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 elevated atmospheric CO2 is a necessary step, but not the only one, for sustaining the terrestrial carbon uptake, but can vary diurnally, seasonally and with duration of CO2 exposure. Hence, we sought to quantify the photosynthetic response of the canopy-dominant species, Quercus robur, in a mature deciduous forest to elevated CO2 (eCO2) (+150 μmol mol−1 CO2) over the first 3 years of a long-term free air CO2 enrichment facility at the Birmingham Institute of Forest Research in central England (BIFoR FACE). Over 3000 measurements of leaf gas exchange and related biochemical parameters were conducted in the upper canopy to assess the diurnal and seasonal responses of photosynthesis during the 2nd and 3rd year of eCO2 exposure. Measurements of photosynthetic capacity via biochemical parameters, derived from CO2 response curves, (Vcmax and Jmax) together with leaf nitrogen concentrations from the pre-treatment year to the 3rd year of eCO2 exposure, were examined. We hypothesized an initial enhancement in light-saturated net photosynthetic rates (Asat) with CO2 enrichment of ≈37% based on theory but also expected photosynthetic capacity would fall over the duration of the study. Over the 3-year period, Asat of upper-canopy leaves was 33 ± 8% higher (mean and standard error) in trees grown in eCO2 compared with ambient CO2 (aCO2), and photosynthetic enhancement decreased with decreasing light. There were no significant effects of CO2 treatment on Vcmax or Jmax, nor leaf nitrogen. Our results suggest that mature Q. robur may exhibit a sustained, positive response to eCO2 without photosynthetic downregulation, suggesting that, with adequate nutrients, there will be sustained enhancement in C assimilated by these mature trees. Further research will be required to understand the location and role of the additionally assimilated carbon.

Introduction

Forest ecosystems cover ~30% of the Earth’s land surface, representing ~50% of terrestrially stored carbon and account 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. As the foundational driver of the carbon cycle of forests (e.g., Bonan 2008), the photosynthetic response to changing atmospheric CO2 is a necessary process 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). The amount of forest C-uptake in the future, and subsequent C sequestration, will be crucial determinants of future atmospheric CO2 concentrations. So, quantifying the photosynthetic response under elevated CO2 (eCO2), especially for mature trees, is critical to understanding the carbon uptake of forests under changing atmospheric composition.

It has been widely observed that eCO2 can have a stimulatory effect on plant photosynthesis, known as photosynthetic enhancement, at least in the short-term (weeks–months) with adequate nutrient and water availability permitting (Brodribb et al. 2020). Long-term (years to decades) photosynthetic responses to eCO2 are less well understood and lower-than-expected responses have been observed (Ainsworth and Long 2005, Ellsworth et al. 2017). Note that, even in studies that report sustained and/or strong stimulation of photosynthesis under eCO2, the additionally assimilated C does not necessarily translate into increased growth stimulation (Bader et al. 2013, Sigurdsson et al. 2013).

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 (Anet) is expected to increase with exposure to eCO2, with greatest photosynthetic enhancement expected at maximum photon flux density (Q) if Rubisco carboxylation is limiting (Sage et al. 2008). Decreases in Anet have been commonly associated with limitations in water and nutrient availability (Nowak et al. 2004, Ainsworth and Rogers 2007). For example, water availability has been found to increase the magnitude of eCO2-induced photosynthetic enhancement in drier years (Nowak et al. 2004, Ellsworth et al. 2012). 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, but not in itself sufficient, for modeling forest productivity (Jiang et al. 2020), and predicting carbon-climate feedbacks (e.g., Cox et al. 2013, Jones et al. 2016).

Despite a significant body of research on the photosynthetic response to eCO2 in tree seedlings and saplings (as reviewed in Medlyn et al. 1999, Ainsworth and Long 2005), fewer studies address the long-term (>1 year) photosynthetic responses in mature plantation trees (Liberloo et al. 2007, Crous et al. 2008, Uddling et al. 2009, Warren et al. 2015) and very few in mature forest-grown trees (Bader et al. 2010, Klein et al. 2016, Ellsworth et al. 2017). 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, generalize, and scale results from young trees in their exponential growth phase to the response of closed-canopy mature forests (Norby et al. 2016). For example, previous work from a long-term natural experiment found CO2 stimulation declined with tree age in Quercus ilex (Hättenschwiler et al. 1997). Therefore, it is plausible that model projections are currently overestimating the photosynthetic responses of mature forests and, thence, the ‘CO2 fertilization’ effect (Zhu et al. 2016). Consequently, 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 (Norby et al. 2016, Jiang et al. 2020).

Free-air CO2 enrichment (FACE) facilities are valuable to understand system-level responses to eCO2 (Ainsworth and Long 2005, Terrer et al. 2019) particularly in forests (Medlyn et al. 2015, Norby et al. 2016). The development of second generation forest FACE experiments focuses 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 to 60%, depending on tree species and environmental factors (as reviewed in Nowak et al. 2004, Ainsworth and Rogers 2007). Of the few studies on closed-canopy-dominant tree species, smaller photosynthetic enhancement to eCO2 have been observed (19–49%) than in studies conducted on younger trees (Sholtis et al. 2004, Liberloo et al. 2007, Crous et al. 2008), but the reasons behind this smaller response remain unclear.

There is evidence of a reduction in photosynthetic activity after long-term eCO2 exposure, known as photosynthetic downregulation (Ainsworth et al. 2004, Crous and Ellsworth 2004), but downregulation is not always observed (Curtis and Wang 1998, Herrick and Thomas 2001). Commonly photosynthetic downregulation under eCO2 exposure is the result of decreases, either directly or indirectly, in Rubisco carboxylation (Vcmax; Feng et al. 2015, Wujeska-Klause et al. 2019b). However, the stimulatory effect of photosynthesis under eCO2 may be reduced but is usually not completely removed (Leakey et al. 2009, Wujeska-Klause et al. 2019b). Photosynthetic downregulation has largely been observed in young plants (Leakey et al. 2009), with some downregulation observed in two aggrading plantation forests (Crous et al. 2008, Warren et al. 2015), commonly as a result of insufficient soil nitrogen supply (Luo et al. 2004). However, photosynthetic downregulation has largely not been observed in mature forests (Bader et al. 2010) and therefore open questions remain concerning the frequency and magnitude of photosynthetic downregulation under eCO2 exposure in mature forests.

To understand the photosynthetic responses in mature temperate deciduous forests, we evaluated the photosynthetic enhancement and potential downregulation in ca. 175-year-old canopy-dominant trees of Q. robur L. exposed to eCO2 for 3 years. Considering that forest FACE experiments aim to operate for 10 years or more, we refer to these CO2 responses as ‘early’ (Griffin et al. 2000). This study is among the oldest trees that have ever been examined under eCO2. 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, that included one pre-treatment year (2015) and 3-treatment years (2016–2019). Our aims were to quantify the photosynthetic response to eCO2 (i.e., ambient +150 μmol mol−1) for mature Q. robur and how light level influences this response, to determine whether photosynthetic downregulation under eCO2 occurred and to establish whether the relationship between leaf N and photosynthetic capacity changed in eCO2. We hypothesized that net photosynthetic gas exchange, Anet, will significantly increase with eCO2 and light levels (Q). 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 would be ≈37% following theory and reasoning in Nowak et al. (2004) (see also Supplemental Appendix 1 available as Supplementary Data at Tree Physiology Online). We also hypothesized that leaf nitrogen (N) will be reduced under eCO2 and that photosynthetic downregulation will be observed under eCO2 as a result of reduced leaf N and/or a decline in either the maximum rate of photosynthetic Rubisco carboxylation (Vcmax, μmol m−2 s−1); and the maximum rate of photosynthetic electron transport (Jmax, μmol m−2 s−1), or both (Griffin et al. 2000).

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), UK. The BIFoR FACE facility is a ‘second generation’ Forest FACE facility, extending the scope of first 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 (Q. robur L.) and a dense understorey comprising mostly of hazel coppice (Corylus avellana L.), sycamore (Acer pseudoplatanus L.), and hawthorn (Crataegus monogyna Jacq.). Quercus 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 30–40 years. Like most established forest of the temperate zone, the BIFoR FACE forest is under-managed.

The study site is situated within the temperature-rainfall climate space occupied by temperate forest (Sommerfeld et al. 2018, Jiang et al. 2020) and is characterized by cool wet winters and warm dry summers with a frost-free growing season from April to October. The mean January and July temperatures were 4 and 17 °C, respectively, and the average annual precipitation for the region is 720 mm (650, 669, 646 and 818 mm, in 2015, 2017, 2018 and 2019, respectively, when the study was conducted; see Figure 1). The total N deposition load at the BIFoR FACE site is ~ 22 Kg N/ha/year (estimate provided by S. Tomlinson at the Centre for Ecology and Hydrology, Edinburgh, UK; MacKenzie et al. 2021), representing ~15% of the total nitrogen nutrition of temperate deciduous trees (Rennenberg and Dannenmann 2015).

Time series showing the daily meteorological data at the BIFoR FACE facility covering the period of 1 January 2015–1 January 2021. Subplots are: (A) maximum, (red), mean (orange) and minimum (blue) daily air temperatures (°C), (B) global downwelling solar radiation (MJ m−2) 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 are 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).
Figure 1.

Time series showing the daily meteorological data at the BIFoR FACE facility covering the period of 1 January 2015–1 January 2021. Subplots are: (A) maximum, (red), mean (orange) and minimum (blue) daily air temperatures (°C), (B) global downwelling solar radiation (MJ m−2) 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 are 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 CO2 (aCO2; ca. 405 μmol mol−1) and three plots supplied with CO2 enriched air, termed elevated CO2 plots (eCO2). The latter plots were operated such that they achieved 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) as 5-min rolling averages (Hart et al. 2020; see Supplementary Figure 1 available as Supplementary Data at Tree Physiology Online). Elevated CO2 is added from dawn (solar zenith angle, sza = −6.5°) to dusk (sza = −6.5°) throughout the growing season. Daytime exposure to eCO2 was almost continuous throughout the growing season (Hart et al. 2020), with exceptions if the 15-min average wind speed was >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 CO2 fumigation thresholds for wind speed and temperature were selected because of the high cost of maintaining eCO2 and the insignificant uptake of carbon under these conditions, respectively. 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).

In each plot, canopy access was gained through a custom-built 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 (see Supplementary Figure 2 available as Supplementary Data at Tree Physiology Online). 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 2 m 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 and June 2019 where replicates were reduced to two 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 focused 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 (ACi) measurements (includes pre-treatment, 2015; 1st year, 2017; and 3rd year, 2019, of CO2 fumigation). Measurements were conducted in all six experimental plots with infrastructure, on one chosen candidate tree per plot. The target tree remained the same for all treatment years (2017, 2018 and 2019) but a different tree was measured during the pre-treatment period in 2015. This change was because the plot infrastructure, which determined the CAS system, was not constructed until 2016.

When reporting treatment effects from the present study, we report the mean enhancement or treatment effect:
(1)
where Ai,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):
(2)
where ca is the aCO2 mixing ratio and Δca 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), caca = 392/150 = 2.61, and we use net photosynthesis instead of GPP. Hence, our theoretical predicted photosynthetic enhancement (Nowak et al. 2004; see Supplemental Appendix 1 available as Supplementary Data at Tree Physiology Online) for the +150 μmol mol−1 increase in CO2 (i.e., ≈37%; 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 (e.g., net CO2 photosynthetic assimilation rates, Anet) 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 3 days to complete, with the exception of September 2018 and June 2019 where only two replicate plots could be measured. A total of four diurnal campaigns were conducted in both 2018 and 2019, providing a total of 3426 data points. Five to six healthy leaves were randomly selected in the same oak tree per plot, every 30–40 min 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 (Tair) 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. Measurements were confined to the first flush of leaves across the season for consistency in leaf age. Expanding leaves, judged from color and texture, were avoided for measurements, as they had not matured 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 stabilization 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 (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 Ca values in the LiCOR chamber head were 390 ± 0.9 and 538 ± 2.7 μmol mol−1, in 2018, and 393 ± 1.0 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–Ci curves

ACi curves were conducted in three growing seasons: pre-treatment year (2015), in the 1st 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). Detached branches were transferred to researchers on the ground immediately after excision, where they were placed in a bucket of water to minimize desiccation. Branches were re-cut under water and allowed to stabilize, before starting measurements. Measurement on detached branches was conducted no longer than 45 min after collection. Previous studies investigating measurements of gas exchange on severed or attached branches found no significant differences between the two methods (Bader et al. 2016, Verryckt et al. 2020). ACi curves were measured at a Q 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. Before each curve, a stabilization period of between 5 and 10 min was used depending on the prevailing environmental conditions and each curve took an average of 40 min. Light-saturated net photosynthesis (Asat) was estimated from ACi curves at growth [CO2]. The CO2 concentrations were changed in 12–14 steps starting at the respective growth [CO2]; every 100 μmol mol−1 down to 50 μmol mol−1 (near the photosynthetic CO2 compensation point), then increasing to 1800 μmol mol−1 in roughly 200 μmol mol−1 increment steps. Five to six replicate ACi curves on different leaves per CO2 treatment were measured per day. Measurements were taken between 09:00–11:00 and 14:00–17:00 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–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 leaf gas exchange. Each leaf was photographed on white graph paper, with a ruler for reference. Leaf area analysis was conducted using imaging software Image J (IMAGE J v1.53, National Institutes of Health, Bethesda, MD, USA) and the fresh weight was recorded. Each leaf was oven dried at 70 °C for at least 72 h, re-weighed for dry weight and the leaf mass per unit area was calculated. Dried leaf fragments were ground and each sample (~2 mg) was enclosed in a tin capsule. Samples were analyzed for δ13C, total C and total N using an elemental analyzer interfaced with an isotope ratio mass spectrometer (Sercon Ltd, Cheshire, UK).

Statistical analysis

All statistical analyses were performed in R version 4.0.3 (R Core Team, 2020). Before statistical analysis, all data were checked for normality by inspection of the QQ plots and Levene’s test, and residuals from model fitting were checked for evidence of heteroscedasticity. Hourly averages of diurnal measurements were analyzed 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), in addition to their interactions. In addition, ‘time of day’ and ‘plot’ were represented as random factors, the latter as individual trees were nested within each experimental plot. Type III F-statistics associated with the mixed model analysis (repeated-measures analysis of variance, ANOVA) were reported. Statistically significant CO2 treatment differences among groups were further tested with Tukey’s post hoc test using the R package ‘emmeans’ (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 vapor pressure deficit (D), D < 2.2 kPa, were sub-divided into four light (Q) categories, each sampled about equally. The Q classes were chosen based on the characteristic response of Anet to light as follows: Q < 250; 250 ≤ Q < 500; 500 ≤ Q < 1000 and Q ≥ 1000 μmol m−2 s−1. CO2 treatment, year and Q category were then used as parameters in the ANOVA.

The photosynthetic CO2 response (ACi) curves were fit with the model of Farquhar et al. (1980) to estimate the apparent maximum rate of photosynthetic Rubisco carboxylation (Vcmax, μmol m−2 s−1) and the apparent maximum rate of photosynthetic electron transport (Jmax, 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 and Ellsworth 2002). We tested for outliers by examining the Jmax/Vcmax ratio, RMSE values and standard errors (SE) for fits of Jmax and Vcmax, all of which indicate violations to the theory for fitting these curves (Sharkey et al. 2007). Visual inspection of each ACi curve with outliers allowed us to identify any incomplete curves and/or mechanical failures and those curves were subsequently removed. This accounted for < 10% of the data, leaving a total of 86 ACi curves across the 3-sampling years in the analysis.

Results

Measurement conditions

Overall, diurnal measurements were conducted on dry, sunny days (Figure 1), and environmental conditions (Q and Tleaf) were consistent between aCO2 and eCO2 across the two growing seasons of diurnal measurements (Figures 2A, B and 3A, B). Q 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.

In situ diurnal measurements of (A) Q (μmol m−2 s−1), (B) hourly mean Tleaf (°C) and (C) hourly mean Anet (μmol m−2 s−1); each fitted with an LOESS regression, 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 and not all time points were replicated. The line types in (A) represent replicate plot pairings of; plots 1 and 3 (dotted), plots 2 and 4 (solid) and plots 5 and 6 (long-dash) and the two colors represent the CO2 treatments of aCO2 (blue) and eCO2 (red).
Figure 2.

In situ diurnal measurements of (A) Q (μmol m−2 s−1), (B) hourly mean Tleaf (°C) and (C) hourly mean Anet (μmol m−2 s−1); each fitted with an LOESS regression, 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 and not all time points were replicated. The line types in (A) represent replicate plot pairings of; plots 1 and 3 (dotted), plots 2 and 4 (solid) and plots 5 and 6 (long-dash) and the two colors represent the CO2 treatments of aCO2 (blue) and eCO2 (red).

In situ diurnal measurements of (A) Q (μmol m−2 s−1), (B) hourly mean Tleaf (°C) and (C) hourly mean Anet (μmol m−2 s−1); each fitted with an LOESS regression, at BIFoR FACE in 2019 from the upper Q. robur canopy. Error bars indicate n = 3, the exception of June where only two replicate plots were measured and not all time points were replicated. The line types in (A) represent replicate plot pairings of; plots 1 and 3 (dotted), plots 2 and 4 (solid) and plots 5 and 6 (long-dash) and the two colors represent the CO2 treatments of aCO2 (blue) and eCO2 (red).
Figure 3.

In situ diurnal measurements of (A) Q (μmol m−2 s−1), (B) hourly mean Tleaf (°C) and (C) hourly mean Anet (μmol m−2 s−1); each fitted with an LOESS regression, at BIFoR FACE in 2019 from the upper Q. robur canopy. Error bars indicate n = 3, the exception of June where only two replicate plots were measured and not all time points were replicated. The line types in (A) represent replicate plot pairings of; plots 1 and 3 (dotted), plots 2 and 4 (solid) and plots 5 and 6 (long-dash) and the two colors represent the CO2 treatments of aCO2 (blue) and eCO2 (red).

Leaf temperature was more stable than Q with lower variability across the diurnal sampling, high similarity between sampling days, and high consistency between CO2 treatments. There were differences of up to 15 °C in midday measurements of Tleaf, between months, suggesting a seasonal influence as would be expected from the site’s mid-latitude location, with differences more prominent in 2019 than 2018. The highest Tleaf values were observed in July with a common seasonal decline after this campaign.

Analysis of the diurnal dataset showed the range of mean daily Anet was similar between years, however the highest mean daily Anet (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 Anet across the growing season observed in 2018 compared with increases in Anet in 2019. In both sampling years, we observed a significant enhancement of Anet when exposed to eCO2 (P < 0.05, Table 1 and Figures 2 and 3). Here, we did not observe any significant effect of either season or sampling year on Anet (Table 1). Therefore, from measurements of Anet collected from the diurnal dataset, a mean eCO2-driven photosynthetic enhancement (i.e., 100.|$\Delta{A}_i/{A}_{i,a}$|⁠) of 23 ± 4% was observed across the 2-year period of this study.

Photosynthesis and variation in photon flux density (Q)

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

Consistent with our hypothesis, we observed mean eCO2-driven photosynthetic enhancement to increase with increasing Q, with the largest enhancement observed at highest Q in both sampling years, 30 ± 9% and 35 ± 13%, for 2018 and 2019, respectively (Figure 4B). In 2018, eCO2-driven photosynthetic enhancement ranged from 7 ± 10%, in the lowest Q class, to 30 ± 9%, in the highest Q class (Figure 4B). A similar positive relationship between eCO2-driven photosynthetic enhancement and Q was present in 2019 with enhancement ranging from 11 ± 6%, in the lowest Q class, to 35 ± 13%, in the highest Q class (Figure 4B). There was no significant effect of year (Table 2) and therefore the mean eCO2-driven photosynthetic enhancement at light saturation (i.e., in the highest Q class) was on average 33 ± 8% across the 2-sampling years. Our results report that the mean eCO2-driven photosynthetic enhancement of light-saturated Anet (Asat) in both sampling years was consistent, within error (using 95% confidence intervals), of the theoretical predicted enhancement based on proportion of CO2 increase (≈37 ± 6%), indicating a sensitivity to eCO2 (Eq. (2), above) of close to unity for Asat.

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 (Vcmax) and apparent maximum electron transport capacity for RuBP regeneration (Jmax; Figure 5) to assess the photosynthetic capacity in the initial years of the long-term experiment. Initially, we tested for differences between the year of sampling and found no statistical difference of either Vcmax or Jmax between the 3-sampling years (2015, 2017 and 2019; Figure 5, see Supplementary Table S2 available as Supplementary Data at Tree Physiology Online). This study found no significant effects of CO2 enrichment on Vcmax or Jmax across the 2 years of CO2 enrichment, i.e., the 1st and 3rd years, and no significant effect of season between the 3 measurement years (Figure 5 and Table 3). However, this study did observe a significant effect of month for the variable Vcmax in 2019, whereby an increase in Vcmax was observed with progression of the growing season (Figure 5A and Table 3). Thus, this study observed no statistical evidence to suggest photosynthetic downregulation of either Vcmax or Jmax under eCO2 across the 3 years of eCO2 exposure in Q. robur.

Table 1

Linear mixed-effects model analysis for photosynthesis with CO2 treatment (CO2) using the diurnal dataset, sampling month (Month) and sampling year (Year) as fixed factors and random effects of ‘plot’ and ‘time’. 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).

ParameterdfP-value
CO210.044*
Month30.14
Year10.31
CO2  * Month30.18
CO2  * Year10.18
Month * Year30.43
CO2  * Month * Year30.079
ParameterdfP-value
CO210.044*
Month30.14
Year10.31
CO2  * Month30.18
CO2  * Year10.18
Month * Year30.43
CO2  * Month * Year30.079
Table 1

Linear mixed-effects model analysis for photosynthesis with CO2 treatment (CO2) using the diurnal dataset, sampling month (Month) and sampling year (Year) as fixed factors and random effects of ‘plot’ and ‘time’. 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).

ParameterdfP-value
CO210.044*
Month30.14
Year10.31
CO2  * Month30.18
CO2  * Year10.18
Month * Year30.43
CO2  * Month * Year30.079
ParameterdfP-value
CO210.044*
Month30.14
Year10.31
CO2  * Month30.18
CO2  * Year10.18
Month * Year30.43
CO2  * Month * Year30.079
(A) The distribution of net photosynthesis (Anet; μmol m−2 s−1) in each of the four photon flux density (Q) categories (Q < 250; 250 ≥ Q < 500; 500 ≥ Q < 1000 and Q ≥ 1000 μmol m−2 s−1) 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 use diurnal gas exchange measurements in the upper canopy oak trees at the BIFoR FACE facility with Tleaf > 18 °C and D < 2.2 kPa, in eCO2 (red) or aCO2 (blue) treatments. Red diamonds indicate the mean Anet values. (B) Boxplots of the enhancement response ratio (A550/A400) (gray) for each year, and predicted enhancement ratio (dashed line) (1.37) following Nowak et al. (2004).
Figure 4.

(A) The distribution of net photosynthesis (Anet; μmol m−2 s−1) in each of the four photon flux density (Q) categories (Q < 250; 250 ≥ Q < 500; 500 ≥ Q < 1000 and Q ≥ 1000 μmol m−2 s−1) 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 use diurnal gas exchange measurements in the upper canopy oak trees at the BIFoR FACE facility with Tleaf > 18 °C and D < 2.2 kPa, in eCO2 (red) or aCO2 (blue) treatments. Red diamonds indicate the mean Anet values. (B) Boxplots of the enhancement response ratio (A550/A400) (gray) for each year, and predicted enhancement ratio (dashed line) (1.37) following Nowak et al. (2004).

Table 2

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

ParameterdfP-value
CO210.016*
Year10.062
Q3<0.001***
CO2  * Year10.97
CO2  *  Q30.011*
Year *  Q30.0078**
CO2  * Year *  Q30.13
ParameterdfP-value
CO210.016*
Year10.062
Q3<0.001***
CO2  * Year10.97
CO2  *  Q30.011*
Year *  Q30.0078**
CO2  * Year *  Q30.13
Table 2

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

ParameterdfP-value
CO210.016*
Year10.062
Q3<0.001***
CO2  * Year10.97
CO2  *  Q30.011*
Year *  Q30.0078**
CO2  * Year *  Q30.13
ParameterdfP-value
CO210.016*
Year10.062
Q3<0.001***
CO2  * Year10.97
CO2  *  Q30.011*
Year *  Q30.0078**
CO2  * Year *  Q30.13
Maximum rates of (A) carboxylation (Vcmax) and (B) electron transport (Jmax), in addition to (C) area-based (Na) and (D) mass based (Nm) 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 indicates the separation of sampling years with campaigns labelled ‘month/year’, as follows: Pre-treatment (‘07/15’); 1st year (‘06/17’) and the 3rd year (‘05/19’–‘08/19’) of CO2 fumigation. Data points may obscure error bars.
Figure 5.

Maximum rates of (A) carboxylation (Vcmax) and (B) electron transport (Jmax), in addition to (C) area-based (Na) and (D) mass based (Nm) 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 indicates the separation of sampling years with campaigns labelled ‘month/year’, as follows: Pre-treatment (‘07/15’); 1st year (‘06/17’) and the 3rd year (‘05/19’–‘08/19’) of CO2 fumigation. Data points may obscure error bars.

Table 3

Linear mixed-effects model analysis for Vcmax, Jmax, net photosynthesis (Anet), area-based leaf nitrogen (Na) and mass-based leaf nitrogen (Nm) with CO2 treatment (CO2) and sampling month (month) as fixed factors and random effects of ‘plot’ and ‘time’. Type III sums of squares computed using restricted maximum likelihood estimates for F-tests. The numerator df for each F-test are shown. Significance is noted in boldface as (*  P < 0.05).

ParameterVcmaxJmaxAnetNmNa
dfP-valueP-valueP-valueP-valueP-value
CO210.700.370.042*0.420.64
Month30.02*0.150.034*0.930.052
CO2  * Month30.200.570.330.690.11
ParameterVcmaxJmaxAnetNmNa
dfP-valueP-valueP-valueP-valueP-value
CO210.700.370.042*0.420.64
Month30.02*0.150.034*0.930.052
CO2  * Month30.200.570.330.690.11
Table 3

Linear mixed-effects model analysis for Vcmax, Jmax, net photosynthesis (Anet), area-based leaf nitrogen (Na) and mass-based leaf nitrogen (Nm) with CO2 treatment (CO2) and sampling month (month) as fixed factors and random effects of ‘plot’ and ‘time’. Type III sums of squares computed using restricted maximum likelihood estimates for F-tests. The numerator df for each F-test are shown. Significance is noted in boldface as (*  P < 0.05).

ParameterVcmaxJmaxAnetNmNa
dfP-valueP-valueP-valueP-valueP-value
CO210.700.370.042*0.420.64
Month30.02*0.150.034*0.930.052
CO2  * Month30.200.570.330.690.11
ParameterVcmaxJmaxAnetNmNa
dfP-valueP-valueP-valueP-valueP-value
CO210.700.370.042*0.420.64
Month30.02*0.150.034*0.930.052
CO2  * Month30.200.570.330.690.11

Consistent with previous research, this study observed a strong positive linear relationship between Jmax and Vcmax, which remained unchanged across CO2 treatments and growing season (r2 = 0.75 ambient; r2 = 0.71 elevated; see Supplementary Figure 3 available as Supplementary Data at Tree Physiology Online). In addition, no eCO2-induced decreases in either area-based foliar nitrogen (Na) or mass-based foliar nitrogen (Nm) were observed (Figure 5C and D and Table 3) across the study period. No change in foliar nitrogen is corroborative of the results in Figure 5 and also suggests the absence of photosynthetic downregulation under eCO2 in mature Q. robur in the first 3 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 Anet (i.e., Asat) values at growth CO2 from the ACi datasets (Figure 6B). There was no significant difference between the measurement years in either Asat or the response ratio suggesting comparability between the instantaneous response ratio and the longer-term response ratio (see Supplementary Table S3 available as Supplementary Data at Tree Physiology Online). A significant treatment effect was observed for Asat (Figure 6A and Table 3) in all 3-sampling years, with a mean eCO2-driven photosynthetic enhancement of 24 ± 2%, 31 ± 7% and 32 ± 11% in 2015, 2017 and 2019, respectively, under eCO2 when compared with aCO2. A significant effect of month on Asat was observed in 2019, with Asat increasing with the progression of the growing season (Table 3 and Figure 6A). The photosynthetic enhancement observed from our ACi curve datasets are consistent with the values obtained in the diurnal dataset (33 ± 8%, Figure 5) but is lower than the theoretical predicted enhancement calculated via CO2 increase (37%) (see Supplemental Appendix 1 available as Supplementary Data at Tree Physiology Online). In summary, the consistency in the two separate measurements (i.e., diurnal and ACi curves) support the finding of sustained eCO2-driven photosynthetic enhancement in mature Q. robur across the first 3 years of the BIFoR FACE experiment.

(A) Net photosynthesis (Anet) at growth CO2 and (B) instantaneous (2015) and longer-term (2017 and 2019) response ratios in the upper oak canopy using the A–Ci 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 (gray 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’), 1st year (‘06/17’) and the 3rd year (‘05/19’–‘08/19’) of CO2 fumigation.
Figure 6.

(A) Net photosynthesis (Anet) at growth CO2 and (B) instantaneous (2015) and longer-term (2017 and 2019) response ratios in the upper oak canopy using the ACi 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 (gray 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’), 1st year (‘06/17’) and the 3rd year (‘05/19’–‘08/19’) of CO2 fumigation.

Discussion

There are ample data on the short-term enhancement of photosynthesis by eCO2 in young trees using a variety of experimental set-ups from tree chambers to FACE experiments (e.g., Ainsworth and Rogers 2007, Crous et al. 2008), but few data for mature forest-grown trees with multi-year CO2 exposure in a FACE setting. For mature trees, available evidence suggests that there are significant increases in light-saturated Anet (Körner et al. 2005, Ellsworth et al. 2017) but there have been mixed results regarding the magnitude of photosynthetic enhancement (range 13–49% per 100 ppm of CO2 increase) and occurrence of photosynthetic downregulation in mature forest-grown trees (Crous et al. 2008, Bader et al. 2010, 2016, Warren et al. 2015, Ellsworth et al. 2017). In this study, we predicted a theoretical Anet enhancement of 37% for the 150 μmol mol−1 increase in CO2 at BIFoR FACE following reasoning in Nowak et al. (2004; see Supplemental Appendix 1 available as Supplementary Data at Tree Physiology Online). After 3 years of eCO2 exposure in mature temperate oak forest, net photosynthetic rates of upper canopy foliage from Q. robur were on average 23 ± 4% higher, based on the diurnal dataset, in the trees exposed to eCO2 when compared with control plots (Figures 24; Tables 1 and 3). The eCO2-driven photosynthetic enhancement observed is substantially lower than the theoretical expected enhancement of 37%, likely due to diurnal and seasonal variation in prevailing environmental conditions such as lower air temperatures, lower light conditions and varying vapor pressure deficits. Only considering light-saturated Anet (Asat) from the diurnal dataset, our mean photosynthetic enhancement is greater than the average diurnal enhancement, at 33 ± 8% rather than 23%. Furthermore, our independent estimate of Asat enhancement based on the ACi curve data is 32 ± 11%, which is comparable within error (using 95% confidence intervals) to both the Asat value from the diurnal measurements and the hypothesized enhancement of 37%. A slight stomatal closure in eCO2 could have contributed to the slightly lower photosynthetic enhancement than the hypothesized enhancement of 37% (see Supplemental Appendix 1 available as Supplementary Data at Tree Physiology Online). However, our average light-saturated photosynthetic enhancement is generally lower than previously reported values in canopy-dominant trees from other forest FACE experiments (Bader et al. 2010, 42–48%; Crous et al. 2008, 40–68%; Liberloo et al. 2007, 49% and Sholtis et al. 2004, 44%), but is somewhat higher than the value of 19% 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 with BIFoR (Crous et al. 2015), although there were other differences such as the tree species and prevailing temperatures that would also affect the magnitude of the photosynthetic enhancement.

The role of environmental conditions for photosynthetic enhancement

Consistent with our initial hypothesis, we observed significantly higher Anet and a 24% higher photosynthetic enhancement under the highest light conditions at BIFoR FACE (i.e., Q > 1000 μmol m−2 s−1) compared with the lowest light category. Thus, a negative linear relationship was observed for both Anet and eCO2-induced photosynthetic enhancement with decreasing light levels. Our results are consistent with previous research on mature trees that observed an effect of light on the magnitude of CO2-driven stimulation of photosynthesis (Bader et al. 2016), suggesting variation in light should be considered when assessing the response to eCO2. Consequently, the relationship of Anet and CO2 treatment effect with light intensity is important when scaling upper canopy data both across diurnal periods of light limitation and extending to the whole canopy, of shaded and sunlit leaves, to avoid overestimating canopy-scale photosynthesis by temperate forests.

It has been previously suggested that larger photosynthetic enhancement may be expected in low light environments (Hättenschwiler 2001, Norby and Zak 2011). For example, deep shaded tree seedlings displayed greater photosynthetic gains than those in moderate shade (photosynthetic enhancement of 97% and 47%, respectively) with exposure to eCO2 (Kitao et al. 2015). In light-limited environments, higher CO2 concentrations can increase the apparent quantum yield and reduce the light compensation point leading to enhanced carbon uptake (Larcher, 2003, Kitao et al. 2015). Hättenschwiler (2001) found large interspecific variability and, in Quercus, that greater photosynthetic responses to CO2 occurred under higher light when compared with low light. However, both Kitao et al. (2015) and Hättenschwiler (2001) studied tree seedlings in contrast to upper canopy leaves of a canopy-dominant species in the present study. Although shade leaves were not measured here, the results here from the top of the tree canopy provide an important benchmark for the magnitude of photosynthetic enhancement by eCO2 in a mature oak forest.

In addition to light intensity, the photosynthetic response of Q. robur varied across the growing season, as has been observed in many other trees (Tissue et al. 1999, Rogers and Ellsworth 2002, Sholtis et al. 2004). Here, Asat (derived from the ACi dataset) in both CO2 treatments increased about 50% from early in the season (May), to the middle of the season (July); yet, the relative response ratio to eCO2 was stable throughout this period at 32%. In addition, when assessing the diurnal dataset, we found contrasting seasonal patterns between 2018 and 2019, with decreases in Anet across the growing season observed in 2018 compared with increases in Anet in 2019, likely due to drier and warmer conditions in 2018. Previous research has identified reductions in photosynthesis across the season is largely associated with drier conditions (Gunderson et al. 2002), which support the results observed in the present study. This suggests that the influence of soil water availability on the seasonal pattern in oak physiology is critical for determining seasonal C-uptake by mature forests and should be further investigated in mature Q. robur to improve longer term carbon-climate models (see Limousin et al. 2013).

Previous research has identified eCO2-driven photosynthetic responses observed in seedlings and saplings may not reflect the photosynthetic responses of mature forest-grown trees (Hättenschwiler et al. 1997). The present study provided a unique opportunity to assess the eCO2-driven photosynthetic responses in 175-year-old canopy-dominant trees and found lower photosynthetic stimulation than the many previous studies on tree seedlings and younger trees (e.g., Curtis and Wang 1998, Sholtis et al. 2004, Ainsworth and Long 2005, Liberloo et al. 2007, Crous et al. 2008). The age dependency of CO2 responsiveness to photosynthesis in trees (Turnbull et al. 1998, Wujeska-Klause et al. 2019a), highlights the importance of long-term experiments, such as the present study and others in understanding potential variable responses across the lifetime of a tree, vital for accurate climate-carbon modeling of forests.

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

In some studies, a time-dependent decline in the magnitude of eCO2-induced photosynthetic enhancement, i.e., photosynthetic downregulation, has been observed (Cure and Acock 1986, Gunderson and Wullschleger 1994). Here, we hypothesized that there may be reductions in Vcmax, Jmax and leaf N, particularly in the 3rd year of eCO2 exposure (Luo et al. 2004). Our analysis of the 86 ACi curves collected in this experiment revealed no decrease in the rate of Vcmax or Jmax, indicating that there were no significant changes in the photosynthetic capacity of Q. robur over the first 3 years of exposure to eCO2. A lack of photosynthetic downregulation has also been found in similar seasonally deciduous species, including the closely related species Quercus petraea (Bader et al. 2010), in addition to Liquidambar styraciflua, Populus spp. and Betula papyrifera (Herrick and Thomas 2001, Sholtis et al. 2004, Liberloo et al. 2007, Uddling et al. 2009). An apparent lack of downregulation has also been observed in other mature forest-grown species (Bader et al. 2010, Ellsworth et al. 2017).

As nitrogen is required for the synthesis and maintenance of photosynthetic proteins, eCO2-driven photosynthetic downregulation has been associated with declines in foliar N (as reviewed in Medlyn et al. 1999) and soil N-limitations (e.g., Rogers and Ellsworth 2002, Crous et al. 2008, Warren et al. 2015). The current study on Q. robur did not find any changes in either mass- or area-based leaf nitrogen across the study period, indicating there are no reductions to photosynthetic capacity (Figure 5). This corroborates the findings from the Vcmax and Jmax parameters, supporting the suggestion for sustained photosynthesis in Q. robur over the first 3 years of exposure to eCO2. Hence, there were no changes to the ratio of Jmax to Vcmax, indicating that the relationship between carboxylation and light-harvesting processes was not affected by CO2 treatment, as found in previous studies (Medlyn et al. 1999, Crous et al. 2008), including the closely related species, Q. petraea (Bader et al. 2010). These results may point to soil nutrient availability not yet limiting the photosynthetic processes in this forest system. The BIFoR FACE site receives moderately high atmospheric N deposition (~22 Kg N/ha/yr) thought to represent 15% of the total nitrogen nutrition of temperate deciduous trees, likely preventing ecosystem N-limitation at present (Rennenberg and Dannenmann 2015). Therefore, with adequate N deposition in the soil, sustained photosynthetic enhancement was observed in the first 3 years of eCO2 exposure at BIFoR FACE.

Conclusions

After 3 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% (mean ± SE) at light saturation, close to the theoretical expectation. The magnitude of photosynthetic enhancement was significantly affected by light conditions with higher enhancement at higher light. 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 a relatively high photosynthetic enhancement under eCO2 conditions, at least to this point in the eCO2 experiment. Much further work remains to determine the movement and allocation of this enhanced C-uptake in the forest. Our results are consistent with a sustained, positive C-uptake response to rising atmospheric CO2 in a mature deciduous forest tree species, provided adequate nutrients are available.

Supplementary data

Supplementary data for this article are available at Tree Physiology Online.

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 Environment Research Council through grant (NE/S015833/1) which also facilitated DSE’s participation. We further gratefully acknowledge advice and field measurement collection in the first CO2 fumigation season from Michael Tausz and Sabine Tausz-Pösch, respectively.

Conflict of interest

None declared.

Author contributions

ARMK, JP and AG designed the study; AG, KYC and DSE collected the data. AG organized 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.

References

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
.

Ainsworth
 
EA
,
Rogers
 
A
(
2007
)
The response of photosynthesis and stomatal conductance to rising [CO2]: mechanisms and environmental interactions
.
Plant Cell Environ
 
30
:
258
270
.

Ainsworth
 
EA
,
Rogers
 
A
,
Nelson
 
R
,
Long
 
SP
(
2004
)
Testing the “source-sink” hypothesis of down-regulation of photosynthesis in elevated [CO2] in the field with single gene substitutions in Glycine max
.
Agric For Meteorol
 
122
:
85
94
.

Bader
 
MKF
,
Leuzinger
 
S
,
Keel
 
SG
,
Siegwolf
 
RTW
,
Hagedorn
 
F
,
Schleppi
 
P
,
Körner
 
C
(
2013
)
Central european hardwood trees in a high-CO2 future: synthesis of an 8-year forest canopy CO2 enrichment project
.
J Ecol
 
101
:
1509
1519
.

Bader
 
MKF
,
Mildner
 
M
,
Baumann
 
C
,
Leuzinger
 
S
,
Körner
 
C
(
2016
)
Photosynthetic enhancement and diurnal stem and soil carbon fluxes in a mature Norway spruce stand under elevated CO2
.
Environ Exp Bot
 
124
:
110
119
.

Bader
 
MKF
,
Siegwolf
 
R
,
Körner
 
C
(
2010
)
Sustained enhancement of photosynthesis in mature deciduous forest trees after 8 years of free air CO2 enrichment
.
Planta
 
232
:
1115
1125
.

Bonan
 
GB
(
2008
)
Forests and climate change: forcings, feedbacks, and the climate benefits of forests
.
Science
 
320
:
1444
1449
.

Brodribb
 
TJ
,
Powers
 
J
,
Cochard
 
H
,
Choat
 
B
(
2020
)
Hanging by a thread? Forests and drought
.
Science
 
368
:
261
266
.

Cox
 
PM
,
Pearson
 
D
,
Booth
 
BB
,
Friedlingstein
 
P
,
Huntingford
 
C
,
Jones
 
CD
,
Luke
 
CM
(
2013
)
Sensitivity of tropical carbon to climate change constrained by carbon dioxide variability
.
Nature
 
494
:
341
344
.

Crous
 
KY
,
Ellsworth
 
DS
(
2004
)
Canopy position affects photosynthetic adjustments to long-term elevated CO2 concentration (FACE) in aging needles in a mature Pinus taeda forest
.
Tree Physiol
 
24
:
961
970
.

Crous
 
KY
,
Ósvaldsson
 
A
,
Ellsworth
 
DS
(
2015
)
Is phosphorus limiting in a mature eucalyptus woodland? Phosphorus fertilisation stimulates stem growth
.
Plant and Soil
 
391
:
293
305
.

Crous
 
KY
,
Reich
 
PB
,
Hunter
 
MD
,
Ellsworth
 
DS
(
2010
)
Maintenance of leaf N controls the photosynthetic CO2 response of grassland species exposed to 9 years of free-air CO2 enrichment
.
Glob Chang Biol
 
16
:
2076
2088
.

Crous
 
KY
,
Walters
 
MB
,
Ellsworth
 
DS
(
2008
)
Elevated CO2 concentration affects leaf photosynthesis-nitrogen relationships in Pinus taeda over nine years in FACE
.
Tree Physiol
 
28
:
607
614
.

Cure
 
JD
,
Acock
 
B
(
1986
)
Crop responses to carbon dioxide doubling: a literature survey
.
Agric For Meteorol
 
38
:
127
145
.

Curtis
 
PS
,
Wang
 
X
(
1998
)
A meta-analysis of elevated CO2 effects on woody plant mass, form, and physiology
.
Oecologia
 
113
:
299
313
.

Duursma
 
RA
(
2015
)
Plantecophys - an R package for analysing and modelling leaf gas exchange data
.
PLoS One
 
10
:e0143346. https://doi.org/10.1371/journal.pone.0143346.

Eaton
 
E
,
Caudullo
 
G
,
Oliveira
 
S
,
de
 
Rigo
 
D
(
2016
) Quercus robur and Quercus petraea in Europe: distribution, habitat, usage and threats. In:
European Atlas of Forest Tree Species, Joint Research Centre (European Commission), Brussels
, pp.
160
163
. ISBN 978-92-79-36740-3, DOI: .

Ellsworth
 
DS
,
Anderson
 
IC
,
Crous
 
KY
 et al. (
2017
)
Elevated CO2 does not increase eucalypt forest productivity on a low-phosphorus soil
.
Nature Climate Change
 
7
:
279
282
.

Ellsworth
 
DS
,
Thomas
 
R
,
Crous
 
KY
,
Palmroth
 
S
,
Ward
 
E
,
Maier
 
C
,
Delucia
 
E
,
Oren
 
R
(
2012
)
Elevated CO2 affects photosynthetic responses in canopy pine and subcanopy deciduous trees over 10 years: a synthesis from Duke FACE
.
Glob Chang Biol
 
18
:
223
242
.

Farquhar
 
GD
,
von
 
Caemmerer
 
S
,
Berry
 
JA
(
1980
)
A biochemical model of photosynthetic CO2 assimilation in leaves of C3 species
.
Planta
 
149
:
78
90
.

Feng
 
Z
,
Rütting
 
T
,
Pleijel
 
H
,
Wallin
 
G
,
Reich
 
PB
,
Kammann
 
CI
,
Newton
 
PCD
,
Kobayashi
 
K
,
Luo
 
Y
,
Uddling
 
J
(
2015
)
Constraints to nitrogen acquisition of terrestrial plants under elevated CO2
.
Glob Chang Biol
 
21
:
3152
3168
.

Friedlingstein
 
P
,
Betts
 
R
,
Bopp
 
L
 et al. (
2006
)
Climate –carbon cycle feedback analysis, results from the C4MIP model intercomparison
.
J Climate
 
19
:
3337
3353
.

Friedlingstein
 
P
,
Jones
 
MW
,
O’Sullivan
 
MA
 et al. (
2019
)
Global carbon budget 2019
.
Earth Syst Sci Data
 
11
:
1783
1838
.

Griffin
 
KL
,
Tissue
 
DT
,
Turnbull
 
MH
,
Whitehead
 
D
(
2000
)
The onset of photosynthetic acclimation to elevated CO2 partial pressure in field-grown Pinus radiata D. don. After 4 years
.
Plant Cell Environ
 
23
:
1089
1098
.

Gunderson
 
CA
,
Sholtis
 
JD
,
Wullschleger
 
SD
,
Tissue
 
DT
,
Hanson
 
PJ
,
Norby
 
RJ
(
2002
)
Environmental and stomatal control of photosynthetic enhancement in the canopy of a sweetgum (Liquidambar styraciflua L.) plantation during 3 years of CO2 enrichment
.
Plant Cell Environ
 
25
:
379
393
.

Gunderson
 
CA
,
Wullschleger
 
SD
(
1994
)
Photosynthetic acclimation in trees to rising atmospheric CO2: a broader perspective
.
Photosynth Res
 
39
:
369
388
.

Hart
 
KM
,
Curioni
 
G
,
Blaen
 
P
 et al. (
2020
)
Characteristics of free air carbon dioxide enrichment of a northern temperate mature forest
.
Glob Chang Biol
 
26
:
1023
1037
.

Hättenschwiler
 
S
(
2001
)
Tree seedling growth in natural deep shade: functional traits related to interspecific variation in response to elevated CO2
.
Oecologia
 
129
:
31
42
.

Hättenschwiler
 
S
,
Miglietta
 
F
,
Raschi
 
A
,
Körner
 
C
(
1997
)
Thirty years of in situ tree growth under elevated CO2: a model for future forest responses?
 
Glob Chang Biol
 
3
:
463
471
.

Hendrey
 
G
,
Ellsworth
 
D
,
Lewin
 
K
,
Nagy
 
J
(
1999
)
A free-air enrichment system for exposing tall forest vegetation to elevated atmospheric CO 2
.
Glob Chang Biol
 
5
:
293
309
.

Herrick
 
JD
,
Thomas
 
RB
(
2001
)
No photosynthetic down-regulation in sweetgum trees (Liquidambar styraciflua L.) after three years of CO2 enrichment at the Duke Forest Face experiment
.
Plant Cell Environ
 
24
:
53
64
.

Jiang
 
M
,
Medlyn
 
BE
,
Drake
 
JE
 et al. (
2020
)
The fate of carbon in a mature forest under carbon dioxide enrichment
.
Nature
 
580
:
227
231
.

Jones
 
CD
,
Ciais
 
P
,
Davis
 
SJ
 et al. (
2016
)
Simulating the Earth system response to negative emissions
.
Environ Res Lett
 
11
:095012. https://doi.org/10.1088/1748-9326/11/9/095012.

Keenan T, Prentice I, Canadell J  et al. (

2016
)
Recent pause in the growth rate of atmospheric CO2 due to enhanced terrestrial carbon uptake
.
Nat Commun
 
7
:
13428
. https://doi.org/10.1038/ncomms13428.

Kitao
 
M
,
Hida
 
T
,
Eguchi
 
N
,
Tobita
 
H
,
Utsugi
 
H
,
Uemura
 
A
,
Kitaoka
 
S
,
Koike
 
T
(
2015
)
Light compensation points in shade-grown seedlings of deciduous broadleaf tree species with different successional traits raised under elevated CO2
.
Plant Biology
 
18
:
31
42
.

Klein
 
T
,
Bader
 
MKF
,
Leuzinger
 
S
,
Mildner
 
M
,
Schleppi
 
P
,
Siegwolf
 
RTW
,
Körner
 
C
(
2016
)
Growth and carbon relations of mature Picea abies trees under 5 years of free-air CO2 enrichment
.
J Ecol
 
104
:
1720
1733
.

Körner
 
C
(
2017
)
A matter of tree longevity
.
Science
 
355
:
130
131
.

Körner
 
C
,
Asshoff
 
R
,
Bignucolo
 
O
,
Hättenschwiler
 
S
,
Keel
 
SG
,
Peláez-Riedl
 
S
,
Pepin
 
S
,
Siegwolf
 
RTW
,
Zotz
 
G
(
2005
)
Ecology: carbon flux and growth in mature deciduous forest trees exposed to elevated CO2
.
Science
 
309
:
1360
1362
.

Larcher W (

2003
)
Physiological Plant Ecology: Ecophysiology and Stress Physiology of Functional Groups
. Springer-Verlag, New York. https://doi.org/10.1007/978-3-662-05214-3.

Leakey
 
ADB
,
Ainsworth
 
EA
,
Bernacchi
 
CJ
,
Rogers
 
A
,
Long
 
SP
,
Ort
 
DR
(
2009
)
Elevated CO2 effects on plant carbon, nitrogen, and water relations: six important lessons from FACE
.
J Exp Bot
 
60
:
2859
2876
.

Liberloo
 
M
,
Tulva
 
I
,
Raïm
 
O
,
Kull
 
O
,
Ceulemans
 
R
(
2007
)
Photosynthetic stimulation under long-term CO2 enrichment and fertilization is sustained across a closed Populus canopy profile (EUROFACE)
.
New Phytol
 
173
:
537
549
.

Limousin
 
JM
,
Bickford
 
CP
,
Dickman
 
LT
,
Pangle
 
RE
,
Hudson
 
PJ
,
Boutz
 
AL
,
Gehres
 
N
,
Osuna
 
JL
,
Pockman
 
WT
,
Mcdowell
 
NG
(
2013
)
Regulation and acclimation of leaf gas exchange in a piñon-juniper woodland exposed to three different precipitation regimes
.
Plant Cell Environ
 
36
:
1812
1825
.

Luo
 
Y
,
Su
 
B
,
Currie
 
WS
 et al. (
2004
)
Progressive nitrogen limitation of ecosystem responses to rising atmospheric carbon dioxide
.
Bio Sci
 
54
:
731
739
.

Luyssaert
 
S
,
Schulze
 
ED
,
Börner
 
A
,
Knohl
 
A
,
Hessenmöller
 
D
,
Law
 
BE
,
Ciais
 
P
,
Grace
 
J
(
2008
)
Old-growth forests as global carbon sinks
.
Nature
 
455
:
213
215
.

MacKenzie
 
R
,
Krause
 
S
,
Hart
 
K
 et al. (
2021
)
BIFoR FACE: water-soil-vegetation-atmosphere research in a temperate deciduous forest catchment, including under elevated CO2
.
Hydrol Process
 
35
:e14096. https://doi.org/10.1002/hyp.14096.

Medlyn
 
BE
,
Badeck
 
FW
,
De Pury
 
DGG
 et al. (
1999
)
Effects of elevated [CO2] on photosynthesis in European forest species: a meta-analysis of model parameters
.
Plant Cell Environ
 
22
:
1475
1495
.

Medlyn
 
BE
,
Zaehle
 
S
,
De Kauwe
 
MG
 et al. (
2015
)
Using ecosystem experiments to improve vegetation models
.
Nat Clim Change
 
5
:
528
534
.

Mölder
 
A
,
Meyer
 
P
,
Nagel
 
RV
(
2019
)
Integrative management to sustain biodiversity and ecological continuity in Central European temperate oak (Quercus robur, Q. petraea) forests: an overview
.
For Ecol Manage
 
437
:
324
339
.

Norby
 
RJ
,
De Kauwe
 
MG
,
Domingues
 
TF
 et al. (
2016
)
Model-data synthesis for the next generation of forest free-air CO2 enrichment (FACE) experiments
.
New Phytol
 
209
:
17
28
.

Norby
 
RJ
,
Zak
 
DR
(
2011
)
Ecological and evolutionary lessons from free air carbon enhancement (FACE) experiments
.
Annu Rev Ecol Evol Syst
 
42
:
181
203
.

Nowak
 
RS
,
Ellsworth
 
DS
,
Smith
 
SD
(
2004
)
Functional responses of plants to elevated atmospheric CO2 - do photosynthetic and productivity data from FACE experiments support early predictions?
 
New Phytol
 
162
:
253
280
.

Ostle
 
NJ
,
Levy
 
PE
,
Evans
 
CD
,
Smith
 
P
(
2009
)
UK land use and soil carbon sequestration
.
Land Use Policy
 
26
:
S274
S283
. https://doi.org/10.1016/j.landusepol.2009.08.006.

Pan
 
Y
,
Birdsey
 
RA
,
Fang
 
J
 et al. (
2011
)
A large and persistent carbon sink in the World’s forests
.
Science
 
333
:
4
. http://science.sciencemag.org/content/333/6045/988#BIBL.

Parsons
 
R
,
Weyers
 
JDB
,
Lawson
 
T
,
Godber
 
IM
(
1998
)
Rapid and straightforward estimates of photosynthetic characteristics using a portable gas exchange system
.
Photosynthetica
 
34
:
265
279
.

Piao
 
S
,
Sitch
 
S
,
Ciais
 
P
 et al. (
2013
)
Evaluation of terrestrial carbon cycle models for their response to climate variability and to CO2 trends
.
Glob Chang Biol
 
19
:
2117
2132
.

Rennenberg
 
H
,
Dannenmann
 
M
(
2015
)
Nitrogen nutrition of trees in temperate forests-the significance of nitrogen availability in the pedosphere and atmosphere
.
Forests
 
6
:
2820
2835
.

Rogers
 
A
,
Ellsworth
 
DS
(
2002
)
Photosynthetic acclimation of Pinus taeda (loblolly pine) to long-term growth in elevated pCO2 (FACE)
.
Plant Cell Environ
 
25
:
851
858
.

Sage
 
RF
,
Way
 
DA
,
Kubien
 
DS
(
2008
)
Rubisco, rubisco activase, and global climate change
.
J Exp Bot
 
59
:
1581
1595
.

Sharkey
 
TD
,
Bernacchi
 
CJ
,
Farquhar
 
GD
,
Singsaas
 
EL
(
2007
)
Fitting photosynthetic carbon dioxide response curves for C3 leaves
.
Plant Cell Environ
 
30
:
1035
1040
.

Sholtis
 
JD
,
Gunderson
 
CA
,
Norby
 
RJ
,
Tissue
 
DT
(
2004
)
Persistent stimulation of photosynthesis by elevated CO2 in a sweetgum (Liquidambar styraciflua) forest stand
.
New Phytol
 
162
:
343
354
.

Sigurdsson
 
BD
,
Medhurst
 
JL
,
Wallin
 
G
,
Eggertsson
 
O
,
Linder
 
S
(
2013
)
Growth of mature boreal Norway spruce was not affected by elevated [CO2] and/or air temperature unless nutrient availability was improved
.
Tree Physiol
 
33
:
1192
1205
.

Sommerfeld
 
A
,
Senf
 
C
,
Buma
 
B
 et al. (
2018
)
Patterns and drivers of recent disturbances across the temperate forest biome
.
Nat Commun
 
9
:4355. https://doi.org/10.1038/s41467-018-06788-9.

Terrer
 
C
,
Jackson
 
RB
,
Prentice
 
IC
 et al. (
2019
)
Nitrogen and phosphorus constrain the CO2 fertilization of global plant biomass
.
Nature Climate Change
 
9
:
684
689
.

Tissue
 
DT
,
Griffin
 
KL
,
Ball
 
JT
(
1999
)
Photosynthetic adjustment in field-grown ponderosa pine trees after six years of exposure to elevated CO2
.
Tree Physiol
 
19
:
221
228
.

Turnbull
 
MH
,
Tissue
 
DT
,
Griffin
 
KL
,
Rogers
 
GND
,
Whitehead
 
D
(
1998
)
Photosynthetic acclimation to long-term exposure to elevated CO2 concentration in Pinus radiata D. don. Is related to age of needles
.
Plant Cell Environ
 
21
:
1019
1028
.

Uddling
 
J
,
Teclaw
 
RM
,
Pregitzer
 
KS
,
Ellsworth
 
DS
(
2009
)
Leaf and canopy conductance in aspen and aspen-birch forests under free-air enrichment of carbon dioxide and ozone
.
Tree Physiol
 
29
:
1367
1380
.

Valentini
 
R
,
Epron
 
D
,
De Angelis
 
P
,
Matteucci
 
G
,
Dreyer
 
E
(
1995
)
In situ estimation of net CO2 assimilation, photosynthetic electron flow and photorespiration in Turkey oak (Q. cerris L.) leaves: diurnal cycles under different levels of water supply
.
Plant Cell Environ
 
18
:
631
640
.

Verryckt
 
LT
,
Van Langenhove
 
L
,
Ciais
 
P
 et al. (
2020
)
Coping with branch excision when measuring leaf net photosynthetic rates in a lowland tropical forest
.
Biotropica
 
52
:
608
615
.

Warren
 
JM
,
Jensen
 
AM
,
Medlyn
 
BE
,
Norby
 
RJ
,
Tissue
 
DT
(
2015
)
Carbon dioxide stimulation of photosynthesis in Liquidambar styraciflua is not sustained during a 12-year field experiment
.
AoB PLANTS
 
7
:plu074. https://doi.org/10.1093/aobpla/plu074.

Wujeska-Klause
 
A
,
Crous
 
KY
,
Ghannoum
 
O
,
Ellsworth
 
DS
(
2019a
)
Leaf age and eCO2 both influence photosynthesis by increasing light harvesting in mature Eucalyptus tereticornis at EucFACE
.
Environ Exp Bot
 
167
:
103857
. https://doi.org/10.1016/j.envexpbot.2019.103857.

Wujeska-Klause
 
A
,
Crous
 
KY
,
Ghannoum
 
O
,
Ellsworth
 
DS
(
2019b
)
Lower photorespiration in elevated CO2 reduces leaf N concentrations in mature eucalyptus trees in the field
.
Glob Chang Biol
 
25
:
1282
1295
.

Zhu
 
Z
,
Piao
 
S
,
Myneni
 
RB
 et al. (
2016
)
Greening of the earth and its drivers
.
Nature Climate Change
 
6
:
791
795
.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Editor: David Whitehead
David Whitehead
Editor
Search for other works by this author on: