Is photosynthetic enhancement sustained through three years of elevated CO₂ exposure in 175-year old Quercus robur?

Abstract

Current carbon cycle models attribute rising atmospheric CO₂ as the major driver of the increased terrestrial carbon sink, but with substantial uncertainties. The photosynthetic response of trees to atmospheric CO₂ is central to sustaining the terrestrial carbon sink, but can vary diurnally, seasonally and with duration of CO₂ exposure. Hence we sought to quantify responses of canopy-dominant species, Quercus robur, in a mature deciduous forest to elevated CO₂ (eCO₂) (+150 µmol mol^-1 CO₂) over the first three years of a long-term free air CO₂ enrichment (FACE) facility in central England. Over three thousand measurements of leaf gas exchange and related biochemical parameters were conducted at the top of the canopy to assess the diurnal and seasonal responses of photosynthesis during the 2^nd and 3^rd year of eCO exposure at the Birmingham Institute of Forest Research (BIFoR) FACE facility. Measurements of photosynthetic capacity and biochemical parameters derived from CO₂ response curves together with leaf nitrogen concentrations from the pre-treatment year to the 3^rd year of CO exposure were examined to assess changes in Q. robur photosynthetic capacity. We expected an enhancement in light-saturated net photosynthetic rates (A_sat) consistent with CO₂ enrichment (≈37%) and that photosynthetic capacity may reduce across over the time of the project. Over the three-year period, A_sat of upper-canopy leaves was 33 ± 8% higher in trees grown in eCO₂ compared with ambient CO₂ (aCO₂), and this enhancement decreased with decreasing light levels. There were also no significant CO₂ treatment effects on photosynthetic capacity measures, nor area- and mass-weighted leaf nitrogen. These results suggest that mature oak trees may exhibit a sustained, positive response to eCO₂ without photosynthetic downregulation, suggesting that, with adequate nutrients, there may be increases in C storage in elevated CO₂.

Introduction

Forest ecosystems cover about 30% of the Earth’s land surface, representing ∼50% of terrestrially stored carbon and accounting for close to 60% of total terrestrial CO₂ fluxes in the global carbon cycle (Luyssaert et al., 2008; Pan et al., 2011). The continual rise in atmospheric CO₂, overwhelmingly due to anthropogenic activity (Friedlingstein et al., 2019), increases the need to understand the terrestrial carbon feedbacks of forests in the global carbon cycle. The removal of atmospheric CO₂ via photosynthesis is a necessary pre-condition for forests to act as long-standing carbon stores with relatively long-lived carbon (C) pools such as wood (Körner, 2017) and soil (Ostle et al., 2009). Forest C-uptake and sequestration in the future will be a crucial determinant of future atmospheric CO₂ concentrations. As the foundational driver of the carbon cycle of forests (Bonan, 2008), the photosynthetic response to changing atmospheric CO₂ is a central part of sustaining the terrestrial carbon feedback of forests in the global carbon cycle. Quantifying the photosynthetic response under eCO₂ is critical to understanding the carbon budget of forests and, thence, to estimating the global land carbon sink under changing atmospheric composition.

It has been widely observed that elevated CO₂ (eCO₂) can have a stimulatory effect on plant photosynthesis, known as photosynthetic enhancement, at least in the short term (weeks to months) with adequate nutrient and water availability permitting (Brodribb et al., 2020). Long-term (years to decades) responses of photosynthesis to eCO₂ are less certain than the short-term responses as lower-than-expected responses have been observed (Ainsworth & Long, 2005; Ellsworth et al., 2017). Additionally, the photosynthetic process and photosynthetic response to eCO₂ is sensitive to changes in environmental variables such as temperature, light, water and availability of nutrients. For example, net photosynthesis (A_net) is expected to increase with exposure to eCO₂, with greatest photosynthetic enhancement expected at maximum photon flux density (PFD) if Rubisco carboxylation is limiting (Kull, 2002; Sage et al., 2008; Wullschleger, 1993). Decreases in A_net have been commonly associated with limitations in water and nutrient availability. For example, water availability has been found to affect the magnitude of eCO₂-induced photosynthetic enhancement more in drier years (Ellsworth et al., 2012; Nowak et al., 2004). Thus, interannual differences in eCO₂–induced photosynthetic enhancement are to be expected as environmental conditions vary. Understanding the photosynthetic response to eCO₂ under different, real-world, environmental conditions provides information essential for modelling, where it has important implications for the determination of forest productivity (Medlyn et al., 2011; Jiang et al. 2020), and for predicting carbon-climate feedbacks (e.g., Cox et al., 2013; Friedlingstein et al., 2006; Jones et al., 2016).

Despite a significant body of research on the photosynthetic response to eCO₂ in tree seedlings and saplings (as reviewed in Ainsworth & Long, 2005; Curtis & Wang, 1998; Medlyn et al., 1999), fewer studies address the long-term (> 1 year) photosynthetic responses in mature forest-grown trees (Crous et al., 2008; Ellsworth et al., 2017; Klein et al., 2016; Zotz et al., 2005). Currently, the dynamic vegetation components of Earth System models, which diagnose vegetation responses to environmental change, have commonly been constructed using data from eCO₂ experiments on young and/or plantation grown trees (Piao et al., 2013). Yet, it is difficult to compare, generalise, and scale results from young trees in their exponential growth phase to the response of closed-canopy mature forests (Norby et al., 2016). It is plausible that model projections are currently overestimating the photosynthetic responses of mature forests and, thence, the ‘CO₂ fertilisation’ effect (Zhu et al., 2016). Therefore, uncertainty remains as to the magnitude of, and environmental constraints on, photosynthetic enhancement under eCO₂ in large, long standing carbon stores, such as mature forests (Jiang et al., 2020; Norby et al., 2016). Reducing these uncertainties will be crucial to quantify the mitigation potential of mature forests under future atmospheric CO₂ and consequently to model confidently future climate projections.

Previously, research has identified growing constraints from seedlings, young plants and trees grown in controlled environments used to deliver eCO₂ to these plants. For example, artificial environments can produce artefacts to eCO₂ responses for growth and physiology, such as nutrient limitations, particularly if plants are grown in pots (Berntson et al., 1993). These artefacts and those associated with chambered plants are avoided by the use of free-air CO₂ enrichment (FACE) technology (Hendrey et al., 1999), which allows plants, including large trees, to be grown and exposed experimentally to eCO₂ for long periods. FACE facilities are valuable to understand system-level responses to eCO₂, (Ainsworth & Long, 2005; Terrer et al., 2016, 2020) particularly in forests (Norby et al., 2016) and the development of 2^nd generation forest FACE experiments additionally allows the unique proposition of experimentation on tall, mature trees grown in their own forest soil (Hart et al., 2020). To date, forest FACE experiments have observed photosynthetic enhancements ranging from 30-60%, depending on tree species and influences of environmental factors such as light, temperature, soil water availability and nutrient supply (as reviewed in Ainsworth & Rogers, 2007; Curtis & Wang, 1998; Nowak et al., 2004). Of the few studies on closed-canopy dominant tree species, smaller photosynthetic enhancement to eCO₂ have been observed (19 to 49%) than in studies conducted on younger trees (Crous et al., 2008; Ellsworth et al., 2017; Liberloo et al., 2007; Sholtis et al., 2004; Zotz et al., 2005).

There is evidence to suggest plants show a reduction in photosynthetic activity after long-term eCO₂ exposure, known as photosynthetic downregulation (Ainsworth et al., 2004; Crous & Ellsworth, 2004), but downregulation is not always observed (Curtis & Wang, 1998; Herrick & Thomas, 2001). Different hypotheses have been proposed to explain photosynthetic downregulation under eCO₂ exposure but commonly it is the result of decreases, either directly or indirectly, in Rubisco carboxylation (V_cmax) (Feng et al., 2014; Wujeska-Klause et al., 2019). Characteristically, downregulation results in a reduction in, but usually not complete removal of, the stimulatory effect of photosynthesis under eCO₂ (Crous et al., 2008; Wujeska-Klause et al. 2019). The occurrence of photosynthetic downregulation has largely been observed in young plants in artificial environments (Leakey et al., 2009), with some exceptions (Crous et al., 2010; Ellsworth et al., 2004), commonly as a result of insufficient nitrogen supplies. Insufficient nitrogen supplies are usually caused by soil nutrient limitations (Luo et al., 2004), therefore changes in leaf nitrogen can indicate changes in photosynthetic capacity. In forest FACE experiments, mixed results have been observed regarding the presence of photosynthetic downregulation after long-term exposure to eCO₂. For example, photosynthetic downregulation was observed at two aggrading plantation forests (Crous et al., 2008; Warren et al., 2015), yet mature forest sites have largely not reported this (Bader et al., 2010; Ellsworth et al., 2017). However, given that mature trees in the field experience such different growing constraints from seedlings and young plants in controlled environments, open questions remain concerning the frequency, importance and outcome of photosynthetic downregulation under eCO₂ exposure in mature forests.

To help understand the photosynthetic responses in mature temperate deciduous forests we evaluated the CO₂ response of photosynthesis in ca. 175-year old canopy dominant trees of Quercus robur L. Since the majority of the work was conducted during the 2^nd and 3^rd year of CO enrichment at the Birmingham Institute of Forest Research (BIFoR) FACE facility, we refer to these responses as ‘early’, considering that forest FACE experiments aim to operate for 10 years or more. The present study aimed to understand the extent of photosynthetic enhancement and potential down-regulation in mature Q. robur at BIFoR FACE, putting this study amongst the oldest such trees that have ever been examined in this regard. To assess the photosynthetic enhancement of the trees on daily and interannual timeframes, measurements of gas exchange and leaf biochemistry were measured in the upper oak canopy over four growing seasons spanning from pre-treatment in 2015 to 2019.

We hypothesized that net photosynthetic gas exchange, A_net, will significantly increase with eCO₂ and light levels (PFD). The greatest enhancement was expected with the highest light levels, as a result of reduced limitations in the light dependent reaction of photosynthesis, and that photosynthetic enhancement will be in proportion to the CO₂ increase (≈37%; Hart et al., 2020). We also hypothesized that leaf nitrogen (N) will be reduced under elevated CO₂ and that photosynthetic downregulation will be observed under eCO₂ as a result of reduced leaf N. Photosynthetic downregulation will be observed by: (a) a reduction in light-saturated A_net (i.e., A_sat); (b) a decline in either the maximum rate of photosynthetic Rubisco carboxylation (V_cmax, μmol m⁻² s⁻¹); and the maximum rate of photosynthetic electron transport (J_max, μmol m⁻² s⁻¹), or both; and (c) reduced foliar N concentration (%w/w N).

In light of the hypotheses we had three major objectives:

1. To quantify the photosynthetic response to eCO₂ (i.e., +150 μmol mol^-1) for mature Q. robur and how light levels influences this response,

2. To determine whether photosynthetic downregulation under eCO₂ occurred in Q.robur,

3. To establish the relationship between leaf N and photosynthetic capacity, and whether this is changed in eCO₂.

Quantifying the magnitude and characteristics of the early-term response of photosynthesis to eCO₂ in BIFoR FACE is necessary to build an accurate picture of the mature forest’s carbon budget (cf. Jiang et al., 2020) and provide an important challenge to dynamic vegetation models seeking to estimate the global land carbon sink reflecting mature forest responses under changing atmospheric composition (Medlyn et al., 2015).

Methods and materials

Site description

This study was conducted at the Birmingham Institute of Forest Research (BIFoR) Free Air CO₂ Enrichment (FACE) facility located in Staffordshire (52.801°N, 2.301°W), United Kingdom. The BIFoR FACE facility is a ‘2^nd generation’ Forest FACE facility, extending the scope of 1^st generation facilities; (see Norby et al., 2016), situated within 19 ha of mature northern temperate broadleaf deciduous woodland having a canopy height of 24-26 m. The woodland consists of an overstorey canopy dominated by English oak (Quercus robur L.) and a dense understorey comprising mostly of hazel coppice (Corylus avellana L.), sycamore (Acer pseudoplatanus L.), and hawthorn (Crataegus monogyna Jacq.). Q. robur (commonly known as pendunculate oak, European oak or English oak) is a common broadleaf species geographically widespread across Europe where it is both economically important and ecologically significant for many biota (Eaton et al., 2016; Mölder et al., 2019). The site was planted with the existing oak standards in the late 1840s and has been largely unmanaged for the past 30 to 40 years. As a mature and unmanaged forest patch, it is typical of established deciduous forest throughout the temperate zone.

The study site is situated within a climate typical of the northern temperate zone, characterized by cool wet winters and warm dry summers with a frost-free growing season from April to October. The site is near the centre of the temperature-rainfall climate space occupied by temperate forest (Sommerfeld et al., 2018). The mean January and July temperatures were 4 and 17°C, respectively, and the average annual precipitation for the region is 720 mm (646 mm and 749 mm, respectively, in 2018 and 2019 when the study was conducted; see Fig 1.)

Figure 1.

Time series showing the daily meteorological data at the BIFoR FACE facility covering the period of 01-01-2018 to 01-12-2019. Subplots are: A) global downwelling solar radiation (MJ/m²), B) mean (orange) and maximum (red) daily air temperatures (°C) and C) total daily precipitation (mm). Vertical dashed lines indicate diurnal sampling days. Clusters of sampling days occurred because different plots were sampled on different days in the same seasonal timeframe. Meteorological data is from RAF Shawbury, located 20 miles west of the BIFoR FACE facility, retrieved from the UK Met Office (https://www.metoffice.gov.uk/research/climate/maps-and-data/historic-station-data).

BIFoR FACE consists of nine approximately circular experimental plots of woodland 30 m in diameter (Hart et al., 2020). Only the six plots with infrastructure were considered in the present study. Each ‘infrastructure plot’ is encircled by steel towers constructed individually to reach 2 m above the local canopy-top height. The facility uses a paired-plot design (Hart et al., 2020): three replicate plots at either ambient CO (aCO) (ca. 405 μmol mol⁻¹) and three plots supplied with CO enriched air, termed elevated CO₂ plots (eCO₂). The latter plots are maintained at a target of +150 μmol mol⁻¹ above the minimum measured in the ambient plots (i.e. concentrations in the elevated plots ca. 555 μmol mol⁻¹). Elevated CO is maintained from dawn (solar zenith angle, sza = −6.5°) to dusk (sza = −6.5°) throughout the growing season. Daytime exposure to eCO was almost continuous throughout the growing season (see Hart et al., 2020), with exceptions if the 15-minute average wind speed was greater than 8 m s^-1, or when canopy-top, 1-min average, air temperature was < 4°C. In the latter case, gas release was resumed when the air temperature was ≥ 5°C. The operation of the FACE system and statistical performance in terms of meeting the target CO₂ concentration in time and space have been described in Hart et al. (2020), who measured the precision (standard deviation) of the applied CO₂ treatment to be less than 6%.

In each plot, canopy access was gained through a bespoke canopy access system (CAS) (Total Access Ltd., UK) that was installed from the central towers with canopy measurements made from a rigged rope access system. This facilitated in situ gas exchange measurements by allowing access to the upper oak canopy. The hoisting system comprises of an electric winch (Harken Power Seat Compact) that lifts a harnessed (Petzl AVAO BOD 5 point harness) user vertically through the air at a predetermined fixed point to a maximum canopy height of 25 m. The system required operation from the ground by trained staff and the user is seated in a Boatswain’s chair. One oak tree per plot was accessible using the CAS system as set up during this study, and all gas exchange measurements were made on unshaded leaves within the top two meters of each tree canopy on dominant trees.

For this study, the sample size used throughout the study (n=3) represents the number of replicate experimental plots at BIFoR FACE and includes within-tree replicates that were averaged per plot before analysis. All the three replicates were sampled for the majority of campaigns, except for September 2018, May 2019, and June 2019 where it was reduced to two, one and two replicates, respectively, due to logistic constraints, weather, and safe tree access.

Gas exchange measurements

All gas exchange measurements were conducted in situ on upper canopy oak leaves using either a Li-6400XT or Li-6800 portable photosynthesis system (LiCOR, Lincoln, NE, USA) to quantify photosynthetic performance at BIFoR FACE. Measurement campaigns focussed on two different types of measurements: i) instantaneous diurnal measurements, at prevailing environmental conditions (2018 and 2019), and ii) net assimilation rate-intercellular CO₂ concentration (A–C_i) measurements (includes pre-treatment, 2015; 1^st year, 2017; and 3^rd year, 2019, of CO fumigation). Measurements were conducted in all six experimental plots with infrastructure, on one chosen candidate tree per plot.

When reporting treatment effects from the present study, we report the mean enhancement or treatment effect: where A_i,x is a measure of gas exchange (i = ‘net’ or ‘sat’, see below) at ambient (a) or elevated (e) CO₂ mixing ratios. When comparing our results with other studies using different eCO₂ treatments, we report the sensitivity to eCO₂, following Keenan et al. (2016): where c_a is the ambient CO₂ mixing ratio and Δc_a is the treatment size (e.g. +150 μmol mol^-1 as in our case). For the conditions of the present study (see ‘Diurnal measurements’ section, below), c_a/Δc_a = 392/150 = 2.61, and we use net photosynthesis instead of GPP. Hence, our expectation of a mean percentage enhancement equal to the size of the CO₂ treatment (i.e., ≈37 ± 6%; using the precision from Hart et al. (2020)) is equivalent to expecting a sensitivity to eCO₂ of unity.

Diurnal measurements

Near the canopy top, in situ diurnal measurements of gas exchange were conducted on upper canopy oak leaves on 11 and 12 separate summer days of 2018 and 2019, respectively. Measurements of gas exchange were made using a Li-6800 equipped with the default clear Propafilm (Innovia Films Inc., Atlanta, GA) window chamber head, which allowed for natural sunlight to illuminate the leaf. Measurements were conducted in one pair of plots (i.e. one eCO₂ plot and its paired aCO₂ plot) on each sampling day. Therefore, each full campaign (n=3) took three days to complete, with the exception of September 2018 and May and June 2019 where only one or two replicate plots could be measured. A total of four and five diurnal campaigns were conducted in 2018 and 2019, respectively, providing a total of 3,552 data points. Five to six healthy leaves were randomly selected in the same oak tree per plot, every 30-40 minutes across the time course of the day for gas exchange measurements, swapping between aCO₂ and eCO₂ plots.

Measurements were made at the respective growth CO₂ of aCO₂ (∼405 μmol mol⁻¹) or +150 μmol mol⁻¹ aCO₂ (∼555 μmol mol⁻¹) for eCO₂ plots, along with other environmental variables such as relative humidity (RH); air temperature (T_air); and quanta of photosynthetically active radiation (PAR). Measurements were confined to the youngest fully expanded leaves of the leader branch within reaching distance of the CAS system. Expanding leaves, judged from colour and texture, were avoided for measurements, as they had not stabilised in terms of chlorophyll and formation of the photosynthetic apparatus. Once a leaf was inside the chamber, the Li-6800 head was gently positioned and held constant at an angle towards the sun. This was to ensure sun exposure on the leaf, to minimize shading of the chamber head on the measured leaf and to reduce variation across the leaf measurements. Measurements were recorded after an initial stabilisation period (typically ∼40 seconds to 1 minute), to meet programmed stability parameters. This allowed for instantaneous steady-state photosynthesis to be captured, yet avoided chamber-related increases in leaf temperature (Long et al., 1996; Parsons et al., 1998). Care was taken to ensure conditions matched those outside the chamber before each measurement was taken. The daily mean RH inside the leaf chamber was between 50% and 77% for all measurements. The mean C_a values in the LiCOR chamber head were 390 ± 0.9 μmol mol⁻¹ and 538 ± 2.7 μmol mol⁻¹, in 2018, and 393 ± 1.0 μmol mol⁻¹ and 545 ± 4.8 μmol mol⁻¹, in 2019, for aCO₂ and eCO₂ respectively. The mean CO₂ treatments were, therefore, +148 ± 2.8 μmol mol⁻¹ in 2018, and +152 ± 4.9 in 2019, and were not statistically different. The gas exchange systems were calibrated before each growing season.

A–C_i curves

A–C_i curves were conducted in three growing seasons: pre-treatment year (2015), in the first year of CO₂ fumigation (2017) and third year of CO₂ fumigation (2019). Measurements were either conducted on attached branches in situ (2015 and 2019) or on detached branches harvested by climbers (2017) using a portable open gas exchange system that incorporated a controlled environment leaf chamber (Li-6400XT and LI-6800, LICOR, Inc., Lincoln, NE, USA). A–C_i curves were measured at a PFD of 1800 μmol m⁻² s⁻¹ (in 2015 and 2019) or 1200 μmol m⁻² s⁻¹ (in 2017) and at a leaf temperature of 25 °C using the LICOR ‘autolog’ function. Before each curve, a stabilisation period of between 5 to 10 minutes was used depending on the prevailing environmental conditions. Net photosynthesis (A_net) were estimated from A–C_i curves at growth [CO₂]. The CO₂ concentrations were changed in 12 to 14 steps starting at the respective growth [CO]; every 100 μmol mol⁻¹ down to 50 μmol mol⁻¹ (near the photosynthetic CO compensation point), then increasing to 1800 μmol mol⁻¹ in roughly 200 μmol mol⁻¹ increment steps. Five to six replicate A–C_i curves on different leaves per CO₂ treatment were measured per day. Measurements were taken between 09:00 and 11:00, and 14:00 and 17:00 to avoid potential midday stomatal closure (Valentini et al., 1995). Measurements were made using the treatment pair arrangement of one aCO₂ and one eCO₂ plot per day (n=3).

Leaf carbon and nitrogen

Oak leaves were collected from the top of the canopy in each month, May to November in 2015 and 2019, by arborist climbers, and stored immediately at −25°C. Two upper canopy leaves, from one tree per plot, were selected for elemental analyses, these trees corresponded to the measurement tree for gas exchange. Each leaf was photographed for leaf area and the fresh weight (FW) was recorded. Each leaf was oven dried at 70°C for at least 72 hours, re-weighed for dry weight (DW) and the leaf mass per unit area (LMA) was calculated. Dried leaf fragments were ground and each sample (∼2 mg) was enclosed in a tin capsule. Samples were analysed for δ¹³C, total C, and total N using an elemental analyser interfaced with an isotope ratio mass spectrometer (Sercon Ltd., Cheshire, UK).

Statistical analysis

All statistical analysis were performed in R version 4.0.3 (http://cran.r-project.org). Before statistical analysis, all data were checked for normality by inspection of Q_Q plots and using Levene’s test, and residuals from model fitting were checked to ensure homoscedasticity.

Hourly averages of diurnal measurements were analysed using a linear mixed effects model (‘lmer’ package). Fixed categorical factors in this model were CO₂ treatment (i.e., aCO₂ or eCO₂), sampling month and sampling year (i.e. 2018 or 2019) with ‘plot’ represented as random factor, as individual trees were nested within each experimental plot. Using type III F-tests, statistically significant CO₂ treatment differences among groups were further tested with Tukey’s post hoc test (P < 0.05 reported as significant). To investigate the dependence of photosynthetic enhancement with variation of light, the diurnal gas exchange data, with leaf temperature, Tleaf >18°C, and vapour pressure deficit, VPD <2.2kPa, were sub-divided into four light (PFD) categories, each sampled about equally. The PFD classes were: PFD < 250; 250 ≤ PFD < 500; 500 ≤ PFD < 1000; and PFD ≥ 1000 µmol m^-2 s^-1. CO₂ treatment, year, and PFD category were then used as parameters in an analysis of variance (ANOVA).

The photosynthetic CO₂ response (A–C_i) curves were fit with the model of Farquhar et al. (1980) to estimate the apparent maximum rate of photosynthetic Rubisco carboxylation (V_cmax, μmol m⁻² s⁻¹) and the apparent maximum rate of photosynthetic electron transport (J_max, μmol m⁻² s⁻¹) using ‘Plantecophys’ package in R (Duursma, 2015). The model-fitting was undertaken to provide insight into photosynthetic capacity and its response to long-term exposure to elevated [CO₂] (Rogers & Ellsworth, 2002). We tested for outliers by examining the J_max/V_cmax ratio, RMSE values (removed if >6), and standard errors (SE) for fits of J_max and V_cmax, all of which indicate violations to the theory for fitting these curves (Sharkey et al., 2007). Those curves that contained outliers were removed, this accounted for < 10% of the data, leaving a total of 86 A–C_i curves across the three sampling years in the analysis.

Results

Measurement conditions

Overall, diurnal measurements were conducted on dry, sunny, days (Fig. 1), and environmental conditions (PFD and T_leaf) were consistent between aCO₂ and eCO₂ across the two growing seasons of diurnal measurements (Figs. 2A, B and 3A, B). PFD levels were largely comparable between CO₂ treatments although cloud and temperature conditions were more variable among sampling days and campaigns in 2018 than in 2019.

Figure 2.

In situ diurnal measurements of A) PFD (µmol m^-2 s^-1); fitted with an LOESS regression B) hourly mean Tleaf (°C) and C) hourly mean A (µmol m^-2 s^-1) at BIFoR FACE in 2018 from the upper Q .robur canopy. Error bars indicate n=3, with the exception of September where only two replicate plots were measured. The line types represent separate sampling days in A) that are averaged in B) and C) and the two colours represent the CO₂ treatments of aCO₂ (blue) and eCO₂ (red).

Leaf temperature was more stable than PFD with lower variability across the diurnal sampling, high similarity between sampling days, and high consistency between CO₂ treatments. There were differences in measured T_leaf between months, suggesting a seasonal influence as would be expected from the site’s mid-latitude location, and the differences were more prominent in 2019 than 2018. The highest T_leaf values were observed in July with a common seasonal decline after this campaign.

The range of mean daily A_net was similar between years, however the highest mean daily A_net (12.2 µmol m^-2 s^-1) was reported in 2018. Contrasting seasonal patterns were observed between the sampling years of 2018 and 2019, with decreases in mean daily A_net across the growing season observed in 2018 compared to increases in A_net in 2019. In both sampling years, we observed a significant enhancement of A_net when exposed to eCO₂ (P <0.05, Table 1 and Figs 2 and 3). Here, we did not observe any significant effect of either season or sampling year on A_net (Table 1.), therefore mean eCO₂ enhancement of A_net (i.e., 100·ΔA_net/A_net,400) of 23 ± 4% was observed across the 2-year period of this study.

Table 1.

Linear mixed-effects model analysis for photosynthesis with CO₂ treatment (CO₂), sampling month (Month) and sampling year (Year) as fixed factors and a random effect of ‘plot’. Type III sums of squares computed using restricted maximum likelihood estimates for F-tests. The numerator degrees of freedom (df) for each F-test are shown. A post-hoc Tukey test was used to determine the significance relationships. Significance of CO₂ treatment is noted in the rightmost column as (*= P < 0.05).

Figure 3.

In situ diurnal measurements of A) PFD (µmol m^-2 s^-1); fitted with an LOESS regression, B) hourly mean T_leaf (°C) and C) hourly mean A_net (µmol m^-2 s^-1) at BIFoR FACE in 2019 from the upper Q. robur canopy. Error bars indicate n=3, the exception of May and June where only one and two replicate plots were measured, respectively. The line types represent separate sampling days in A) that are averaged in B) and C) and the two colours represent the CO₂ treatments of aCO₂ (blue) and eCO₂ (red).

Photosynthesis and variation in photo flux density (PFD)

This study analysed the role of measurement PFD affecting A_net and its response to eCO₂ in separate growing seasons to investigate photosynthetic enhancement values at different light conditions. In each light category (see Methods, above), the light conditions between the CO₂ treatments were statistically comparable (Figure 4, Supplementary table 1: S1). Mean, median, and interquartile range of A_net increased with increasing PFD class for both sampling years and CO₂ treatments (Fig 4A, Table 2). We observed no significant effect of year for A_net in this study, but we did observe a larger variation in A_net in 2019 when compared to 2018 (Table 2, Fig 4A). Values of mean A_net ranged from 4.6 ± 0.3 µmol m^-2 s^-1, at the lowest PFD level of a mean of 150 µmol m^-2 s^-1, to 11.5 ± 0.7 µmol m^-2 s^-1 at highest PFD (mean PFD of 1360 µmol m^-2 s^-1). Additionally, in both sampling years A_net was significantly higher under eCO₂ conditions when compared to aCO₂ (P < 0.05, Table 2, Fig 4A).

Table 2.

Linear mixed-effects model parameters for prediction of A_net with variation in photo flux density (PFD). Type III sums of squares computed using restricted maximum likelihood estimates for F-tests. The numerator degrees of freedom (DF) for each F-test are shown. A post-hoc Tukey’s test and xx was used to determine the significance relationships. Significance of CO₂ treatment is noted in the rightmost column as (*** = P <0.001; ** = P < 0.01; *= P < 0.05)

Figure 4.

A) The distribution of net photosynthesis (A_net) (µmol m^-2 s^-2) in each of the four photon flux density (PFD) categories (PFD < 250; 250 ≥ PFD < 500; 500 ≥ PFD < 1000; and PFD ≥ 1000 µmol m s) for years 2018 (left) and 2019 (right). Whiskers denote the 5 %ile and 95 %ile; outliers are plotted as individual points (filled circles). The box denotes the interquartile range and the bar denotes the median with the number of data points above each boxplot. The mean is also plotted as a diamond symbol. Data uses diurnal gas exchange measurements in the upper canopy oak trees at the BIFoR FACE facility with Tleaf >18°C and vapour pressure deficit (VPD) <2.2kPa, in eCO₂ (red) or aCO₂ (blue) treatments. Red diamonds indicate the mean A_net values. B) Boxplots of the enhancement response ratio (eCO₂/aCO₂) (grey) for each year, and predicted enhancement ratio (dashed line) (1.37) following Nowak et al (2004).

Consistent with our hypothesis, we observed mean photosynthetic enhancement to increase with increasing PFD, with the largest enhancement observed at highest PFD in both sampling years, 30 ± 9%, and 35 ± 13%, for 2018 and 2019 respectively (Fig 4B). In 2018, photosynthetic enhancement ranged from 7 ± 10%, in the lowest PFD class, to 30 ± 9%, in the highest PFD class (Fig 4B). A similar positive relationship between photosynthetic enhancement and PFD was present in 2019 with photosynthetic enhancement ranging from 11 ± 6%, in the lowest PFD class, to 35 ± 13%, in the highest PFD class (Fig 4, B). There was no significant effect of year (Table 2) and the mean photosynthetic enhancement at light saturation (i.e. in the highest PFD class) was on average 33 ± 8 % across the two sampling years. Therefore, an important finding in this analysis was that photosynthetic enhancement of light-saturated A_net (A_sat) in both sampling years was consistent, within error, of the predicted enhancement based on proportion of CO₂ increase (≈37 ± 6%), indicating a sensitivity to eCO₂ (equation 2, above) of close to unity for A_sat.

Photosynthetic capacity and foliar nitrogen

The seasonal and interannual biochemical changes in Q. robur were assessed via differences in leaf apparent maximum CO₂ carboxylation capacity (V_cmax) and apparent maximum electron transport capacity for RuBP regeneration (J_max) (Fig 5.) to examine if there were effects on the long term photosynthetic capacity with exposure to eCO₂. Initially, we tested for differences between the year of sampling and found no statistical difference of either V_cmax or J_max between the three sampling years (2015, 2017 and 2019) (Fig 5, Supplementary table 2: S2). Furthermore, this study found no significant effects of CO₂ enrichment on V_cmax or J_max across the two years of CO₂ enrichment, i.e. the 1^st and 3^rd years, and no significant effect of season between the three measurement years (Fig 5; A and B, Table 3.). However, this study did observe a significant effect of month for the variable V_cmax in 2019, whereby an increase in V_cmax was observed with progression of the growing season (Fig 5A, Table 3.). Thus, this study observed no statistical evidence to suggest photosynthetic down-regulation of either V_cmax or J_max under elevated CO₂ across the three years of eCO₂ exposure in Q. robur.

Table 3.

Linear mixed-effects model analysis for V_cmax, J_max, net photosynthesis (A_net), area-based leaf nitrogen (Na) and mass-based leaf nitrogen (N_m) with CO₂ treatment (CO₂) and sampling month (Month) as fixed factors and a random effect of ‘plot’. Type III sums of squares computed using restricted maximum likelihood estimates for F-tests. The numerator degrees of freedom (DF) for each F-test are shown. Significance is noted in boldface as (* P < 0.05)

Figure 5.

Maximum rates of (A) carboxylation (V_cmax) and (B) electron transport (J_max), in addition to (C) area based (N_a) and D) mass based (N_m) leaf nitrogen of upper canopy Q. robur from 2015 to 2019 at BIFoR FACE. Means (±SE) of whole-plot averages (n=3) for ambient (blue circles) and elevated (red triangles) CO₂ treatments. Dashed line indicate the separation of sampling years with campaigns labelled ‘month/year’, as follows: Pre-treatment (‘07/15’); 1^st Year (‘06/17’); and the 3^rd year (‘05/19’ -‘08/19’) of CO fumigation. Data points may obscure error bars.

Furthermore, this study observed a strong positive linear relationship between J_max and V_cmax, which remained unchanged across CO treatments and growing season (R² = 0.75 ambient; R² = 0.71 elevated) (Supplementary Figure S1). The regressions for both treatments were statistically significant (P < 0.001 ambient, P < 0.001 elevated), with similar slopes (joint slope of R² = 0.72) and intercepts (intercept = 1.52; see Figure S1). Additionally, no eCO₂-induced decreases in either area-based foliar nitrogen (N_a) or mass-based foliar nitrogen (N_m) were observed (Fig 5; C and D, Table 3.) across the study period. No change in foliar nitrogen is corroborative of the results in Fig 5; A and B and also suggest the absence of photosynthetic downregulation under eCO₂ in mature Q. robur in the first three years of the long-term experiment.

The instantaneous response ratio (2015) and the longer-term response ratio (2017 and 2019) were calculated using the light-saturated A_net (i.e., A_sat) values at growth CO₂ from the A–C_i datasets (Fig 6B). There was no significant difference between the measurement year in either A_sat or the response ratio suggesting comparability between the instantaneous response ratio and the longer-term response ratio (Supplementary table 3: S3). A significant treatment effect was observed for A_sat (Fig. 6A, Table 3.) in all three sampling years, with a mean photosynthetic enhancement of 24 ± 2%, 31 ± 7% and 32 ± 11% in 2015, 2017 and 2019, respectively, under elevated CO₂ when compared to aCO₂. A significant effect of month on A_sat was observed in 2019, with A_sat increasing with the progression of the growing season (Table 3, Fig 6A). The photosynthetic enhancement observed from our A–C_i curve datasets are consistent with the values obtained in the diurnal dataset (33 ± 8%, Fig 5) and with the predicted enhancement calculated via CO₂ increase (37%). In summary, the consistency in the two separate measurements (i.e. diurnal and A–C_i curves) support the finding of sustained photosynthetic enhancement in mature Q. robur across the first three years of the BIFoR FACE experiment.

Figure 6.

A) Net photosynthesis (A_net) at growth CO₂ and B) Instantaneous (2015) and longer term (2017 and 2019) response ratios in the upper oak canopy using the A–C_i curve data. Means (± SD) of the plots per treatment are shown across six sampling campaigns for aCO₂ (blue circles), eCO₂ (red triangles) and either the instantaneous (grey squares) or longer term response ratio (grey circles). Dashed line indicate the separation of sampling years with campaigns labelled as follows; Pre-treatment (‘07/15’), 1^st Year (‘06/17’) and the 3^rd year (‘05/19’-’08/19’) of CO₂ fumigation.

Discussion

There is ample data on the short-term enhancement of photosynthesis by eCO₂ in trees using a variety of experimental set-ups from tree chambers to FACE experiments (Ainsworth & Rogers, 2007, Crous et al. 2013, others used above), but few data for mature forest-grown trees with multi-year CO₂ exposure in a FACE setting, and none for aged trees like presented here. For mature trees, available evidence suggests that there are significant increases in light-saturated A_net (Ellsworth et al., 2017; Körner et al., 2005) but there have been mixed results regarding the magnitude of photosynthetic enhancement and occurrence of photosynthetic downregulation in mature forest-grown trees (Bader et al., 2010; Crous et al., 2008; Ellsworth et al., 2017; Warren et al., 2015). In this study, after three years of eCO₂ exposure in mature temperate oak forest, net photosynthetic rates of upper canopy sunlit foliage from Q. robur were on average 23 ± 4% higher in the trees exposed to elevated CO₂ when compared to trees in control plots (Figs 2, 3 and 4; Tables 1 and 3). Light-saturated photosynthetic rates are found to be 33 ± 8%, higher when compared to aCO₂ trees, i.e., enhanced in the same proportion as the CO₂.

We predicted a theoretical A_net enhancement of 37 ± 6% for the 150 μmol mol^-1 increase in CO₂ at BIFoR FACE following reasoning in Nowak et al. (2004). Our mean annual eCO₂ enhancement of 23 ± 4% was based on our diurnal dataset of 3,552 samples, which included large variation in environmental conditions across the diurnal and seasonal profiles. Our result is somewhat lower than the expected enhancement of 37 ± 6%, which may be due to inclusion of the diurnal and seasonal variations in prevailing environmental conditions that include lower air temperatures, lower light conditions, and varying relative humidity. When we consider only light-saturated A_net (A_sat), from the diurnal dataset, our mean eCO₂ enhancement is 33 ± 8%. Our independent estimate of A_sat enhancement, using the A–C_i curve data, gives 32 ± 11 %, which is comparable within error to both the A_sat value from the diurnal measurements and the expected hypothesized enhancement of 37 ± 6%. Our average light-saturated eCO₂-induced photosynthetic enhancement from the A–Ci curves, 32 ± 11%, is generally lower than previously reported values in canopy dominant trees from other forest FACE experiments (Sholtis et al. 2004, 44%; Zotz et al. 2005, 36-49%; Liberloo et al. 2007, 49% Ainsworth & Rogers 2007, 46%; Crous et al. 2008, 40-68%), but is somewhat higher than the value of 19% in an analyses from the EucFACE experiment on mature Eucalyptus trees (Ellsworth et al. 2017). The lower photosynthetic enhancement observed at EucFACE was likely due to lower nutrient availability compared to BIFoR (Crous et al., 2015), although there were other differences such as the tree species and prevailing temperatures that would also affect this photosynthetic enhancement. We conclude that temperate deciduous forests similar to that in BIFoR FACE offer a continuing strong sensitivity to CO₂, maintaining enhanced gross primary productivity in these forests. Whether this equates to a sustained and continuing land carbon sink for these forests requires further work to capture the complete carbon cycle.

The role of environmental conditions for photosynthetic enhancement

In this study, we found that diurnal and seasonal environmental variables influenced the magnitude of photosynthetic enhancement under elevated CO₂. Consistent with our initial hypothesis, we observed significantly higher A_net and the highest eCO₂-induced photosynthetic enhancement under the highest light conditions at BIFoR FACE (i.e., PFD > 1000 µmol m^-2 s^-1). A negative linear relationship was observed for both A_net and eCO₂-induced photosynthetic enhancement with decreasing light level. This study observed a mean difference in eCO₂-indced photosynthetic enhancement of 24% between the highest light and lowest light category. Therefore, if the response to eCO₂ with variations in light are not considered, the difference in enhancement would likely translate to a significant over-estimation of potential carbon uptake by the tree. Our findings support previous research that observed cloud cover, and therefore reduced light availability, to limit carbon gain of canopy leaves by 25% in tropical tree species (Graham et al., 2003). Consequently, the relationship of A_net and CO₂ treatment effect with light intensity is important when scaling upper canopy data temporally — across diurnal periods of light limitation— and spatially — to the whole canopy of shaded and unshaded leaves— and helps to avoid overestimating the carbon uptake by temperate forests.

It has been previously suggested that larger eCO₂-enhancement is expected in shaded leaves as a result of a reduced light compensation point under eCO₂ exposure (Hättenschwiler, 2001; Norby & Zak, 2011). Yet, Hättenschwiler (2001) did find large interspecific variability of responses to eCO₂ with light, and in Quercus found that a greater CO₂ response occurred under higher light when compared to low light. These results support the present work as they suggest an increased carbon acquisition under eCO₂ at higher light, rather than for low light. Nevertheless, in the present study only upper canopy leaves were measured and therefore the results cannot be generalised to shade-adapted leaves i.e. lower in the canopy. Future work should include measurements on lower canopy (i.e. shade-adapted) leaves to see if the response to eCO₂ changes between the canopy layers (Crous et al., 2020). Understanding and quantifying the effects of light limitation, in both sunlit and shaded leaves, on the magnitude of the response to eCO₂ exposure will help improve canopy-scale photosynthesis models and will be particularly useful in understanding the longer-term response of mature forests to eCO₂.

In addition to light intensity (PFD), we observed interannual differences in the quantity and frequency distribution of precipitation (Fig 1.) across this study. In 2018, the United Kingdom (UK) experienced a combination of low precipitation and high temperatures that led to a widespread prolonged drought and heatwave event across the summer. The 2018 drought reported ground-level air temperatures as the highest on record (UK Met Office, 2020) and resulted in 2018 being a notably drier year than 2019 (see site description, above). Previous research suggests that greater eCO₂-induced photosynthetic enhancement is expected under drier conditions (Ellsworth et al., 2012; Nowak et al., 2004), when compared to wet conditions, as a result of the shape of the fundamental photosynthetic CO₂ response of C3 plants (Strain & Bazzaz, 1983). Greater eCO₂-induced photosynthetic enhancement under drought has been found before in Morgan et al. (2004) and McCarthy et al. (2010) but is not always evident (Ellsworth et al., 2012). In our study, we found no interannual differences in the eCO₂-induced photosynthetic enhancement in Q. robur, despite the notably drier year in 2018 (Table 1, Table 2, Fig 4). Therefore, our results do not support previous research suggesting that under drier conditions, larger photosynthetic enhancements may persist. Sustained photosynthetic enhancement in Q. robur with elevated CO₂ during dry years may be especially relevant for the southern geographical distribution of this species, such as in Mediterranean locations (Eaton et al., 2016), where drier environments are present or are expected to become more prevalent in the future.

The photosynthetic response of Q. robur was found to vary across the growing season, as has been observed in many other trees (Rogers & Ellsworth, 2002; Sholtis et al., 2004; Tissue et al., 1999). In this study, the A_sat (derived from the A–C_i dataset) in both CO₂ treatments increased about 50% from low, early in the season, to high, in the middle of the season; however, the relative response ratio to eCO₂ was maintained throughout this period. Furthermore, when assessing the diurnal dataset we found contrasting seasonal patterns between 2018 and 2019, with decreases in A_net across the growing season observed in 2018 compared to increases in A_net in 2019. Previous work has identified the reductions in photosynthesis across the season are largely associated with drier conditions (Gunderson et al., 2002). The contrasting seasonal patterns of A_net observed in this study between the sampling years may have been a result of the drier year in 2018. Data regarding the environmental influence of water availability on seasonal patterns will be important in determining seasonal C-uptake by mature forests and should be further investigated in mature Q. robur to improve longer term carbon-climate models.

Did changes to photosynthetic capacity or leaf biochemistry occur under eCO₂?

Seasonal and interannual biochemical changes are important in the regulation of A_net and allow us to understand any changes to photosynthetic capacity under eCO₂ in the long term. In some studies, a time-dependent decline in the magnitude of eCO₂-induced photosynthetic enhancement, i.e. photosynthetic downregulation, has been observed (Cure & Acock, 1986; Gunderson & Wullschleger, 1994). Here, it was investigated whether photosynthetic downregulation was observed in mature Q. robur after several years of eCO₂ exposure. As nitrogen is required for the synthesis and maintenance of photosynthetic proteins, downregulation of photosynthesis under eCO₂ exposure, commonly towards the end of the growing season, has been widely associated with leaf N declines and can therefore be a common observation in sites with limited soil N availability (Oren et al., 2001). For example at the N-limited site Duke FACE, declines of V_cmax and J_max resulted in reduced photosynthetic enhancement under eCO₂ exposure (Crous et al., 2008; Rogers & Ellsworth, 2002). We hypothesized that there may be reductions in V_cmax, J_max and leaf N, particularly in the 3^rd year of eCO₂ exposure and towards the end of the growing season, as a result of depleting soil nutrients with progression of the study and season, respectively. Instead, this study identified no significant changes in photosynthetic capacity of Q.robur over the first three years of exposure to elevated CO₂.

Our analysis of the 86 A–C_i curves collected in this experiment revealed no decrease in the rate of Rubisco carboxylation capacity (V_cmax) or RuBP regeneration mediated by electron transport (J_max), indicating that there were no changes in the photosynthetic capacity and that an acclimation to elevated CO₂ did not occur across the span of this study in mature Q. robur. Similar results have been found in other mature forest trees, particularly in deciduous forest species (Bader et al., 2010), as well as in a variety of plantation grown deciduous species (Herrick & Thomas, 2001; Sholtis et al., 2004). Additionally, this study did not find any changes in either mass- or area-based leaf nitrogen across the study period. No changes in foliar N with exposure to eCO₂ is also an indication that no reductions to the photosynthetic capacity were observed in Q. robur and corroborates the results obtained from the biochemical capacity (V_cmax and J_max).

Multiple studies have previously correlated the presence of photosynthetic downregulation with declines in foliar nitrogen (as reviewed in Medlyn et al., 1999) but this is not always the case. For example, no evidence of photosynthetic downregulation was observed in Liquidambar styraciflua (L.styraciflua.L) despite a reduction in foliar N under eCO₂ (Sholtis et al., 2004), as the leaves did not experience foliar N deficiency (Blinn & Buckner, 1989). Alternatively, in some studies changes in N_m were observed, yet no changes to N_a were, as a result of changes in LMA under eCO₂ (Curtis, 1996; Curtis & Wang, 1998; Medlyn et al., 1999; Norby et al., 1999). In our study, the absence of reduced N_m and N_a are supported by the absence of change in LMA under eCO₂ (data not shown). That is to say that if changes to LMA did occur with the longer-term exposure to eCO₂, this may result in changes to N_a in this species, but this would need to be revisited after 8-10 years of eCO₂ exposure. As a result, we observed no evidence to suggest the presence of photosynthetic downregulation occurring after three years of eCO₂ exposure and our results do not support the hypothesis that extended CO₂ enrichment induces photosynthetic downregulation. Instead, our results suggest that photosynthetic enhancement is sustained in mature Q. robur.

The ratio of J_max to V_cmax has been suggested to increase in plants exposed to eCO₂ as a result of a reallocation of leaf N from carboxylation processes to the light-harvesting processes (Medlyn et al., 1999; Onoda et al., 2005). In contrast, this study found no changes to the ratio of J_max to V_cmax, indicating that the relationship between carboxylation and light-harvesting processes was not affected by CO₂ treatment. This supports previous research in tree species that similarly observed no change in the J_max to V_cmax ratio under eCO₂ exposure (Crous et al., 2008; Kubiske et al., 2002; Medlyn et al., 1999). Our results suggests that, as no N reallocation occurred in the first three years, any increased N demand that may occur with eCO₂ exposure was instead met by increased N uptake (Johnson et al., 2004).

Photosynthetic downregulation has more often been observed in both young and mature gymnosperms, primarily in the older tissues (Griffin et al., 2000; Rogers & Ellsworth, 2002; Tissue et al., 2001; Tissue et al., 1999). Differences in phenology between angiosperms and gymnosperms may attribute to why photosynthetic downregulation has not been observed in species of deciduous angiosperms such as in Q.robur in this study. Seasonally deciduous trees have only current-year foliage and have specific growth and dormancy periods across the annual timeframe. By contrast, gymnosperms can maintain foliage across multiple years and continue growth throughout the year. These phenological differences result in changes in nutrient supply and demand for the tree and may result in lower occurrences of downregulation in deciduous angiosperm species such as Q. robur. Despite this, photosynthetic downregulation has been observed in a small number of deciduous angiosperm trees exposed to long-term eCO₂ (Juurola, 2003; Kubiske et al., 2002), noting that Juurola (2003) used an eCO₂ level at 2,000 µmol mol ⁻¹ and both studies were conducted on tree seedlings. Most often, the presence of photosynthetic downregulation has been correlated to soil N-limitations. As the present study observed no photosynthetic downregulation or decreases in leaf nitrogen, this suggest that the soil nutrient availability at this site is not yet limiting the photosynthetic processes in this forest system for Q. robur. However, as our results are from the initial three years of a 10-year study, the continuation of measurements will provide further insight into photosynthetic capacity after longer-term eCO₂ exposure.

Conclusions

After three years of eCO₂ exposure in a temperate deciduous forest at the BIFoR FACE facility, photosynthetic enhancement, of mature Q. robur leaves at the top of the canopy, was sustained across all years and was 33 ± 8%. The magnitude of photosynthetic enhancement was significantly affected by light conditions and so these results may be useful to extrapolate the carbon uptake of upper canopy data, both temporally and spatially, to the whole canopy, particularly during periods of light limitation, to avoid over estimating the carbon uptake of mature forests. We found no evidence of photosynthetic downregulation under eCO₂ and no declines in leaf nitrogen in the upper canopy. The lack of evidence for downregulation suggest there are sufficient soil nutrients for Q. robur to maintain increased photosynthetic enhancement under eCO₂ conditions, at least to this point in the eCO₂ experiment. The results suggest that mature deciduous forest species, such as those dominant in many lowland temperate forests, might exhibit a sustained, long-term positive C uptake response to rising atmospheric CO₂ provided adequate nutrients are available.

Conflict of interest

None declared

Author contributions

ARMK, JP, and AG designed the study; AG, KYC and DSE collected the data. AG organised the datasets under the supervision of DSE, with input from ARMK; AG and DSE designed and performed the statistical analyses, with input from KYC and ARMK. AG and DSE wrote the first draft of the paper. All authors contributed to the manuscript revision, and read and approved the submitted version.

Supplementary Information

Supplementary Figure 1.

Linear relationship between maximum rates of electron transport (J_max)and maximum rate of Rubisco carboxylation(V_cmax) in upper canopy Q.robur at BIFoR FACE. Vcmax and Jmax were derived as best-fit estimates from individual A—Ci curves. Data points represent trees that were exposed to either ambient (blue circles) or elevated (red triangles) CO₂ concentrations in 2018 and 2019. A single regression line is presented because the regressions generated for each treatment were not statistically different. The regression for the combined data was statistically significant (P < 0.0001). Individual regressions for ambient, , and elevated, , concentrations are:, and .

Supplementary Table S1. Anova to compare light categories used in Figure 4.

Supplementary Table S2. Anova to compare sampling years in Figure 5.

Supplementary Table S3. Anova to compare sampling years in Figure 6.

Acknowledgments

We thank the BIFoR technical team for canopy access operations and Ian Boomer for technical support with leaf elemental analysis. AG gratefully thanks Agnieszka Wujeska-Klause for guidance with statistical analysis in the early stages of the manuscript. AG gratefully acknowledges a studentship provided by the John Horseman Trust and the University of Birmingham. The BIFoR FACE facility is supported by the JABBS foundation, the University of Birmingham and the John Horseman Trust. ARMK acknowledges support from the Natural Research Council through grant (NE/S015833/1). We further gratefully acknowledge advice and field measurement collection in the first CO₂ fumigation season from Michael Tausz and Sabine Tausz-Pösch, respectively.

Abbreviations

[CO₂]

CO₂ concentration of the atmosphere

A

photosynthesis

A–C_i

curve Photosynthetic CO₂ response curve

aCO₂

CO₂ at ambient Ca (∼405 ppm)

A_net

Net photosynthetic rates.

A_sat

Light-saturated net photosynthesis

C

Carbon

CAS

Canopy access system

C_i

CO₂ concentration of the intercellular leaf space

DW

dry weight

eCO₂

CO₂ at elevated Ca (+150 ppm ambient)

FACE

Free air carbon dioxide enrichment

FW

fresh weight

J_max

Maximal photosynthetic electron transport rate (a proxy for ribulose-1,5-bisphosphate regeneration)

LMA

Leaf mass per unit area

MAP

mean annual precipitation

MAT

mean annual temperature

N

Nitrogen

N_a

Area-based foliar Nitrogen

N_m

Mass-based foliar Nitrogen

NSC

non-structural carbohydrates

PAR

photosynthetically active radiation

PFD

photon flux density

RH

relative humidity

T

temperature

T_air

Air temperature

T_leaf

Leaf temperature

SE

Standard error of the mean

V_cmax

Maximal carboxylation rate of Rubisco

VPD

vapour pressure deficit of the atmosphere

δ¹³C

ratio of ¹³C to ¹²C stable carbon isotopes

δ¹⁵N

ratio of ¹⁵N to¹⁴ N stable carbon isotopes