 86% higher than in the aCO2 tamarack (Supplementary Fig. 9 and Supplementary Table 1), suggesting an overall CO2-induced increase in short-term temperature sensitivity (Supplementary Fig. 2). In contrast, in black spruce, CO2 had no effect on b in the temperature control treatments (+0). However, b marginally increased (p = 0.067) with warming in the eCO2-grown trees, such that it was 68% higher in eCO2 than in AC in the warmest plot (+9 °C) (Supplementary Fig. 9 and Supplementary Table 1), suggesting an eCO2-induced increase in the temperature sensitivity of net photosynthesis as it gets warmer (Supplementary Fig. 3).
Net photosynthetic rates at the T optA and growth temperature
Thermal acclimation of net photosynthesis can also be assessed by examining the extent to which net photosynthetic rates at the thermal optimum (Aopt) and at prevailing growth temperature are affected by warming17. In tamarack, Aopt was constant across the warming treatments but with overall higher rates in eCO2 trees compared to their aCO2 counterparts (Fig. 4a and Supplementary Table 1). By contrast, in black spruce, there was an interaction of warming and elevated CO2 such that Aopt significantly increased with warming in eCO2 trees, while it was constant across warming in aCO2 trees (Fig. 4b and Supplementary Table 1). Moreover, net photosynthetic rates estimated at mean (Ag) growth temperature exhibited similar responses to Aopt in both species (Fig. 5 and Supplementary Table 1). These results suggest that, overall, the two species were able to maintain their carbon uptake at prevailing growth temperatures. We further estimated net CO2 assimilation at growth temperature conditions for 2 years (2016 and 2017), representing the entire acclimation period to temperature in this study. The results show that net CO2 assimilation rates were not negatively affected by warming in either species throughout the growth seasons of both 2016 and 2017. In tamarack, Ag was constant across warming and CO2 treatments throughout the growth seasons of the 2 years (Supplementary Fig. 10 and Supplementary Table 5). In black spruce, Ag was largely constant across warming treatments in both years for aCO2 trees, while for eCO2 trees, Ag commonly increased with warming (Supplementary Fig. 11 and Supplementary Table 5).
Discussion
We report findings, to our knowledge, from the first field study assessing responses of the short-term temperature sensitivity of photosynthesis to long-term exposure to whole-ecosystem warming (2 years) combined with elevated atmospheric CO2 (1 year) in mature trees (~45 years old). These results provide a benchmark for our understanding of the impacts of these climate change variables (and their potential interaction) on the thermal sensitivity of photosynthesis in long-lived trees that are experiencing gradual increases in temperature and atmospheric CO2 in their natural environment.
We show that the thermal optimum of net photosynthesis (ToptA) increased by 0.26–0.35 °C per °C warming in mature boreal conifers (Fig. 1). These results are comparable to those from a long-term (3 years) field-based warming study with boreal and temperate seedlings, which reported a rise in ToptA of ~0.38 °C per °C warming19. However, our ToptA values were largely exceeded by mean daytime growth temperature under current atmospheric CO2 conditions (Fig. 3), suggesting that shifts in ToptA in mature trees of boreal conifers growing in the natural field conditions may not fully adjust to compensate for increases in ambient air temperatures. Therefore, exceedance of ToptA by prevailing mean air temperatures across treatments implies that the frequent and severe heat stress predicted under climate change will further constrain carbon uptake in boreal forest conifers.
Until now, knowledge of the thermal acclimation of ToptA and its underlying processes was largely based on short-term studies with seedlings grown in artificial growth environments (e.g., pots) and in controlled environmental conditions (e.g., humidity, light). It was thus unclear whether those results would hold for mature trees growing in the field. Observed shifts in ToptA in our study are at the lower end of the spectrum (0.35–0.8 °C per 1 °C) reported for lab-based experimental studies with seedlings22, 30,50, but are comparable to mean values reported by recent meta-analyses for C3 plants (0.3431 and 0.3811 °C per 1 °C), indicating that while seedlings may have a greater ability to acclimate photosynthesis to warming than mature trees, average responses of photosynthetic thermal acclimation can be broadly used. Furthermore, the shift in ToptA with warming in our field study is much lower than that from a recent global compilation (0.62 °C per 1 °C) that estimated shifts in ToptA using seasonal changes in temperature (i.e., acclimatization). Therefore, we suggest that the use of temperature sensitivity parameters derived from ‘acclimatization studies’ should be used with caution when predicting the acclimation of forests to warming in global vegetation models. We also show that thermal acclimation of ToptA is strongly driven by concomitant adjustments of the thermal optima of photosynthetic biochemical processes (Fig. 2), and not changes in stomatal conductance or respiration (Supplementary Table 3a, b), findings that agree with prior work on controlled experiments in seedlings29,30,51, field warming experiments19,21,52, and a recent acclimatization study8. These results imply that changes in photosynthetic biochemical processes strongly underlie the adjustment of photosynthesis to long-term changes in growth temperature, regardless of experimental approach or tree life stage, although stomatal limitations are likely to play a greater role in limiting photosynthesis in water-stressed trees.
Most studies that have examined thermal acclimation of photosynthesis did so on ambient CO2-grown trees. Elevated CO2 is expected to influence the thermal acclimation of photosynthetic biochemistry (i.e., maximum Rubisco carboxylation rate, Vcmax, and maximum electron transport rates, Jmax) mainly due to its suppressive effect on photorespiration53,54 and its direct effects on Rubisco carboxylation35,36,37, both of which are temperature dependent processes5. However, we show that elevated CO2 does not largely affect the thermal optima or activation energies of Vcmax or Jmax (Supplementary Fig. 3 and Supplementary Table 1). These findings with field-grown mature boreal trees agree with an earlier, short-term study with seedlings of the same species30, suggesting that regardless of the experimental approach, life stage, and leaf habit, elevated CO2 does not have strong effects on the thermal sensitivity of photosynthetic biochemical processes, such as Vcmax and Jmax, in boreal conifers. Since Vcmax and Jmax are key parameters for representing carbon uptake within TBMs6, our findings imply that potential interactive effects of elevated CO2 on temperature sensitivity parameters of Vcmax and Jmax (i.e., their activation energies and thermal optima) can be ignored in TBMs. Our findings also suggest that temperature response functions of these parameters, developed mainly from ambient CO2-grown plants8,46 and currently employed in all TBMs6,7,48 might accurately represent carbon uptake for trees growing in both current and projected elevated CO2 conditions in future climates. However, further research on tree species from other biomes and plant functional types (e.g., broadleaved tree and shrub species) are still needed to validate this conclusion for broad use.
We show that the b parameter was generally increased by elevated CO2 for both species, suggesting that elevated CO2 increases the thermal sensitivity of net photosynthesis, a result in line with a shift to photosynthesis being more RuBP-regeneration limited at high CO2 concentrations9. In addition, elevated CO2 did affect the ToptA, but these effects were species dependent. In tamarack, the ToptA was higher in elevated CO2, which likely reflects a direct suppression of photorespiration5,9,43,54. In contrast, there was no effect of elevated CO2 on the ToptA in black spruce, and these results contrast prior findings in black spruce seedlings30. The reasons behind this lack of elevated CO2 effect on ToptA in mature black spruce are unclear since, similar to tamarack, the needle cohorts that were measured developed in prevailing environmental conditions across treatments. However, the magnitude of suppression of photorespiration by elevated CO2 may vary across species or plant functional types—or in our case differences in leaf habit (evergreen versus deciduous). In our study, we cannot make a solid conclusion on the main cause for this, but two possibilities are differences in stomatal (Supplementary Fig. 13 and Supplementary Table 1) and mesophyll conductance between the species. Our data shows little differences in intracellular CO2 concentration between the two species across the two CO2 treatments (Supplementary Fig. 14 and Supplementary Table 1), indicating that stomatal limitations are unlikely to underlie the difference in how ToptA responds to elevated CO2. This leaves mesophyll conductance as a possible factor, as higher mesophyll conductance in tamarack could enhance CO2 supply to Rubisco for a given unit of intercellular CO2. However, without mesophyll conductance measurements we cannot directly prove this, and future research is needed to investigate this possibility.
Even though prevailing air temperatures largely exceeded ToptA, our findings show that photosynthesis acclimated such that at the prevailing daytime mean air temperature (between 9 a.m. and 3 p.m.), net carbon fixation remained constant or even increased (in eCO2 black spruce trees) across the warming treatments (Fig. 5 and Supplementary Figs. 10 and 11). Therefore, our findings imply that warming alone may have little negative impacts on leaf-level carbon uptake in these cold-adapted mature boreal conifers when soil moisture is not limiting55, as is the case at our current study site56. However, ongoing climate change and the increased frequency of strong heat and dry spell events that will accompany it will likely reduce the ability of forests dominated by these species to fix and sequester carbon57. Moreover, increased autotrophic respiration, which is temperature-dependent, has also been indicated as another factor that will release carbon sequestered in these North American boreal forests58. Our previous work from this experiment support this, where we showed that foliar dark respiration did not thermally acclimate in these boreal conifers56, suggesting that although carbon fixation may not be negatively impacted by warming, thermal effects on autotrophic respiration will further reduce the carbon sequestration potential of these forests59.
In summary, our study has implications for the understanding of climate warming effects on carbon uptake of mature boreal conifers growing in field conditions, and for improving the representation of photosynthesis in TBMs. First, we show that although thermal acclimation of ToptA is limited and does not fully match increases in air temperature, photosynthetic carbon fixation is maintained at the prevailing growth conditions through a combination of photosynthetic acclimation and changes in instantaneous temperature responses of photosynthetic processes. Second, our study provides an improved framework for modeling photosynthesis in TBMs considering both warming and elevated CO2, because we provide support for ignoring effects of elevated CO2 on the thermal sensitivity of photosynthetic biochemical parameters (thermal optima and activation energies of Vcmax and Jmax)22. However, we show that it is important to account for effects of elevated CO2 on the ToptA and on the overall thermal sensitivity of net photosynthesis (b parameter).
Methods
Site description and experimental design
This study was conducted at the Oak Ridge National Laboratory’s SPRUCE (Spruce and Peatland Responses Under Changing Environments) project site at the U.S. Forest Service’s Marcell Experimental Forest, in Minnesota, USA (47°30.476’ N; 93°27.162’ W). The details of the study site and experimental design are provided in recent studies from this experiment56,60,61,62. But briefly, this forest grows naturally in a bog located at the southern limit of the boreal peatland forests. The forest is approximately 50 years old as it regenerated following canopy tree removal in 1969 and 197463. The dominant canopy species is Picea mariana (Mill.) B.S.P. (black spruce) mixed with less abundant Larix laricina (Du Roi) K. Koch (tamarack). The understory vegetation is dominated by ericaceous shrubs Rhododendron groenlandicum (Oeder) Kron & Judd and Chamaedaphne calyculata (L.) Moench. The experiment comprises five temperature treatments (ambient or +0, which serves also as the control, +2.25, +4.5, +6.75, and +9 °C above the ambient) established in a regression-based design64. This experiment uses 10 large octagonal open-top enclosures with an interior surface area of 114.8 m2, and a sampling area of 66.4 m2. Five enclosures have an ambient-CO2 atmosphere, while the other five have an elevated CO2 atmosphere varying between +430 and 500 ppm above the ambient. The heating treatments started August 15, 2015, and CO2 treatments were initiated a year later, on June 15, 2016. The targeted temperature treatments and CO2 concentrations were largely achieved (Supplementary Fig. 12).
Plant material sampling and gas exchange measurements
Field measurements were conducted between June 18–30 and August 15–30, 2017. The daytime temperatures (4:00 a.m.–8:30 p.m.) during June and August were 18.97 and 18.02 °C, respectively. We studied the two mixed-age (up to ~45 years old) canopy tree species at SPRUCE, Picea mariana (Mill.) B.S.P. (black spruce) and Larix laricina (Du Roi) K. Koch (tamarack). For black spruce, one branchlet for each, randomly selected tree and in each plot was harvested and 1-year needle cohorts (i.e., developed in growth season of 2016) from each branch was measured. For tamarack, fully expanded current year foliage was used. In the June field campaign, three trees in each plot were randomly sampled, while in the August campaign, only two trees were used. For tamarack, we used the same number of branchlets from different trees in each plot, except in one plot (in ambient CO2 and +0) where only one tamarack tree was available to be sampled. All measurements were made on sun-exposed branchlets cut using a pruning pole. After cutting, branchlets were put in water, and recut under water to avoid xylem transport disruption and stomatal closure. The branches were harvested between 4 and 5 a.m. of the measurement day, placed in water bottles inside a plastic cooler, and transported from the field site in Marcell, Minnesota to the walk-in growth chambers at the University of Minnesota in St. Paul, where the measurements were conducted. The branchlets were re-cut again before starting the measurements. The effect of cutting and the time lag between cutting and gas exchange measurements has been shown not to have significant effect on stomatal conductance in conifers65. Gas exchange measurements were conducted between 10:00 and 20:00 using 7 portable photosynthesis systems (Li-COR 6400 XT, 6400-18 RGB light source, and 6400-22 opaque conifer chamber; LI-COR Biosciences, Lincoln, NE, USA). Net CO2 assimilation rates (A) were measured at a pre-determined saturating light (1800 µmol m−2 s−1) and eleven different air CO2 concentrations (to generate so-called A–Ci curves) in the following order: 400, 300, 200, 50, 400, 500, 600, 800, 1200, 1600, and 2000 µmol mol−1. The A–Ci curves were conducted at five different leaf temperatures (Tleaf): 15, 25, 32.5, 40, and 45 °C. In order to achieve each targeted Tleaf, all measurements were completed inside the growth chamber, allowing the entire branch to be exposed to the desired temperature for at least 30 min before starting measurements at that temperature. As the gas exchange systems were also inside the chamber, this method minimized the measurement error driven by the internal thermal gradient that was recently reported for the LI-6400 instruments66. Since the vapor pressure of the air (VPDair) increases with increasing temperature, resulting in decreased stomatal conductance15, we moistened the soda lime column of the gas exchange systems to reduce stomatal closure associated with high VPDs at high measurement temperatures (>32.5 °C). In total, we present results of 96 A–Ci temperature response curves. After gas exchange measurements, projected leaf area of the measured needles was determined using ImageJ 1.51 software (NH, Bethesda, MD, USA). We, thereafter, corrected for the total leaf area before the analyses.
Parameterization
The FvCB (Farquhar, von Caemmerer, and Berry) C3 photosynthesis model67 was used to derive Vcmax and Jmax from the A–Ci curves using the fitacis function from the plantecophys 1.4-6 R package68 and using the bilinear fitting method. We maintained the default temperature dependencies of the CO2 compensation point in the absence of mitochondrial respiration (Γ*) and the Michaelis–Menten constants for CO2 and O2 (Kc and Ko) from Bernacchi et al.69. The leaf mesophyll conductance for CO2 was not measured, therefore apparent Vcmax and Jmax based on intercellular CO2 concentrations (Ci), rather than the CO2 concentration at the site of carboxylation (Cc) in the chloroplast, were estimated. The temperature sensitivity parameters of Vcmax (ToptV and EaV) and Jmax (ToptJ and EaJ) were derived using the modified Arrhenius function outlined in the following Eq. 170:
where kopt is the process rate (i.e., Vcmax or Jmax; µmol m−2 s−1) at the optimum temperature (Vcmaxopt, Jmaxopt), Hd (kJ mol−1) is the deactivation energy term that describes the decline in enzyme activity at higher temperature, Ea (kJ mol−1) is the activation energy term that describes the exponential increase in enzyme activity with an increase in temperature, R is the universal gas constant (8.314 J mol−1 K−1), and Topt and Tk are the optimum and given temperatures of the process rate (i.e., Vcmax or Jmax; µmol m−2 s−1). The value of Hd was fixed at 200 kJ mol−1 to avoid over-parameterization70,71.
Net photosynthesis data at the tree growth CO2 (400 or ~800 ppm, for ambient CO2 and elevated CO2 treatments, respectively) were extracted from the A–Ci curves. Thereafter, the temperature response of A was fitted using the following Eq. 216 to estimate the ToptA:
where A (T) is the A (µmol m−2 s−1) at a given air temperature T (°C), Aopt is the A at the optimum temperature (Topt), and the b parameter represents the breadth of the photosynthetic temperature response curve; larger values of b indicates that A (T) has greater sensitivity to changes in T. After fitting b and ToptA, we used Eq. 2 to model net photosynthesis at prevailing growth temperature conditions using mean and maximum air temperature (9–4 a.m.) for each plot for 10 days preceding measurement of each tree/species, as well as for the entire growing season period (June–September) of both 2016 and 2017.
In order to estimate to what extent stomatal conductance may have affected the shifts in ToptA, we re-calculated net photosynthesis at a Ci/Ca ratio of 0.7 (A70; with a final Ci of 280 or 560 ppm for ambient and elevated CO2, respectively) using the parameterized Vcmax, Jmax, Rday, and TPU (triose phosphate use) from the plantecophys 1.4-6 R package in the following equations44:
where O the intercellular concentrations of O2, Kc and Ko are the Michaelis–Menten coefficients of Rubisco activity for CO2 and O2, respectively, and Γ* is the CO2 compensation point in the absence of mitochondrial respiration. Values at 25 °C and temperature sensitivities of Γ*, Kc and Ko were taken from Bernacchi et al.69.
A70 was considered as the minimum of Ac, Aj, and ATPU, and ToptA of A70 was estimated using Eq. 2.
Statistical tests
In order to evaluate the effect of elevated CO2 on the thermal acclimation of the photosynthetic parameters, we used a mixed-effects regression model where warming and elevated CO2 treatment were the fixed effects, and the month in which the campaign was done was the random effect. All analyses were run on the plot means with n = 1–4 trees/plot. The selection of the final statistical model was done in two steps following the protocol proposed by Zuur et al.72. We first evaluated whether a random factor was required by comparing the model with the random intercept (i.e., month) with the model without any random structure using the gls function of the nlme 3.1.162 R Package73 and the method set to the Restricted maximum likelihood (REML). We did not include the model with a random slope and intercept structure since preliminary analyses indicated that the statistical model was over-parameterized. Thereafter, the model with the adequate random structure was selected based on the lowest AIC (Akaike Information Criterion) using the R anova function. After, the selection of the adequate random structure, we then selected for the adequate fixed effect structure between the structure with just main effects (i.e., warming and elevated CO2) without interaction and with interaction. The latter selection was done by comparing these two fixed effect structures using the maximum likelihood—ML method within the gls function. Similarly, the best fixed effect structure was selected based on the lowest AIC value. But because our sample size is relatively small, we then computed the AICc using AICmodavg 2.3-2 R package74 (Supplementary Tables 6 and 7). We also run ANOVA tests to examine the effects of temperature and elevated CO2 treatments on delta-mean temperature growth (ΔMeanTg; Supplementary Table 4). All analyses were conducted in R 3.6.1 software. (R Core Team, 2019), except for unpaired t Tests (Supplementary Table 3a, b) that were performed using statistical package in Excel 16.74 software.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
The raw and processed (i.e., mean values used to generate each figure in the paper) photosynthesis data generated in this study have been deposited in the figshare database and can be accessed at https://doi.org/10.6084/m9.figshare.2264503075. The complete leaf gas exchange data, including the data used in this paper, are also available through the SPRUCE project website at https://doi.org/10.25581/spruce.056/145513876.
Code availability
The R codes used for analyses for each figure included in this paper can be accessed at https://doi.org/10.6084/m9.figshare.2264503075.
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Acknowledgements
Research was sponsored by the Biological and Environmental Research Program in the Office of Science, U.S. Department of Energy managed by UT- Battelle, LLC, for the U.S. Department of Energy under contract DEAC05-00OR22725. M.E.D., J.M.W., E.J.W., D.A.M., A.W.K. and P.J.H. were supported under this contract. E.J.W. also acknowledges support from USGS Climate Research and Development Program. P.B.R., A.S., R.B., and R.A.M acknowledge funding support by the U.S. NSF Biological Integration Institutes grant DBI-2021898. D.A.W. acknowledges funding from the NSERC Discovery and Strategic programs (RGPIN/04677-2019 and STPGP/521445-2018), the Research School of Biology at the Australian National University, and the U.S. Department of Energy contract No. DE-SC0012704 to Brookhaven National Laboratory. Notice: This manuscript has been authored by UT-Battelle, LLC, under contract DE-AC05-00OR22725 with the US Department of Energy (DOE). The US government retains and the publisher, by accepting the article for publication, acknowledges that the US government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for US government purposes. DOE will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan).The DOI link for the dataset used in this paper can be accessed at https://doi.org/10.25581/spruce.056/1455138 and https://doi.org/10.6084/m9.figshare.22645030. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government.
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M.E.D., J.M.W., E.J.W., P.J.H., and D.A.W. designed the research; M.E.D., J.M.W., E.J.W., B.K.M., A.S., R.B., M.C., D.A.M., and A.W.K. collected the data; M.E.D. analyzed the data; M.E.D. wrote the manuscript with significant contributions from J.M.W., P.B.R., and D.A.W.; E.J.W., B.K.M., A.S., R.B., M.C., D.A.M., A.W.K., R.A.M., and P.J.H. provided editorial advice. All co-authors commented on versions of the manuscript.
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Dusenge, M.E., Warren, J.M., Reich, P.B. et al. Boreal conifers maintain carbon uptake with warming despite failure to track optimal temperatures. Nat Commun 14, 4667 (2023). https://doi.org/10.1038/s41467-023-40248-3
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DOI: https://doi.org/10.1038/s41467-023-40248-3
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