TY - JOUR
AU - Way, Danielle A
AB - Abstract Climate warming is increasing the frequency of climate-induced tree mortality events. While drought combined with heat is considered the primary cause of this mortality, little is known about whether moderately high temperatures alone can induce mortality, or whether rising CO2 would prevent mortality at high growth temperatures. We grew tamarack (Larix laricina) under ambient (400 p.p.m.) and elevated (750 p.p.m.) CO2 concentrations combined with ambient, ambient +4 °C and ambient +8 °C growth temperatures to investigate whether high growth temperatures lead to carbon (C) limitations and mortality. Growth at +8 °C led to 40% mortality in the ambient CO2 (8TAC) treatment, but no mortality in the elevated CO2 treatment. Thermal acclimation of respiration led to similar leaf C balances across the warming treatments, despite a lack of photosynthetic acclimation. Photosynthesis was stimulated under elevated CO2, increasing seedling growth, but not leaf C concentrations. However, growth and foliar C concentrations were lowest in the +8 °C treatments, even with elevated CO2. Dying 8TAC seedlings had lower needle C concentrations and lower ratios of photosynthesis to respiration than healthy 8TAC seedlings, indicating that C limitations were likely the cause of seedling mortality under high growth temperatures. Introduction With atmospheric CO2 concentrations increasing at ~2.0 p.p.m. per year, global temperatures are projected to increase 2.0–4.5 °C by the year 2100 (Cramer et al. 2014). Warming is most extreme in high northern latitudes, which could experience temperature increases of more than 8 °C by the end of the century (Serreze et al. 2000, Oppenheimer et al. 2014). These increased temperatures and atmospheric CO2 concentrations have already intensified climatic stress on vegetation, leading to greater tree mortality globally. Since 1970, there have been over 88 documented large-scale tree mortality events, and tree mortality has been identified as a major contributor to future vegetation shifts (Allen et al. 2010, 2015). Many forest mortality events have been linked to global change-related droughts, where high temperatures and drought occur simultaneously. Tree die-offs have therefore been largely attributed to water stress causing either hydraulic failure (i.e., catastrophic xylem cavitation) or carbon (C) starvation (where low stomatal conductance suppresses photosynthetic C gains, but respiratory C losses remain high) (Allen et al. 2010, Mcdowell and Sevanto 2010, Anderegg et al. 2012, Sevanto et al. 2014, Adams et al. 2017, Hartmann et al. 2018). Tree die-offs are already proving to be detrimental to the boreal biome. In high latitude regions in North America, boreal tree species had mortality rate increases of up to 4.7% per year between 1963 and 2008 (Peng et al. 2011), likely due to climate change. But while warming was positively correlated with mortality rates for all plots in Peng et al. (2011), water deficits were only positively correlated with mortality rates in western Canada, indicating that temperature, and not drought, may be the main driver of mortality, a finding supported by Luo and Chen (2015). This raises the question of whether warming may directly increase tree mortality risk through C starvation, even when water supplies are ample, an idea that has received little attention. High temperatures may cause C starvation if they reduce the ratio of photosynthesis to respiration sufficiently such that tree C stores are depleted without being replenished. However, if this is true, high CO2 concentrations predicted over the next few decades may counteract this warming effect by stimulating photosynthesis, meaning that tree mortality rates would not increase under future warming combined with elevated CO2. Photosynthesis, a key determinant of plant C balance, is sensitive to both CO2 concentrations and temperature. Photosynthesis is stimulated by short-term exposure to high CO2 concentrations, as ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is substrate-limited under current CO2 concentrations (Ainsworth and Rogers 2007). Thus, high CO2 conditions increase substrate availability for photosynthesis and suppress photorespiration (Drake et al. 1997). However, plants will often downregulate net CO2 assimilation rates (Anet) after long-term exposure to elevated CO2 to cope with sink limitations related to low nitrogen (N) availability (Tjoelker et al. 1998, Ainsworth and Rogers 2007). In contrast, the temperature response of Anet is curvilinear, with Anet peaking near the growth temperature experienced by the plant (Sage and Kubien 2007). Above this thermal optimum, Anet declines due to increases in respiration and photorespiration, and often also due to declines in stomatal conductance (Sage and Kubien 2007, Lin et al. 2012). Plants acclimate to warming by shifting the photosynthetic temperature optimum toward higher temperatures (Way and Yamori 2014). However, thermal acclimation can result in increased, similar or even lower rates of Anet at the new growth temperature compared with a control plant (Way and Yamori 2014), meaning that even in plants that thermally acclimate, C availability may be reduced in warm-grown plants. Photosynthetic capacity, a measure of the maximum capacity of a leaf to fix CO2, can also be used to quantify photosynthetic thermal acclimation (Way and Yamori 2014). Photosynthetic capacity consists of both the maximum rate of electron transport, Jmax, and the maximum rate of Rubisco carboxylation, Vcmax. Elevated growth temperatures and growth CO2 concentrations both affect Jmax and Vcmax. Plants can decrease their photosynthetic capacity in warm growth conditions to maintain a similar rate of Anet as that achieved by cool-grown plants, thus maintaining C uptake while reducing leaf N costs (Dusenge et al. 2020). In contrast, an increase in photosynthetic capacity at warmer growth temperatures will maximize Anet in conditions where water and nutrient availability are not limiting (Way and Yamori 2014). Thus, both a decrease and an increase in Vcmax and Jmax in warm-grown plants may represent photosynthetic thermal acclimation (Way and Yamori 2014). Photosynthetic capacity can also be affected by growth under elevated CO2. Long-term exposure of plants to elevated CO2 often results in a decrease in photosynthetic capacity (Moore et al. 1999, Ainsworth and Rogers 2007, Albert et al. 2011). Under elevated CO2, plants are able to maintain high photosynthetic rates with less investment in Rubisco, since CO2 substrate availability is high. Leaf N can act as a proxy for Rubisco concentrations (Reich et al. 1998), and changes in leaf N concentrations in plants grown at high CO2 are often used as indicators of changes in photosynthetic capacity (Crous et al. 2008, Kattge et al. 2009). The other main determinant of plant C balance is respiration. Over minutes to hours, respiration increases exponentially with increasing temperatures (Atkin and Tjoelker 2003), but respiration is relatively insensitive to short-term changes in CO2 concentrations (Amthor et al. 2001). Under longer-term exposure to elevated temperatures, thermal acclimation of respiration often results in a decrease in respiration at a common measurement temperature, which mitigates plant C losses (Atkin and Tjoelker 2003, Slot and Kitajima 2015). Long-term exposure to elevated CO2 can also increase respiration in both herbaceous and woody species (Davey et al. 2004, Jauregui et al. 2015, Way et al. 2015), though this is not always the case (Tissue et al. 2001, Haworth et al. 2016). To avoid C starvation, plants must be able to acclimate both photosynthesis and respiration in a manner that ensures a sufficient supply of carbohydrates for growth and metabolism. If plants are unable to balance the ratio of photosynthesis to respiration under elevated growth temperatures and CO2 concentrations, they will be at risk for growth reductions and mortality from C starvation in future climate conditions. Tamarack is a deciduous conifer that is widely distributed across North America and contributes to the boreal forest’s C sequestration potential. Deciduous conifers are considered to be highly phenotypically plastic as they produce new needles each spring that can maximize photosynthetic capacity (Gower and Richards 1990). However, there are also trade-offs associated with tamarack’s growth strategy, such as reduced water-use efficiency (Dusenge et al. 2021), that may impact the survival of this species under future climatic stresses. In this study, we grew tamarack at either ambient or elevated CO2 concentrations combined with ambient temperatures or a +4 °C or +8 °C warming treatment to simulate future climate scenarios. In a recent paper from our group, tamarack seedlings had high levels of mortality under 8 °C warming when coupled with ambient CO2 (Dusenge et al. 2020). We hypothesized that C starvation caused this high mortality in tamarack, since: (i) seedlings were well watered and (ii) seedlings grown under the same +8 °C temperature regime, but with elevated CO2, had no mortality. The main objectives of our study were therefore to evaluate (i) C fluxes, growth and performance of healthy tamarack seedlings across all six treatments and (ii) C fluxes, growth and performance of dying tamarack seedlings. The overarching goal was to determine if differences in leaf C balance between dying and healthy seedlings grown under elevated temperatures and ample water imply that heat stress from high growth temperatures can directly induce C starvation. Materials and methods Experimental design Tamarack seeds were sown on 12 May 2017 in 11.3 l pots filled with Promix HP mycorrhizal growing medium (Premier Tech Horticulture, Rivieire-du-Loup, QC, Canada) with slow-release fertilizer (Slow Release Plant Food, 12-4-8, Miracle Grow, The Scotts Company, Mississauga, ON, Canada). Seeds were ordered from the Canadian National Seed Tree Center (provenance from Finch Township, ON, 45.133°N, 75.083°W) to match the seed collection site with ambient growing season temperatures and photoperiods of London, ON, where the experiment was performed (Western University, 43.0096°N, 81.2737°W). While tamarack grows across the northern latitudes of North America (Brandt 2009), and thus experiences low mean annual temperatures, growing season temperatures are more moderate, with mean maximum August temperatures of 26.2 °C over the years 2012–16 (Environment Canada) for the collection site of the seeds used in this experiment. Forty pots with five seeds per pot were randomly assigned to each of six climate-controlled glasshouses at the University of Western Ontario’s Biotron Experimental Climate Change Research Centre (N = 240 pots). Once seedlings were established, seedlings were thinned to one per pot. Each 6.0 m × 3.4 m × 4.3 m glasshouse had a different temperature × CO2 treatment. Seedlings were grown under either ambient CO2 (AC, 400 p.p.m.) or elevated CO2 (EC, 750 p.p.m.) concentrations with either ambient (0T, ambient control temperatures), ambient +4 °C (4T) or ambient +8 °C (8T) day and night temperatures (Figure 1). The temperature treatments were chosen to represent a realistic range of possible future temperatures: mean annual air temperature is expected to warm up to 5.8 °C under the Representative Concentration Pathway (RCP) 4.5 and up to 9.0 °C under RCP8.5 by 2100 in the Great Lakes Basin (McDermid et al. 2014), where the tamarack seeds used in the experiment were collected. The 0T temperature regime was determined from hourly temperature averages for each day of the growing season (using data from 2012 to 2016) from the London, ON, airport meteorological station (Environment Canada) and varied daily over the experiment accordingly. CO2 concentrations were measured in each glasshouse every 10 min with an infrared gas analyzer in the Argus control system (Argus Control Systems, Surrey, Canada) and were controlled by injecting pure CO2 as needed to maintain the EC treatment (Figure 1b). The growth irradiance inside the glasshouses is approximately ~35% of outdoor light irradiance (Kurepin et al. 2018), varying with naturally fluctuating sunlight. Humidity was controlled at 60%, and seedlings were watered as needed to maintain a moist growth medium (see Figure S1 available as Supplementary data at Tree Physiology Online). Figure 1. Open in new tabDownload slide Daily temperature and CO2 levels across all six glasshouses over the duration of the experiment. Day 0 indicates when seeds were potted (12 May) and Day 140 indicates when seedlings were harvested (28 September). Temperature and CO2 readings were taken daily at 12:00 a.m. Circles, ambient temperature (0T); triangles, +4 °C warming (4T); squares, +8 °C warming (8T). White symbols, ambient growth CO2 (AC); black symbols, elevated growth CO2 (EC). Once seedlings were established and thinned, stem height and health ratings were recorded for all seedlings every 14 days. Health was rated on a scale of 1–5 based on the percent of brown needles (Figure 2). Volumetric soil water content was measured in each pot every 14 days (HH2 Moisture Meter, Delta-T Devices, Cambridge, UK). Figure 2. Open in new tabDownload slide Representative seedlings showing the seedling health scale. (1) Needles are 100% green; (2) seedling has <50% brown needle tissue; (3) seedling has approximately 1-to-1 brown to green leaf tissue; (4) seedling has >50% brown needle tissue; (5) seedling is 100% brown. Physiological measurements Shoot gas exchange measurements were taken in August and September 2017 (when the maximum average daytime air temperature in the 0T treatment was 25 °C) on fully expanded needles using a portable photosynthesis system with the opaque conifer chamber (Li-cor 6400XT and 6400-22L, Li-cor Biosciences, Lincoln, NE, USA) and plasticine was used around the gaskets where stems entered the cuvette to prevent any leakage. First, six healthy seedlings from each treatment were measured to establish treatment effects (N = 36). Then, to compare gas exchange across seedlings of varying health, six healthy and six dying (health rating = 2–4) seedlings were measured in the 8TAC treatment. Needles from dying seedlings measured in the opaque conifer chamber included those with variable but minor degrees of browning when necessary. The Anet was quantified at a range of intracellular CO2 concentrations (i.e., A/Ci curves) of 50, 200, 300, 400, 500, 600, 750, 1200, 1600 and 2000 p.p.m. A/Ci curves were measured at a standard leaf temperature of 25 °C for all treatments and at the respective growth temperatures (25 °C for AT; 29 °C for 4T; 33 °C for 8T), where the growth temperature was taken as the mean of the daily maximum air temperature at the beginning of the measurements. The Anet was assessed at light saturation (1200 μmol photons m−2 s−1) and a relative humidity (RH) of 30–65%. Relative humidity was held constant at ~65% at the 25 °C measurement temperature; however, RH decreased with increasing measurement temperature despite the use of an inline humidifier for the photosynthesis system. After the last A/Ci measurement was recorded at 2000 p.p.m. CO2, the cuvette CO2 was set to 400 p.p.m. and the sample was dark acclimated for 20 min. Shoot dark respiration (Rshoot) was then measured at 400 p.p.m. in all seedlings, as there is no short-term effect of CO2 on Rshoot (Amthor et al. 2001). The Rshoot was measured at 25 °C (Rshoot-25) and 35 °C (Rshoot-35), and these data were used to calculate Q10 values, defined as the temperature sensitivity of respiration rates over a 10 °C temperature increase (Atkin and Tjoelker 2003), using the approach of Loveys et al. (2003): $$\begin{equation} {Q}_{10}=\left(\frac{\ {R}_{\mathrm{shoot}-35}}{\ {R}_{\mathrm{shoot}-25}}\right). \end{equation}$$(1) Using the Q10 and Rshoot-25 values, shoot respiration at the growth temperature (Rshoot-growth) was calculated for each seedling. Shoot growth temperatures were based on the mean daily maximum air temperature in the treatments at the beginning of the measurements. Maximum rates of electron transport (J1200) and Rubisco carboxylation (Vcmax) were estimated from the A/Ci curves using the Farquhar–von Caemmerer–Berry model of C3 photosynthesis (Farquhar et al. 1980) as described in Medlyn et al. (2002) and used in the R package ‘plantecophys’ by Duursma (2015). We used the default values for the Km (710.3 μmol mol−1, which is given by Kc (1 + Oi/Ko), where Kc and Ko are the Michaelis–Menten constants for Rubisco carboxylation (Kc) and oxygenation (Ko), respectively, and Oi is the intercellular O2 concentration) and the photorespiratory CO2 compensation point (Γ*, 42.75 μmol mol−1) in Duursma (2015), and incorporated temperature corrections for these parameters as determined in Bernacchi et al. (2001). The reported rates of photosynthetic capacity are apparent J1200 and apparent Vcmax, as mesophyll conductance was not measured (given that approaches for reliably measuring mesophyll conductance in short-needled conifers are lacking), and photosynthetic capacity values were based on data from intracellular CO2 (Ci) concentrations rather than CO2 concentrations at the site of carboxylation (CC). Once gas exchange measurements were complete, needles in the cuvette were removed and photographed to determine projected leaf area (LA) using ImageJ (US National Institutes of Health, Bethesda, MD, USA). The needles were dried at 65 °C for 48 h and weighed for biomass to determine leaf mass area (LMA; i.e., needle biomass divided by LA) and then used for C and N analyses. Biomass After gas exchange measurements were completed, the remaining 4-month old seedlings in the experiment were harvested and dried to a constant mass at 65 °C. All seedlings, including those used for gas exchange (n = 40), were divided into roots, shoots and leaves, and each tissue was weighed individually. Carbon and nitrogen analysis A subset of the dried leaf tissue was ground using a Wiley mill (Thomas Scientific, Swedesboro, NJ, USA) and analyzed for C and N concentrations using an elemental analyzer (NCS 2500, Carlo Erba, Peypin, France). Non-structural carbohydrate (NSC) concentrations (glucose, fructose, sucrose and starch) were extracted according to Landhäusser et al. (2018). Sugars were extracted from dried, ground needle tissue using 80% ethanol, and the residual tissue pellet was used for starch determination. Soluble sugars were determined through the addition of enzymes (glucose hexokinase, GHK, assay reagent for glucose; phosphoglucose isomerase for fructose; and invertase for sucrose; Sigma-Aldrich Co., St Louis, MO, USA) to sugar extracts, and the absorbance was read at 340 nm after 75 min of incubation on a shaker at room temperature (Versa max microplate reader, Molecular Devices, S/N BN02815, Molecular Devices Corporation, Sunnyvale, CA, USA). Residual tissue pellets were digested through the addition of α-amylase and amyloglucosidase (Sigma-Aldrich). The resulting glucose hydrolysate was converted to glucose with the addition of GHK, and the absorbance was read at 340 nm after 75 min of incubation on a shaker at room temperature. Using a standard glucose curve, concentrations of glucose, fructose, sucrose and starch were determined. In samples where sucrose was undetectable, a value of zero was reported. Statistics R software (R Foundation for Statistical Computing, Vienna, Austria) was used for modeling and statistical analyses. The R package ‘plantecophys’ was used to estimate J1200 and Vcmax (Duursma 2018). The R package ‘tidyverse’ was used for all statistical analyses (Wickham 2019). Response variables of healthy seedlings from all six treatments were analyzed using two-way ANOVAs, considering growth temperature, growth CO2 and their interaction. Orthogonal polynomial contrasts were specified for the three levels of the temperature variable and thus type III sum of squares were used for the model. The R package ‘nlme’ was used to compute type III ANOVA tests (Pinheiro et al. 2013). Log transformation was used to normalize the biomass and height data before the two-way ANOVA was performed. A post hoc Tukey test was used when significant treatment effects were found. The R package ‘emmeans’ was used to compute post-hoc tests (Lenth et al. 2021). The comparison of variables between healthy and dying 8TAC seedlings was done with a two-sample t-test. The treatments were each imposed in a single greenhouse, raising the concern of pseudoreplication. However, our study builds on an earlier study from our group (Dusenge et al. 2020) that grew the same species in these glasshouses and treatments in a separate year, and similarities in the results of common measurements between the two studies (e.g., dark respiration rates at a common temperature, Q10, and growth patterns across the treatments) provide confidence that our conclusions are robust. Results Temperature and CO2 effects on healthy tamarack seedlings Carbon fluxes and photosynthetic capacity When comparing Anet under common conditions of 400 p.p.m. CO2 and 25 °C (A25), there was no difference in A25 across the treatments (Figure 3a, Table 1). Under these common measurement conditions, there was also no significant treatment effect on stomatal conductance (gs.25), the ratio of intracellular CO2 to ambient CO2 (Ci/Ca25) or transpiration (E25; Tables 1 and 2). The Rshoot-25 decreased with increasing growth temperature, but the Q10 of shoot respiration was not significantly altered by the treatments (Figure 3b, Tables 1 and 2). The ratio of A25 to Rshoot-25 (A/R25), an index of shoot-level C balance, therefore increased with increasing growth temperature, but there was no evidence for a CO2 effect on A/R25 (Figure 3c, Table 1). Figure 3. Open in new tabDownload slide Thermal acclimation of respiration led to similar leaf carbon balances across the warming treatments, despite a lack of photosynthetic acclimation in healthy seedlings. Photosynthetic and respiratory responses to elevated CO2 and temperature treatments measured at 25 °C and 400 p.p.m. CO2 (presented on the left) and growth conditions (25 °C for 0T, 29 °C for 4T, 33 °C for 8T; 400 p.p.m. CO2 for AC, 750 p.p.m. CO2 for EC) (presented on the right). (a, d) Net CO2 assimilation rate (A25°C, Agrowth); (b, e) shoot dark respiration rate (Rshoot-25°C, Rshoot-growth); (c, f) the ratio of net CO2 assimilation rate to respiration rate (A/R25°C, A/Rgrowth). White, 0T; light gray, 4T; dark gray, 8T. The horizontal line of the boxplot represents the mean; the box edges indicate the 25th and 75th percentiles; the whiskers display the minimum and maximum values; n = 6. Different letters above boxplots denote a significant difference between treatments (P < 0.05). T, growth temperature; CO2, growth CO2 concentration; n/s, non-significant. *P |$\le$| 0.05, **P < 0.01 and ***P < 0.001. Table 1 Summary of ANOVA statistics for response of gas exchange parameters measured at 25 °C and 400 p.p.m. CO2 (denoted by ‘25’) and at growth conditions (denoted by ‘growth’), as well as leaf biochemistry and growth, to the treatments. Parameters include net CO2 assimilation rate (A25, Agrowth); shoot dark respiration rate (Rshoot-25, Rshoot-growth); the ratio of net CO2 assimilation rate to shoot dark respiration rate (A/R25, A/Rgrowth); the Q10 of shoot respiration (Q10-Rshoot); stomatal conductance (gs-25, gs-growth); the ratio of intracellular to ambient CO2 (Ci/Ca-25, Ci/Ca-growth); transpiration rate (E25, Egrowth); the maximum rate of Rubisco carboxylation (Vcmax-25, Vcmax-growth); the maximum rate of electron transport (J1200-25, J1200-growth); and the ratio of J1200 to Vcmax (J1200-25/Vcmax-25, J1200-growth/Vcmax-growth); needle percent carbon (%C); needle percent nitrogen (%N); the ratio of C/N; needle soluble sugar concentrations; needle starch concentrations; needle total non-structural carbohydrate (TNC) concentrations; total biomass (BiomassTotal); leaf biomass (BiomassLeaf); stem biomass (BiomassStem); root biomass (BiomassRoot); the root/shoot ratio (BiomassRoot/Shoot); and tree height. T, growth temperature; CO2, growth CO2 concentration; CO2 × T, interaction between growth temperature and CO2 concentration. P-values that are statistically significant (P |$\le$| 0.05) are bolded.   . T . CO2 . CO2 × T .   . F-ratio . P-value . F-ratio . P-value . F-ratio . P-value . Gas exchange parameters A25 0.05 0.95 0.05 0.83 0.21 0.81 Rshoot-25 7.09 <0.05 1.27 0.27 0.11 0.89 A/R25 4.05 <0.05 1.54 0.22 0.04 0.96 Agrowth 2.90 0.07 52.81 <0.0001 1.58 0.22 Rshoot-growth 0.18 0.84 2.17 0.15 0.59 0.56 A/Rgrowth 1.16 0.22 9.48 <0.01 1.02 0.37 Q10-Rshoot 1.38 0.27 0.001 0.98 3.18 0.06 gs-25 0.02 0.98 0.84 0.37 0.80 0.46 gs-growth 0.37 0.70 0.09 0.77 1.78 0.19 Ci/Ca-25 0.29 0.75 0.34 0.56 0.013 0.99 Ci/Ca-growth 0.27 0.76 7.67 <0.01 0.15 0.86 E25 0.01 0.99 0.20 0.66 0.26 0.77 Egrowth 6.64 <0.01 0.17 0.68 0.71 0.50 Photosynthetic capacity Vcmax-25 0.10 0.90 0.12 0.73 0.08 0.92 J1200-25 0.52 0.60 0.86 0.36 0.07 0.93 J1200-25/Vcmax-25 4.65 <0.05 29.85 <0.0001 0.07 0.93 Vcmax-growth 13.30 <0.0001 0.59 0.45 0.05 0.95 J1200-growth 0.56 0.58 0.30 0.59 1.07 0.35 J1200-growth/Vcmax-growth 280.60 <0.0001 8.14 <0.001 13.15 <0.0001 Leaf biochemistry %N 2.98 0.06 8.54 <0.01 2.09 0.14 %C 8.61 <0.01 1.48 0.23 0.65 0.53 C/N 1.58 0.22 10.02 <0.01 2.12 0.14 Soluble sugars 4.01 <0.05 0.70 0.41 1.54 0.23 Starch 0.19 0.83 0.07 0.79 1.50 0.24 TNC 3.71 <0.05 0.59 0.45 1.70 0.20 Growth BiomassTotal 18.75 <0.0001 9.57 <0.01 0.94 0.39 BiomassLeaf 20.33 <0.0001 8.94 <0.01 0.30 0.74 BiomassStem 24.68 <0.0001 9.51 <0.01 0.87 0.42 BiomassRoot 14.38 <0.0001 9.87 <0.01 2.30 0.10 BiomassRoot/Shoot 2.15 0.12 6.47 <0.05 7.22 <0.0001 Tree height 52.15 <0.0001 8.47 <0.01 4.45 <0.05   . T . CO2 . CO2 × T .   . F-ratio . P-value . F-ratio . P-value . F-ratio . P-value . Gas exchange parameters A25 0.05 0.95 0.05 0.83 0.21 0.81 Rshoot-25 7.09 <0.05 1.27 0.27 0.11 0.89 A/R25 4.05 <0.05 1.54 0.22 0.04 0.96 Agrowth 2.90 0.07 52.81 <0.0001 1.58 0.22 Rshoot-growth 0.18 0.84 2.17 0.15 0.59 0.56 A/Rgrowth 1.16 0.22 9.48 <0.01 1.02 0.37 Q10-Rshoot 1.38 0.27 0.001 0.98 3.18 0.06 gs-25 0.02 0.98 0.84 0.37 0.80 0.46 gs-growth 0.37 0.70 0.09 0.77 1.78 0.19 Ci/Ca-25 0.29 0.75 0.34 0.56 0.013 0.99 Ci/Ca-growth 0.27 0.76 7.67 <0.01 0.15 0.86 E25 0.01 0.99 0.20 0.66 0.26 0.77 Egrowth 6.64 <0.01 0.17 0.68 0.71 0.50 Photosynthetic capacity Vcmax-25 0.10 0.90 0.12 0.73 0.08 0.92 J1200-25 0.52 0.60 0.86 0.36 0.07 0.93 J1200-25/Vcmax-25 4.65 <0.05 29.85 <0.0001 0.07 0.93 Vcmax-growth 13.30 <0.0001 0.59 0.45 0.05 0.95 J1200-growth 0.56 0.58 0.30 0.59 1.07 0.35 J1200-growth/Vcmax-growth 280.60 <0.0001 8.14 <0.001 13.15 <0.0001 Leaf biochemistry %N 2.98 0.06 8.54 <0.01 2.09 0.14 %C 8.61 <0.01 1.48 0.23 0.65 0.53 C/N 1.58 0.22 10.02 <0.01 2.12 0.14 Soluble sugars 4.01 <0.05 0.70 0.41 1.54 0.23 Starch 0.19 0.83 0.07 0.79 1.50 0.24 TNC 3.71 <0.05 0.59 0.45 1.70 0.20 Growth BiomassTotal 18.75 <0.0001 9.57 <0.01 0.94 0.39 BiomassLeaf 20.33 <0.0001 8.94 <0.01 0.30 0.74 BiomassStem 24.68 <0.0001 9.51 <0.01 0.87 0.42 BiomassRoot 14.38 <0.0001 9.87 <0.01 2.30 0.10 BiomassRoot/Shoot 2.15 0.12 6.47 <0.05 7.22 <0.0001 Tree height 52.15 <0.0001 8.47 <0.01 4.45 <0.05 Open in new tab Table 1 Summary of ANOVA statistics for response of gas exchange parameters measured at 25 °C and 400 p.p.m. CO2 (denoted by ‘25’) and at growth conditions (denoted by ‘growth’), as well as leaf biochemistry and growth, to the treatments. Parameters include net CO2 assimilation rate (A25, Agrowth); shoot dark respiration rate (Rshoot-25, Rshoot-growth); the ratio of net CO2 assimilation rate to shoot dark respiration rate (A/R25, A/Rgrowth); the Q10 of shoot respiration (Q10-Rshoot); stomatal conductance (gs-25, gs-growth); the ratio of intracellular to ambient CO2 (Ci/Ca-25, Ci/Ca-growth); transpiration rate (E25, Egrowth); the maximum rate of Rubisco carboxylation (Vcmax-25, Vcmax-growth); the maximum rate of electron transport (J1200-25, J1200-growth); and the ratio of J1200 to Vcmax (J1200-25/Vcmax-25, J1200-growth/Vcmax-growth); needle percent carbon (%C); needle percent nitrogen (%N); the ratio of C/N; needle soluble sugar concentrations; needle starch concentrations; needle total non-structural carbohydrate (TNC) concentrations; total biomass (BiomassTotal); leaf biomass (BiomassLeaf); stem biomass (BiomassStem); root biomass (BiomassRoot); the root/shoot ratio (BiomassRoot/Shoot); and tree height. T, growth temperature; CO2, growth CO2 concentration; CO2 × T, interaction between growth temperature and CO2 concentration. P-values that are statistically significant (P |$\le$| 0.05) are bolded.   . T . CO2 . CO2 × T .   . F-ratio . P-value . F-ratio . P-value . F-ratio . P-value . Gas exchange parameters A25 0.05 0.95 0.05 0.83 0.21 0.81 Rshoot-25 7.09 <0.05 1.27 0.27 0.11 0.89 A/R25 4.05 <0.05 1.54 0.22 0.04 0.96 Agrowth 2.90 0.07 52.81 <0.0001 1.58 0.22 Rshoot-growth 0.18 0.84 2.17 0.15 0.59 0.56 A/Rgrowth 1.16 0.22 9.48 <0.01 1.02 0.37 Q10-Rshoot 1.38 0.27 0.001 0.98 3.18 0.06 gs-25 0.02 0.98 0.84 0.37 0.80 0.46 gs-growth 0.37 0.70 0.09 0.77 1.78 0.19 Ci/Ca-25 0.29 0.75 0.34 0.56 0.013 0.99 Ci/Ca-growth 0.27 0.76 7.67 <0.01 0.15 0.86 E25 0.01 0.99 0.20 0.66 0.26 0.77 Egrowth 6.64 <0.01 0.17 0.68 0.71 0.50 Photosynthetic capacity Vcmax-25 0.10 0.90 0.12 0.73 0.08 0.92 J1200-25 0.52 0.60 0.86 0.36 0.07 0.93 J1200-25/Vcmax-25 4.65 <0.05 29.85 <0.0001 0.07 0.93 Vcmax-growth 13.30 <0.0001 0.59 0.45 0.05 0.95 J1200-growth 0.56 0.58 0.30 0.59 1.07 0.35 J1200-growth/Vcmax-growth 280.60 <0.0001 8.14 <0.001 13.15 <0.0001 Leaf biochemistry %N 2.98 0.06 8.54 <0.01 2.09 0.14 %C 8.61 <0.01 1.48 0.23 0.65 0.53 C/N 1.58 0.22 10.02 <0.01 2.12 0.14 Soluble sugars 4.01 <0.05 0.70 0.41 1.54 0.23 Starch 0.19 0.83 0.07 0.79 1.50 0.24 TNC 3.71 <0.05 0.59 0.45 1.70 0.20 Growth BiomassTotal 18.75 <0.0001 9.57 <0.01 0.94 0.39 BiomassLeaf 20.33 <0.0001 8.94 <0.01 0.30 0.74 BiomassStem 24.68 <0.0001 9.51 <0.01 0.87 0.42 BiomassRoot 14.38 <0.0001 9.87 <0.01 2.30 0.10 BiomassRoot/Shoot 2.15 0.12 6.47 <0.05 7.22 <0.0001 Tree height 52.15 <0.0001 8.47 <0.01 4.45 <0.05   . T . CO2 . CO2 × T .   . F-ratio . P-value . F-ratio . P-value . F-ratio . P-value . Gas exchange parameters A25 0.05 0.95 0.05 0.83 0.21 0.81 Rshoot-25 7.09 <0.05 1.27 0.27 0.11 0.89 A/R25 4.05 <0.05 1.54 0.22 0.04 0.96 Agrowth 2.90 0.07 52.81 <0.0001 1.58 0.22 Rshoot-growth 0.18 0.84 2.17 0.15 0.59 0.56 A/Rgrowth 1.16 0.22 9.48 <0.01 1.02 0.37 Q10-Rshoot 1.38 0.27 0.001 0.98 3.18 0.06 gs-25 0.02 0.98 0.84 0.37 0.80 0.46 gs-growth 0.37 0.70 0.09 0.77 1.78 0.19 Ci/Ca-25 0.29 0.75 0.34 0.56 0.013 0.99 Ci/Ca-growth 0.27 0.76 7.67 <0.01 0.15 0.86 E25 0.01 0.99 0.20 0.66 0.26 0.77 Egrowth 6.64 <0.01 0.17 0.68 0.71 0.50 Photosynthetic capacity Vcmax-25 0.10 0.90 0.12 0.73 0.08 0.92 J1200-25 0.52 0.60 0.86 0.36 0.07 0.93 J1200-25/Vcmax-25 4.65 <0.05 29.85 <0.0001 0.07 0.93 Vcmax-growth 13.30 <0.0001 0.59 0.45 0.05 0.95 J1200-growth 0.56 0.58 0.30 0.59 1.07 0.35 J1200-growth/Vcmax-growth 280.60 <0.0001 8.14 <0.001 13.15 <0.0001 Leaf biochemistry %N 2.98 0.06 8.54 <0.01 2.09 0.14 %C 8.61 <0.01 1.48 0.23 0.65 0.53 C/N 1.58 0.22 10.02 <0.01 2.12 0.14 Soluble sugars 4.01 <0.05 0.70 0.41 1.54 0.23 Starch 0.19 0.83 0.07 0.79 1.50 0.24 TNC 3.71 <0.05 0.59 0.45 1.70 0.20 Growth BiomassTotal 18.75 <0.0001 9.57 <0.01 0.94 0.39 BiomassLeaf 20.33 <0.0001 8.94 <0.01 0.30 0.74 BiomassStem 24.68 <0.0001 9.51 <0.01 0.87 0.42 BiomassRoot 14.38 <0.0001 9.87 <0.01 2.30 0.10 BiomassRoot/Shoot 2.15 0.12 6.47 <0.05 7.22 <0.0001 Tree height 52.15 <0.0001 8.47 <0.01 4.45 <0.05 Open in new tab In contrast, Anet measured at the growth CO2 and temperature (Agrowth) was 49–69% higher in EC seedlings compared with AC plants, although Agrowth showed no significant response to growth temperature (Figure 3d, Table 1). Stomatal conductance at growth conditions (gs-growth) showed little response to the treatments, while transpiration measured at growth conditions (Egrowth) increased with warming and the higher measurement vapor pressure deficit (VPD, i.e., the difference in vapor pressure inside and outside of the leaf) associated with high measurement temperatures (Tables 1 and 2). The ratio of intracellular CO2 to ambient CO2 at growth conditions (Ci/Ca-growth) was higher in EC than AC seedlings (Tables 1 and 2). Shoot dark respiration rates at growth temperature (Rshoot-growth) showed little response to either growth temperature or CO2 (Figure 3E, Table 1). The ratio of Agrowth to Rgrowth (A/Rgrowth) was stimulated by EC (Figure 3F, Table 1). When measured at 25 °C, the maximum rate of Rubisco carboxylation (Vcmax-25) and the maximum rate of electron transport (J1200-25) were not significantly affected by the treatments (Figure 4a and b, Table 1). The ratio of J1200-25/Vcmax-25 decreased from 0T to 8T and increased with EC (Figure 4c, Table 1). When measured at the growth conditions, the maximum rate of Rubisco carboxylation (Vcmax-growth) was increased by warming (Figure 4d, Table 1), but the maximum rate of electron transport (J1200-growth) was not significantly affected by the treatments (Figure 4E, Table 1). The ratio of J1200-growth/Vcmax-growth therefore decreased with warming and also increased with EC (Figure 4F, Table 1). Figure 4. Open in new tabDownload slide Photosynthetic capacity did not acclimate to either elevated CO2 or warming in healthy seedlings. Responses of photosynthetic capacity at 25 °C and 400 p.p.m. CO2 (presented on the left) and growth conditions (25 °C for 0T, 29 °C for 4T, 33 °C for 8T; 400 p.p.m. CO2 for AC, 750 p.p.m. CO2 for EC) (presented on the right) to treatments. (a, d) Maximum rate of Rubisco carboxylation (Vcmax-25, Vcmax-growth); (b, e) maximum rate of electron transport (J1200-25, J1200-growth); (c, f) the ratio of J1200 to Vcmax (J1200-25/Vcmax-25, J1200-growth/Vcmax-growth). White, 0T; light gray, 4T; dark gray, 8T. The horizontal line of the boxplot represents the mean; the box edges indicate the 25th and 75th percentiles; the whiskers display the minimum and maximum values; n = 6. Different letters above boxplots denote significant differences across all treatments (P < 0.05). T, growth temperature; CO2, growth CO2 concentration; n/s, non-significant. *P |$\le$| 0.05, **P < 0.01 and ***P < 0.001. Growth response Seedling growth was affected by both growth temperature and CO2 (Figure 5, Table 1). As growth temperature increased from 0T to 4T, total seedling biomass was constant, but biomass decreased at 8T (Figure 5E, Table 1). There was also greater biomass in EC compared with AC seedlings. The ratio of root/shoot biomass was similar across the warming treatments in AC seedlings, but higher in 8T than 0T in EC seedlings due to increased root biomass (Figure 5b and c, Table 1). Tree height was increased by EC only in the 0T seedlings, and 8T seedlings were shorter than 0T and 4T plants in both CO2 treatments (Figure 5F, Table 1). Figure 5. Open in new tabDownload slide Warming suppressed seedling growth while high CO2 stimulated growth in healthy seedlings. Growth responses to treatments: (a) leaf biomass; (b) root biomass; (c) the root/shoot ratio; (d) stem biomass; (e) total biomass and (f) tree height. White, 0T; light gray, 4T; dark gray, 8T. The horizontal line of the boxplot represents the mean; the box edges indicate the 25th and 75th percentiles; the whiskers display the minimum and maximum values; n = 40. Different letters above boxplots denote significant differences across all treatments (P < 0.05). T, growth temperature; CO2, growth CO2 concentration; n/s, non-significant. *P|$\le$|0.05, **P < 0.01 and ***P < 0.001. Tissue biochemistry Leaf %N was lower in EC than AC seedlings, but there was little effect of growth temperature on leaf %N (Figure 6a, Table 1). In contrast, as growth temperature increased, needle %C declined, with no apparent effect of growth CO2 on leaf %C (Figure 6b, Table 1). The ratio of C/N was increased by EC but did not respond to growth temperature (Figure 6c, Table 1). Needle soluble sugar and total NSC concentrations decreased with warming but were not significantly affected by growth CO2 (Figure 6D and F, Table 1), while needle starch concentrations were similar across all treatments (Figure 6E, Table 1). Figure 6. Open in new tabDownload slide Needle biochemical responses to the treatments in healthy seedlings. (a) Needle nitrogen (N) concentrations; (b) carbon (C) concentrations; (c) the C/N ratio of needles; (d) soluble sugar concentrations; (e) starch concentrations; (f) total non-structural carbohydrate (TNC) concentrations. The NSCs (D–F) were measured in g of glucose equivalents g−1 (w/w). White, 0T; light gray, 4T; dark gray, 8T. The horizontal line of the boxplot represents the mean; the box edges indicate the 25th and 75th percentiles; the whiskers display the minimum and maximum values; n = 6. Different letters above boxplots denote significant differences across all treatments (P < 0.05). T, growth temperature; CO2, growth CO2 concentration; n/s, non-significant. *P |$\le$| 0.05, **P < 0.01 and ***P < 0.001. Comparison of dying versus healthy seedlings in the 8TAC treatment Overall, 8TAC seedlings had a 40% mortality rate compared with 0% mortality in all other treatments. Similar to the quantification of C fluxes of healthy seedlings across all treatments, the same traits were examined in 8TAC dying and healthy seedlings at their growth temperature of 33 °C. There was no significant difference in Agrowth, Rshoot-growth, J1200-growth, Vcmax-growth or J1200/Vcmax-growth between healthy and dying seedlings, nor was there any significant difference in the ratio of Agrowth to Rshoot-growth (A/Rgrowth) (Figure 7, Table 3). Needle %C was lower in the dying seedlings, although the needle %N and the ratio of C/N were similar between all 8TAC seedlings (Figure 8A–c, Table 3). The NSC concentrations were also similar across the two groups of seedlings (Figure 8D–F, Table 3). However, there were significant negative correlations between the health rating of the seedlings and both the needle %C and the ratio of A/Rgrowth (Figure 9). Figure 7. Open in new tabDownload slide Photosynthetic and respiration measurements were similar in healthy and dying seedlings. Comparison of (a) net CO2 assimilation rate (Agrowth), (b) shoot dark respiration rate (Rshoot-growth), (c) the ratio of Agrowth to Rshoot-growth (A/Rgrowth), (d) maximum rate of electron transport (J1200-growth), (e) maximum rate of Rubisco carboxylation (Vcmax-growth) and (f) the ratio of J1200-growth to Vcmax-growth (J1200/Vcmax-growth) between dying (gray) and healthy (white) seedlings grown in the 8TAC treatment. The horizontal line of the boxplot represents the mean; the box edges indicate the 25th and 75th percentiles; the whiskers display the minimum and maximum values; n = 6. Different letters above boxplots denote significant differences between groups (P < 0.05). Table 2 Response of gas exchange parameters measured at 25 °C and 400 p.p.m. CO2 (denoted by ‘25’) and under growth conditions (denoted by ‘growth’) to the treatments. Parameters include stomatal conductance (gs-25, gs-growth; mol H2O m−2 s−1); the ratio of intracellular to atmospheric CO2 (Ci/Ca-25, Ci/Ca-growth); transpiration rate (E25, Egrowth; mmol H2O m−2 s−1) and Q10 values of shoot respiration of seedlings from different growth treatments. 0TAC, ambient temperature combined with ambient CO2; 4TAC, +4 °C warming combined with ambient CO2; 8TAC, +8 °C warming combined with ambient CO2; 0TEC, ambient temperature combined with elevated CO2; 4TEC, +4 °C warming combined with elevated CO2; 8TEC, +8 °C warming combined with elevated CO2. Means ± SE, n = 6. There were no differences between groups across all six growth treatments, so letters were not used to denote significant differences.   . 0TAC . 4TAC . 8TAC . 0TEC . 4TEC . 8TEC . gs-25 0.16 ± 0.02 0.15 ± 0.01 0.15 ± 0.02 0.15 ± 0.01 0.16 ± 0.01 0.17 ± 0.01 gs-growth 0.16 ± 0.02 0.15 ± 0.01 0.14 ± 0.02 0.14 ± 0.01 0.15 ± 0.01 0.17 ± 0.01 Ci/Ca-25 0.76 ± 0.01 0.76 ± 0.01 0.75 ± 0.02 0.76 ± 0.02 0.77 ± 0.01 0.76 ± 0.02 Ci/Ca-growth 0.76 ± 0.01 0.74 ± 0.01 0.74 ± 0.02 0.78 ± 0.02 0.78 ± 0.01 0.78 ± 0.02 E25 1.97 ± 0.24 1.81 ± 0.17 1.86 ± 0.26 1.89 ± 0.07 2.00 ± 0.13 1.97 ± 0.19 Egrowth 1.97 ± 0.24 2.34 ± 0.15 2.76 ± 0.25 2.02 ± 0.12 2.65 ± 0.17 2.61 ± 0.21 Q10 1.70 ± 0.06 1.71 ± 0.02 1.70 ± 0.02 1.63 ± 0.02 1.71 ± 0.05 1.80 ± 0.05   . 0TAC . 4TAC . 8TAC . 0TEC . 4TEC . 8TEC . gs-25 0.16 ± 0.02 0.15 ± 0.01 0.15 ± 0.02 0.15 ± 0.01 0.16 ± 0.01 0.17 ± 0.01 gs-growth 0.16 ± 0.02 0.15 ± 0.01 0.14 ± 0.02 0.14 ± 0.01 0.15 ± 0.01 0.17 ± 0.01 Ci/Ca-25 0.76 ± 0.01 0.76 ± 0.01 0.75 ± 0.02 0.76 ± 0.02 0.77 ± 0.01 0.76 ± 0.02 Ci/Ca-growth 0.76 ± 0.01 0.74 ± 0.01 0.74 ± 0.02 0.78 ± 0.02 0.78 ± 0.01 0.78 ± 0.02 E25 1.97 ± 0.24 1.81 ± 0.17 1.86 ± 0.26 1.89 ± 0.07 2.00 ± 0.13 1.97 ± 0.19 Egrowth 1.97 ± 0.24 2.34 ± 0.15 2.76 ± 0.25 2.02 ± 0.12 2.65 ± 0.17 2.61 ± 0.21 Q10 1.70 ± 0.06 1.71 ± 0.02 1.70 ± 0.02 1.63 ± 0.02 1.71 ± 0.05 1.80 ± 0.05 Open in new tab Table 2 Response of gas exchange parameters measured at 25 °C and 400 p.p.m. CO2 (denoted by ‘25’) and under growth conditions (denoted by ‘growth’) to the treatments. Parameters include stomatal conductance (gs-25, gs-growth; mol H2O m−2 s−1); the ratio of intracellular to atmospheric CO2 (Ci/Ca-25, Ci/Ca-growth); transpiration rate (E25, Egrowth; mmol H2O m−2 s−1) and Q10 values of shoot respiration of seedlings from different growth treatments. 0TAC, ambient temperature combined with ambient CO2; 4TAC, +4 °C warming combined with ambient CO2; 8TAC, +8 °C warming combined with ambient CO2; 0TEC, ambient temperature combined with elevated CO2; 4TEC, +4 °C warming combined with elevated CO2; 8TEC, +8 °C warming combined with elevated CO2. Means ± SE, n = 6. There were no differences between groups across all six growth treatments, so letters were not used to denote significant differences.   . 0TAC . 4TAC . 8TAC . 0TEC . 4TEC . 8TEC . gs-25 0.16 ± 0.02 0.15 ± 0.01 0.15 ± 0.02 0.15 ± 0.01 0.16 ± 0.01 0.17 ± 0.01 gs-growth 0.16 ± 0.02 0.15 ± 0.01 0.14 ± 0.02 0.14 ± 0.01 0.15 ± 0.01 0.17 ± 0.01 Ci/Ca-25 0.76 ± 0.01 0.76 ± 0.01 0.75 ± 0.02 0.76 ± 0.02 0.77 ± 0.01 0.76 ± 0.02 Ci/Ca-growth 0.76 ± 0.01 0.74 ± 0.01 0.74 ± 0.02 0.78 ± 0.02 0.78 ± 0.01 0.78 ± 0.02 E25 1.97 ± 0.24 1.81 ± 0.17 1.86 ± 0.26 1.89 ± 0.07 2.00 ± 0.13 1.97 ± 0.19 Egrowth 1.97 ± 0.24 2.34 ± 0.15 2.76 ± 0.25 2.02 ± 0.12 2.65 ± 0.17 2.61 ± 0.21 Q10 1.70 ± 0.06 1.71 ± 0.02 1.70 ± 0.02 1.63 ± 0.02 1.71 ± 0.05 1.80 ± 0.05   . 0TAC . 4TAC . 8TAC . 0TEC . 4TEC . 8TEC . gs-25 0.16 ± 0.02 0.15 ± 0.01 0.15 ± 0.02 0.15 ± 0.01 0.16 ± 0.01 0.17 ± 0.01 gs-growth 0.16 ± 0.02 0.15 ± 0.01 0.14 ± 0.02 0.14 ± 0.01 0.15 ± 0.01 0.17 ± 0.01 Ci/Ca-25 0.76 ± 0.01 0.76 ± 0.01 0.75 ± 0.02 0.76 ± 0.02 0.77 ± 0.01 0.76 ± 0.02 Ci/Ca-growth 0.76 ± 0.01 0.74 ± 0.01 0.74 ± 0.02 0.78 ± 0.02 0.78 ± 0.01 0.78 ± 0.02 E25 1.97 ± 0.24 1.81 ± 0.17 1.86 ± 0.26 1.89 ± 0.07 2.00 ± 0.13 1.97 ± 0.19 Egrowth 1.97 ± 0.24 2.34 ± 0.15 2.76 ± 0.25 2.02 ± 0.12 2.65 ± 0.17 2.61 ± 0.21 Q10 1.70 ± 0.06 1.71 ± 0.02 1.70 ± 0.02 1.63 ± 0.02 1.71 ± 0.05 1.80 ± 0.05 Open in new tab Table 3 Summary of two-sample t-test statistics for parameters comparing dying and healthy 8TAC seedlings: net photosynthesis at growth conditions (Agrowth); shoot dark respiration at growth conditions (Rshoot-growth); ratio between Agrowth and Rshoot-growth (A/Rgrowth); maximum rate of electron transport (J1200-growth); maximum rate of Rubisco carboxylation (Vcmax-growth); ratio between J1200-growth and Vcmax-growth (J1200-growth/Vcmax-growth); percent needle carbon (%C); percent needle nitrogen (%N); the ratio of needle C/N; needle soluble sugar concentrations; needle starch concentrations and needle total non-structural carbohydrate (TNC) concentrations. DF, degrees of freedom. P-values that are statistically significant (P < 0.05) are bolded.   . Healthy vs dying .   . DF . T-stat . P-value . Agrowth 10 0.35 0.73 Rshoot-growth 10 0.64 0.54 A/Rgrowth 10 1.87 0.09 Vcmax-growth 10 0.60 0.56 J1200-growth 10 1.11 0.30 J1200-growth/Vcmax-growth 10 0.62 0.55 % N 10 1.30 0.22 % C 10 2.45 <0.05 C/N 10 0.91 0.38 Soluble sugar 10 1.74 0.11 Starch 10 0.64 0.54 TNC 10 1.98 0.08   . Healthy vs dying .   . DF . T-stat . P-value . Agrowth 10 0.35 0.73 Rshoot-growth 10 0.64 0.54 A/Rgrowth 10 1.87 0.09 Vcmax-growth 10 0.60 0.56 J1200-growth 10 1.11 0.30 J1200-growth/Vcmax-growth 10 0.62 0.55 % N 10 1.30 0.22 % C 10 2.45 <0.05 C/N 10 0.91 0.38 Soluble sugar 10 1.74 0.11 Starch 10 0.64 0.54 TNC 10 1.98 0.08 Open in new tab Table 3 Summary of two-sample t-test statistics for parameters comparing dying and healthy 8TAC seedlings: net photosynthesis at growth conditions (Agrowth); shoot dark respiration at growth conditions (Rshoot-growth); ratio between Agrowth and Rshoot-growth (A/Rgrowth); maximum rate of electron transport (J1200-growth); maximum rate of Rubisco carboxylation (Vcmax-growth); ratio between J1200-growth and Vcmax-growth (J1200-growth/Vcmax-growth); percent needle carbon (%C); percent needle nitrogen (%N); the ratio of needle C/N; needle soluble sugar concentrations; needle starch concentrations and needle total non-structural carbohydrate (TNC) concentrations. DF, degrees of freedom. P-values that are statistically significant (P < 0.05) are bolded.   . Healthy vs dying .   . DF . T-stat . P-value . Agrowth 10 0.35 0.73 Rshoot-growth 10 0.64 0.54 A/Rgrowth 10 1.87 0.09 Vcmax-growth 10 0.60 0.56 J1200-growth 10 1.11 0.30 J1200-growth/Vcmax-growth 10 0.62 0.55 % N 10 1.30 0.22 % C 10 2.45 <0.05 C/N 10 0.91 0.38 Soluble sugar 10 1.74 0.11 Starch 10 0.64 0.54 TNC 10 1.98 0.08   . Healthy vs dying .   . DF . T-stat . P-value . Agrowth 10 0.35 0.73 Rshoot-growth 10 0.64 0.54 A/Rgrowth 10 1.87 0.09 Vcmax-growth 10 0.60 0.56 J1200-growth 10 1.11 0.30 J1200-growth/Vcmax-growth 10 0.62 0.55 % N 10 1.30 0.22 % C 10 2.45 <0.05 C/N 10 0.91 0.38 Soluble sugar 10 1.74 0.11 Starch 10 0.64 0.54 TNC 10 1.98 0.08 Open in new tab Figure 8. Open in new tabDownload slide Needle carbon concentrations were lower in dying seedlings than in healthy seedlings, although other biochemical indicators were similar in both groups. Biochemical responses between dying (gray) and healthy (white) seedlings grown in the 8TAC treatment. (a) Needle nitrogen (N) concentrations; (b) carbon (C) concentrations; (c) the C/N ratio of needles; (d) soluble sugar concentrations; (e) starch concentrations; (f) total non-structural carbohydrate (TNC) concentrations. The horizontal line of the boxplot represents the mean; the box edges indicate the 25th and 75th percentiles; the whiskers display the minimum and maximum values; n = 6. Different letters above boxplots denote significant differences between groups (P < 0.05). Figure 9. Open in new tabDownload slide Healthy seedlings had higher ratios of photosynthesis to respiration and higher leaf carbon concentrations than less healthy seedlings. Relationship between seedling health rating and (a) the ratio of net CO2 assimilation rate to shoot dark respiration rate at growth conditions (A/Rgrowth); and (b) the percent needle carbon (%C). Points represent individual seedlings, n = 12. Solid line, linear regression. Discussion Carbon balance and photosynthetic capacity There was surprisingly little evidence for photosynthetic acclimation across 8 °C of warming and a 350 p.p.m. increase in growth CO2, indicating that tamarack has considerable capacity for maintaining C uptake under future climates. Many studies have shown that plants grown at elevated CO2 tend to have reduced photosynthetic capacity (Moore et al. 1999, Ainsworth and Long 2005, Albert et al. 2011), but our data support the idea that conifers may be less sensitive to rising CO2 than other plant functional types (Ellsworth 1999, Medlyn et al. 2001, Ainsworth and Rogers 2007). While less is known about how deciduous conifers will respond to elevated CO2 compared with evergreen conifers, Dusenge et al. (2020) also found that photosynthetic capacity was unresponsive to changing CO2 in tamarack seedlings. Photosynthetic acclimation to temperature is also common in the literature (Tjoelker et al. 1998, Yamori et al. 2005, 2014, Way and Oren 2010, Kroner and Way 2016). However, the response of Anet to warming is variable in boreal tree species. Previous studies have found that Picea asperata, Abies faxoniana and Pinus sylvestris seedlings increase Agrowth with warming, whereas Picea abies and Picea mariana seedlings decrease Agrowth with warming (Tjoelker et al. 1998, Yin et al. 2008, Kurepin et al. 2018). While it is unusual to find the degree of photosynthetic stability across such a broad range of growth temperatures as seen in our data, thermal acclimation can result in a similar Agrowth across different growth temperatures (Way and Yamori 2014). Stomatal conductance was also constant across the warming treatments, which may have contributed to the similar Agrowth observed here. Despite high growth temperatures at 8T, the volumetric water content maintained in the soil would have allowed the seedlings to maintain similar rates of photosynthesis and transpiration compared with the 0T and 4T warming treatments. The lack of photosynthetic acclimation was also correlated with the maintenance of relatively similar concentrations of needle N, implying that warming had little effect on photosynthetic enzyme and protein concentrations across the treatments. Since gs was also relatively insensitive to warming and CO2, there was higher Ci/Ca-growth under EC and therefore higher Agrowth in the EC seedlings. At current CO2 concentrations, Rubisco is substrate-limited; by increasing intracellular CO2 concentrations, photosynthetic rates increase and photorespiration is suppressed (Sage and Kubien 2007). Stimulation of photosynthesis by EC in mature conifers is common in the absence of sink limitations (DeLucia et al. 1999, Ainsworth and Rogers 2007, Ryan 2013, Dusenge et al. 2020), as these limitations can feed back to instigate a downregulation of photosynthesis (Ainsworth and Long 2005, Leakey et al. 2009). Dusenge et al. (2020) and Tjoelker et al. (1999a) also found that warming led to a constant Agrowth, but that EC stimulated Agrowth in tamarack, indicating that photosynthetic rates of tamarack are highly responsive to changes in elevated CO2, even when stomatal conductance is not. While photosynthetic capacity at 25 °C was unaffected by the treatments, the ratio of J1200-25/Vcmax-25 was slightly reduced with warming. Meta-analyses have noted that the effect of warming on photosynthetic capacity measured at 25 °C is variable (Way and Oren 2010, Way and Yamori 2014), and we still lack a general understanding of how Vcmax-25 and J1200-25 will be affected by a warming world. Tamarack seedlings in Dusenge et al. (2020) had decreased photosynthetic capacity with warming and associated decreases in foliar N concentrations (%N), indicative of a lower investment in photosynthetic enzymes (Reich et al. 1998). Comparatively, in our data, photosynthetic capacity did not acclimate to warming, and there was a similar %N across temperature treatments, indicating there was likely no large change in the relative amounts of Rubisco across the treatments. However, the decline in J1200-25/Vcmax-25 in warm-grown plants is common (Yamori et al. 2005, Kattge and Knorr 2007, Dusenge et al. 2020) and is thought to indicate a shift in N partitioning within the photosynthetic apparatus from RuBP carboxylation to RuBP regeneration (Hikosaka et al. 2006). Under low temperatures, plants invest more N into RuBP regeneration, resulting in a higher ratio of cytochrome f to Rubisco and subsequently a higher ratio of J1200-25/Vcmax-25(Onoda et al. 2005), with the opposite occurring at warm temperatures. The shift in J1200-25/Vcmax-25 found here was small, though significant, implying that there may have been subtle shifts in N allocation between electron transport and carboxylation capacity across the treatments. When measured at growth conditions, J1200-growth was constant across the treatments, while Vcmax-growth increased from 0T to 8T. Rising leaf temperatures generally stimulate both Vcmax and J1200 (Kattge and Knorr 2007, Way and Oren 2010), which makes the J1200 data somewhat surprising. However, the ‘co-ordination hypothesis’ predicts that Vcmax and Jmax should be co-limiting (Hikosaka et al. 2006, Maire et al. 2012, Togashi et al. 2018). Given this, the stimulation of Vcmax-growth with warming may allow plants to match higher Rubisco activity with higher photosynthetic rates (Togashi et al. 2018). Higher rates of RuBP carboxylation by Rubisco would maintain Agrowth across the warming treatments despite the greater photorespiratory losses expected with increased temperatures. Both J1200-25/Vcmax-25 and J1200-growth/Vcmax-growth increased in EC seedlings, a result linked to increased efficiency of Rubisco carboxylation under high CO2 and a resultant rebalancing of allocation toward J1200 (Crous et al. 2008). Thermal acclimation of respiration mitigated C losses across the warming treatments, as seen in other studies (Reich et al. 1998, Tjoelker et al. 1999a, Atkin and Tjoelker 2003, Loveys et al. 2003, Slot and Kitajima 2015, Dusenge et al. 2020). Overall, thermal acclimation of respiration led to similar rates of Rgrowth and similar A/Rgrowth from 0T to 8T. In comparison, photosynthetic stimulation by EC increased A/Rgrowth. By acclimating respiration, tamarack was able to effectively minimize C losses under +8 °C and maintain similar leaf C balances in healthy seedlings across all warming treatments. Growth, biomass allocation and C/N dynamics While the leaf C flux data were not indicative of warming stress, tree biomass and height decreased at 8T compared with 0T and 4T (a growth result similar to that in our earlier study of Dusenge et al. 2020). These growth reductions were largely offset by EC, implying that the decline in growth is related to plant C dynamics. In support of this hypothesis, 8TAC seedlings had lower root-to-shoot ratios than 8TEC seedlings, indicating increased allocation to aboveground tissues (i.e., photosynthetic tissue) to compensate for limited C availability (Poorter et al. 2012). The biomass allocation patterns of different conifer species to warming and EC are variable (Yin et al. 2008). However, other work has confirmed that tamarack and other tree species increase allocation to leaf tissue under warming (Way and Oren 2010, Dusenge et al. 2020). Plants with low belowground biomass allocation prioritize C gain over water uptake, which is beneficial under C stress conditions, but could put these trees at greater risk under drier climates in the future (Way and Oren 2010). As tamarack seedlings in this study were well watered and fertilized, 8TAC seedlings were able to invest in aboveground tissues to maximize C gains without experiencing water or nutrient stress, but this may not be true for tamarack that experience warming in the field over the coming decades. Warming also reduced foliar C concentrations (%C). Foliar %C has been estimated at ~50% in conifers (Ma et al. 2018), so a reduction by 5% in 8T seedlings is considerable and one indication of C limitation. Surprisingly, these decreases in %C were not offset by EC, a pattern also seen in Tjoelker et al. (1999a). The changes in leaf %C were mirrored by changes in foliar soluble sugar and total NSC concentrations, providing further evidence that warming may have led to a decline in whole tree C balance (Dietze et al. 2014). A meta-analysis by Du et al. (2020) also noted a reduction in soluble sugar concentrations in plants exposed to warmer temperatures. Decreased starch concentrations are also often prevalent under C stress (Sevanto et al. 2014, Wiley et al. 2017, Du et al. 2020), though this was not apparent in the tamarack in our study. Higher C availability from stimulated Agrowth under EC was apparently allocated to growth over storage, given the strong effect of EC on plant growth. Prioritization of growth over storage is common in conifer seedlings (Dietze et al. 2014), but the small C stores often found in seedlings may make them more vulnerable to C stress under this C allocation strategy. Rising CO2 concentrations can affect foliar N content (%N) through a direct suppression of Rubisco production (Crous et al. 2008) or a growth dilution effect (Taub and Wang 2008). A synthesis of 62 plant species by Yin (2002) found that the proportional decline in %N with EC is highest in deciduous woody species, like tamarack. While our EC seedlings had lower leaf %N than the AC tamarack, EC also led to higher Agrowth as these seedlings were able to invest less in photosynthetic enzymes and still attain higher photosynthetic rates than AC plants. Elevated CO2 in future climates will likely be beneficial for tamarack C gain and growth, even when combined with moderate warming, as seen here in 0TEC and 4TEC seedlings. Mortality in 8TAC seedlings One of the main objectives of this study was to investigate whether C starvation was the cause of mortality in 8TAC seedlings. The few studies that have measured tree mortality have found higher respiration in seedlings experiencing C starvation (Sevanto et al. 2014, Wiley et al. 2017). These studies also found depletions in plant C after long durations of C stress. While we found no significant difference in respiration (or photosynthetic) rates between healthy and dying seedlings, leaf %C was lower in dying 8TAC seedlings compared with healthy seedlings, providing evidence of C limitations, and there was a trend (P = 0.08) toward lower total NSC concentrations in the dying 8TAC seedlings. Additionally, both the ratio of A/Rgrowth and leaf %C were negatively correlated with the health ratings, providing evidence that C balance was depleted as seedling health deteriorated. In measuring healthy 8TAC seedlings, it is important to consider that a survivor effect (i.e., that we only measured healthy seedlings that had survived the treatment) may have biased our estimates of C fluxes; however, this would indicate that our 8TAC C balance results were conservative, given that seedlings that had already died likely had lower photosynthetic rates, higher respiration rates and lower C stores than the surviving seedlings we sampled. While differences between the mean values of these parameters for the healthy and dying seedlings may also have been obscured by large variation in individual seedlings (especially because dying seedlings were drawn from three different health ratings), our data imply that warming, even without water stress, can lead to C stress and tree mortality. Despite maintaining constant air humidity and volumetric soil water content, some atmospheric water stress may have occurred under +8 °C warming, as the VPD would have been ~0.7 kPa higher in the 8T glasshouses than the 0T glasshouse. Given that gs was unresponsive to warming, this higher VPD in the 8T treatments would lead to higher rates of transpiration in 8T seedlings than those from 0T. This was unlikely to have led to significant water stress in the 8T plants, since they were watered daily and had large soil volumes to hold water compared with their very small root masses. With future warming, relative humidity is expected to stay constant, but as temperatures increase, so will VPD (Dessler and Sherwood 2009, Trenberth 2011, Zhang et al. 2017). Under these ecologically realistic conditions, an inability to acclimate stomatal conductance to warming may be detrimental to tamarack as droughts become more frequent with climate change. The benefits that tamarack and other larch species experience from a deciduous leaf economic strategy, i.e., higher photosynthetic rates, come at a cost compared with the conservative evergreen leaf economic strategy (Kloeppel et al. 2000). The prioritization of C allocation to growth over storage in deciduous conifers results in higher respiratory losses (Tjoelker et al. 1999a) and smaller initial seasonal NSC stores compared with co-occurring evergreens (Hoch et al. 2003). However, over a growing season, NSC pools of deciduous conifers can become similar to or even greater than some evergreen conifer species (Tjoelker et al. 1999b). The consequence of initially smaller C stores and higher respiratory costs is that deciduous conifers may have a smaller buffer against climatic stresses that limit photosynthesis and thus may only be able to withstand short periods of C limitation before survival is impacted. Evergreen conifers are considered to be more conservative, with a greater allocation to C storage under stress (Weber et al. 2019) and higher water-use efficiencies (So et al. 2019), which may increase their likelihood of survival. For example, the study from our group that first noted tamarack mortality under 8TAC observed no mortality in black spruce (P. mariana) grown under the same experimental treatments (Dusenge et al. 2020). In addition to plant functional types, intraspecific variation will also impact the future composition of the boreal forest. While population variation in tamarack in response to drought or heat stress has not been well studied, mature white spruce (Picea glauca) has increased resilience to drought in populations from drier geographical locations compared with those from more humid locations (Depardieu et al. 2020). Adaptive genetic variation will likely be a strong determinant for thermal plasticity and survival, and it would be useful to compare different genotypic responses of tamarack populations to climatic stresses in future studies. Conclusions Reduced growth and high mortality were linked to decreases in leaf %C and the balance between photosynthesis and respiration in tamarack seedlings grown under +8 °C warming coupled with ambient CO2. In healthy seedlings, thermal acclimation of respiration minimized C losses under warming, resulting in similar leaf C balances across the temperature treatments. While moderate warming (i.e., +4 °C) combined with EC may be beneficial to tamarack seedling growth, +8 °C warming was detrimental to growth even when combined with EC. To reach a warming of +8 °C in high latitudes, atmospheric levels of CO2 will have to rise, which should help prevent the high mortality observed in our 8TAC seedlings. Additionally, trees in the field may be better equipped than seedlings against high growth temperature-induced C stress as they have larger C stores, which is one of the greatest determinants of survival against C starvation (Hartmann and Trumbore 2016). Regardless, our results indicate that high temperature-induced C stress can ultimately lead to reduced growth and mortality in the absence of water stress, which may be detrimental to the future functioning of tamarack and potentially other boreal tree species as warming continues. Acknowledgments We thank Kristyn Bennett for assistance with samples, and Carrie Hamilton and Steve Bartlett for setting up and monitoring the glasshouses. We also thank the National Tree Seed Center for providing seeds for this experiment. Funding B.K.M. was supported by the Queen Elizabeth II Graduate Scholarship for Science and Technology. D.A.W. acknowledges the support of a Natural Sciences and Engineering Research Council of Canada Discovery Grant, support from the Canadian Foundation for Innovation, an Ontario Early Researcher Award, and the United States Department of Energy contract No. DE-SC0012704 to Brookhaven National Laboratory. Conflict of interest None declared. References Adams HD , Zeppel MJB, Anderegg WRL et al. ( 2017 ) A multi-species synthesis of physiological mechanisms in drought-induced tree mortality . Nat Ecol Evol 1 : 1285 – 1291 . Google Scholar Crossref Search ADS PubMed WorldCat Ainsworth EA , Long SP ( 2005 ) What have we learned from 15 years of free-air CO2 enrichment (FACE)? A meta-analytic review of the responses of photosynthesis, canopy properties and plant production to rising CO2 . New Phytol 165 : 351 – 372 . 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TI - Warming and elevated CO2 alter tamarack C fluxes, growth and mortality: evidence for heat stress-related C starvation in the absence of water stress
JO - Tree Physiology
DO - 10.1093/treephys/tpab077
DA - 2021-12-04
UR - https://www.deepdyve.com/lp/oxford-university-press/warming-and-elevated-co2-alter-tamarack-c-fluxes-growth-and-mortality-orb0drKTUb
SP - 2341
EP - 2358
VL - 41
IS - 12
DP - DeepDyve
ER -