Abstract

To determine the effect of growth under elevated CO2 partial pressures (pCO2) on photosynthetic electron transport and photoprotective energy dissipation, we examined light-saturated net photosynthetic CO2 assimilation (Asat), the capacity for photosynthetic O2 evolution, chlorophyll fluorescence emission and the pigment composition of upper-canopy loblolly pine needles in the eighth year of exposure to elevated pCO2 (20 Pa above ambient) at the free-air CO2 enrichment facility in the Duke Forest. During the summer growing season, Asat was 50% higher in current-year needles and 24% higher in year-old needles in elevated pCO2 in comparison with needles of the same age cohort in ambient pCO2. Thus, photosynthetic down-regulation at elevated pCO2 was observed in the summer in year-old needles. In the winter, Asat was not significantly affected by growth pCO2. Reductions in Asat, the capacity for photosynthetic O2 evolution and photosystem II (PSII) efficiency in the light-acclimated and fully-oxidized states were observed in the winter when compared to summer. Growth at elevated pCO2 had no significant effect on the capacity for photosynthetic O2 evolution, PSII efficiencies in the light-acclimated and fully-oxidized states, chlorophyll content or the size and conversion state of the xanthophyll cycle, regardless of season or needle age cohort. Therefore, we observed no evidence that photosynthetic electron transport or photoprotective energy dissipation responded to compensate for the effects of elevated pCO2 on Calvin cycle activity.

Introduction

Exposure to elevated atmospheric CO2 concentrations (pCO2) generally results in significant stimulation (20–80%) in light-saturated rates of photosynthetic CO2 assimilation (Asat; reviewed in Curtis and Wang 1998, Saxe et al. 1998, Norby et al. 1999, Ellsworth et al. 2004, Long et al. 2004, Ainsworth and Long 2005, Ainsworth and Rogers 2007). After long-term exposure (i.e., seasons) to elevated pCO2, the magnitude of this stimulation can be attenuated in trees by photosynthetic down-regulation (reviewed in Gunderson and Wullschleger 1994, Rogers and Humphries 2000, Ellsworth et al. 2004, Ainsworth and Rogers 2007). Meta-analyses of field studies indicate that, in comparison to trees grown at ambient pCO2, down-regulatory reductions in CO2-saturated photosynthetic capacity (Amax) of 10–20% are typical in trees grown at elevated pCO2 (Medlyn et al. 1999, Rogers and Humphries 2000, Ainsworth and Long 2005). Exposure to abiotic environmental stresses such as low temperatures and soil nutrient deficiency tend to increase the magnitude of photosynthetic down-regulation to elevated pCO2 (Gunderson and Wullschleger 1994, Medlyn et al. 1999, Ainsworth and Long 2005). Leaf age may also affect the degree of photosynthetic down-regulation to elevated pCO2. For example, older needles of evergreen trees generally exhibit greater photosynthetic down-regulation than younger needles in elevated pCO2 (Wang et al. 1995, Turnbull et al. 1998, Griffin et al. 2000, Jach and Ceulemans 2000, Tissue et al. 2001, Rogers and Ellsworth 2002, Crous and Ellsworth 2004).

Alterations in Asat at elevated pCO2 could necessitate compensatory alterations in the rate of photosynthetic electron transport to balance ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity with the rate of ribulose-1,5-bisphosphate (RuBP) regeneration (Sage et al. 1989, Pammenter et al. 1993). For example, using the data derived from the analyses of the relationship between net photosynthetic CO2 assimilation (A) and internal pCO2 (Ci) (i.e., ACi curves) of ponderosa pine needles, a 36% reduction in maximum apparent carboxylation rate of Rubisco (Vcmax) was observed in trees grown at elevated pCO2 in comparison to those grown at ambient pCO2, along with a 21% reduction in the maximum apparent rate of electron transport mediated by the rate of regeneration of RuBP (Jmax; Tissue et al. 1999). The difference in the magnitude of the reductions in Vcmax and Jmax may be due to the increased efficiency of carboxylation by Rubisco in elevated pCO2, whereas there is no such enhancement in the efficiency of photosynthetic electron transport (Wullschleger 1993, Gunderson and Wullschleger 1994, Sage 1994, Medlyn et al. 1999, Norby et al. 1999). Therefore, maintaining the balance between RuBP carboxylation and regeneration during photosynthetic down-regulation in elevated pCO2 may involve a relatively greater decrease in Vcmax than in Jmax.

Alterations in the rate of photosynthetic electron transport under elevated pCO2 may necessitate alterations in the size and composition of light harvesting complexes, along with alterations in the allocation of absorbed light to electron transport and other processes. Plants in most environments routinely absorb more light than they can utilize to support photosynthesis and reductive nitrogen and sulfur assimilation (Demmig-Adams and Adams 1996, Müller et al. 2001, Ort 2001). This so-called excess light can decrease the efficiency of photosynthetic light utilization by leading to the oxidation of essential constituents of photosynthetic electron transport, in particular the D1 protein of photosystem II (PSII; Melis 1999). This effect is commonly referred to as ‘photoinhibition’. Environmental variables that affect Asat will affect the level of excess light absorption in an opposing manner, because the level of excess light absorption is a function of both incident light intensity and photosynthetic light utilization. Therefore, enhanced Asat in response to elevated pCO2 could decrease the levels of excess light, whereas photosynthetic down-regulation after long-term growth in elevated pCO2 may raise the levels of excess light back to those observed under ambient pCO2.

Plants are protected against photoinhibition by biochemical processes termed ‘photoprotection’, including energy dissipation, which safely converts excess absorbed light energy to heat. Energy dissipation requires de-epoxidized carotenoids (typically zeaxanthin, a member of the xanthophyll cycle) and a trans-thylakoid membrane proton gradient (reviewed in Demmig-Adams and Adams 1996, Niyogi 1999, Logan 2006). During the growing season, the level of energy dissipation can be modulated diurnally via enzyme-catalyzed conversions among the carotenoids of the xanthophyll cycle or by changes in the magnitude of the trans-thylakoid membrane proton gradient such that only excess light is dissipated and photochemical light utilization is unaffected. The size of the overall pool of xanthophyll cycle carotenoids acclimates over a timescale of days to seasons and influences the capacity for energy dissipation (Logan et al. 1998a, 1998b). In the winter, long-lived evergreen plants typically exhibit seasonally-sustained energy dissipation that results in reductions in the efficiency of photosynthetic light utilization that are not modulated diurnally (reviewed in Adams et al. 2004). These seasonal reductions in light use efficiency are photoprotective in nature, although they have been interpreted by some researchers as photoinhibition because both phenomena result in decreased PSII quantum yield, as measured by chlorophyll fluorescence emission (discussed in Adams et al. 1995).

The impact of elevated pCO2 on Asat, Vcmax, Jmax and Rubisco enzyme kinetics has been well described for loblolly pine (Tissue et al. 1993, 1996, 1997, Lewis et al. 1996). At the Duke free-air CO2 enrichment (FACE) site, elevated pCO2 has consistently stimulated Asat in the summer (typically > 30%) over the 9 years of operation (Myers et al. 1999, Rogers and Ellsworth 2002, Crous and Ellsworth 2004, Springer et al. 2005, Crous et al. 2008, Maier et al. 2008). Stimulation of Asat tended to be greater in sun-acclimated needles compared with shade-acclimated needles (Springer et al. 2005) and greater in current-year needles than in year-old needles (Rogers and Ellsworth 2002, Crous and Ellsworth 2004, Crous et al. 2008). Photosynthetic down-regulation has also been observed in loblolly pine at the Duke FACE site, with reductions (typically 15–30%) in Asat, Vcmax and Jmax occurring in year-old needles, but not in current-year needles (Rogers and Ellsworth 2002, Crous and Ellsworth 2004, Crous et al. 2008). However, the response of photosynthetic electron transport and photoprotective processes such as energy dissipation to elevated pCO2 is less well understood (Myers et al. 1999). Furthermore, much of the research examining the effect of elevated pCO2 on photosynthetic electron transport depends on the estimates of Jmax, which is an indirect measurement modeled from an ACi curve. Measurement of the capacity of photosynthetic O2 evolution is the most direct means of assessing the rate of photosynthetic electron transport, as this quantifies O2 production resulting from water oxidation by the oxygen-evolving complex associated with PSII. Water oxidation provides electrons for photosynthetic linear electron transport.

In this study, conducted in the eighth year of exposure to elevated pCO2 at the Duke FACE site, we coupled analyses of the capacity for photosynthetic O2 evolution, chlorophyll fluorescence emission and photosynthetic pigment composition with measurements of Asat to examine the effect of elevated pCO2 on electron transport and energy dissipation in loblolly pine. We took measurements in both summer and winter on year-old and current-year needles to capture potential seasonal and needle age impacts on photosynthetic performance.

Materials and methods

Site description

Measurements were conducted at the Duke Forest FACE facility which is located in an even-aged plantation of loblolly pine (Pinus taeda L.) established in 1983 in Orange County, NC (35°58′ N and 79°08′ W). The temperate forest overstory is dominated by loblolly pine and to a lesser extent by sweetgum (Liquidambar styraciflua L.), whereas the understory consists of more than 50 woody and herbaceous species (Springer et al. 2005). Mean annual precipitation is 1145 mm, distributed evenly throughout the year, and mean annual temperature is 15.5 °C. The growing season generally occurs from March to mid-October. Summers are warm and humid, whereas winters are cooler and drier. Soils at the site are clay loam (Enon series), acidic (soil pH ~ 6.0) and of moderately low fertility, considered to be N-limited (Oren et al. 2001).

The FACE array consists of six 30-m-diameter plots with three replicate plots in ambient atmospheric pCO2 and three replicate plots in pCO2 elevated 20 Pa above ambient. Between August 1996 and 2002, CO2 enrichment was provided 24 h per day when air temperature was above 6 °C and wind speed was below 5 m s−1; see Hendrey et al. (1999) for complete details on FACE operation and protocol. In 2003, the enrichment protocol was modified such that enrichment was maintained during day-time hours only according to the same temperature and wind speed thresholds.

Photosynthetic CO2 assimilation and chlorophyll fluorescence

In the summer (July 2004) and in the winter (January 2005) of the eighth year of exposure of loblolly pine to elevated pCO2, we simultaneously measured light-saturated rates of photosynthesis (Asat), stomatal conductance (gs) and chlorophyll fluorescence emission using a portable photosynthesis system fitted with a fluorometer chamber (LI 6400F; Li-Cor Biosciences, Lincoln, NE). Measurements were conducted on attached, intact, sun-lit, upper-canopy needles on three trees in the center of each ring; current-year and year-old needles were measured in the summer, but only current-year needles were available in the winter. During measurements, needles in the chamber were exposed to saturating light (1500 μmol photons m−2 s−1), ambient vapor pressure deficit (1.36 ± 0.28 kPa in July and 0.62 ± 0.07 kPa in January (mean ± standard deviation)), ambient air temperature (26.4 ± 1.2 °C in July and 9.8 ± 1.5 °C in January) and growth pCO2 (38 Pa in ambient pCO2 rings and 58 Pa in elevated pCO2 rings). To determine whether photosynthetic down-regulation occurred, we also measured gas exchange at 38 Pa pCO2 on needles on elevated pCO2 trees. Gas exchange measurements were recorded when the readings were stable and the coefficient of variation was < 1%, typically 5–10 min after needle placement in the chamber. Needle photosynthetic rates were expressed on the basis of total needle surface area estimated as in Lewis et al. (1996).

Measurements of chlorophyll fluorescence emission provided a nondestructive means of determining the status of PSII and the allocation of light energy absorbed by PSII antennae to photochemistry and other processes (Schreiber et al. 1986, 1995, Genty et al. 1989, Bilger and Björkman 1990, Demmig-Adams et al. 1996, Logan et al. 2007). We measured steady-state fluorescence from light-acclimated needles (Fs), maximal fluorescence from light-acclimated needles during transient exposure to super-saturating light intensities (Fm′) and minimal fluorescence from light-acclimated needles upon transient darkening (Fo′). These measured variables were used to determine Fv′/Fm′ (where Fv′ = Fm′ − Fo′), which represents the intrinsic efficiency of PSII in the fully-oxidized state, and (Fm′ − Fs)/Fm′, which is the actual PSII efficiency under ambient light conditions (ΦPSII; Schreiber et al. 1986, 1995, Genty et al. 1989).

Photosynthetic O2 evolution

The rate of O2 evolution under CO2- and light-saturated conditions is a common and direct measure of the capacity for photosynthetic electron transport (Evans and Terashima 1987, Ottander and Öquist 1991, Grace and Logan 1996). Photosynthetic O2 evolution was measured on needles in a fascicle adjacent to the fascicle used for measurements of gas exchange, fluorescence and pigments. Detached needles were wrapped in a moist paper towel and transported to the laboratory where measurements were initiated < 10 min after collection. Measurements were conducted at 1500 μmol photons m−2 s−1 at 25 °C using a gas-phase O2 electrode (model LD-2 equipped with a LS-2 light source, Hansatech, King’s Lynn, UK) according to the protocol in Delieu and Walker (1981). The air in the chamber was gently replaced with expired air before measurements to achieve CO2 saturation, as in Li et al. (2000) and Wheelwright and Logan (2004). Needle segments were arranged so that their outer (convex) surfaces were exposed to the light source and the projected areas were determined from geometric measurements.

Needle pigment composition

Immediately after the determination of needle gas exchange and fluorescence emission under growth conditions, the portions of needles that were exposed to Li-Cor 6400 chamber conditions were harvested and frozen in liquid N2 for analyses of chlorophyll and carotenoid composition. Chlorophylls were extracted in acetone according to the method described by Adams and Demmig-Adams (1992) and quantified using high performance liquid chromatography as described by Gilmore and Yamamoto (1991) using an Agilent 1100 series HPLC (Agilent Technologies, Palo Alto, CA) equipped with a YMC Carotenoid™ C-30 reverse phase column (Waters Corp., Milford, MA).

Statistical analyses

The effects of growth pCO2 and needle age during the growing season were examined by two-way analyses of variance (ANOVA) of the data collected in the summer (July 2004). Year-old needles senesced in the autumn and thus were not available for the winter (January 2005) measurements. Therefore, ANOVAs designed to examine the effects of season along with needle age cohort or growth pCO2 or both would be unbalanced. Instead, seasonal effects on current-year needles were examined by paired t test. The effects of growth pCO2 in winter on current-year needles were examined by t test. Three trees from each of three rings for each growth pCO2 were examined. Mean values for each ring were determined and the ring was considered the experimental unit of observation, yielding n = 3 for each growth pCO2. All statistics were performed using Statview 5.0.1 (SAS Institute, Cary, NC).

Results

Photosynthetic CO2 assimilation

In the summer, Asat was 50% higher in current-year needles and 24% higher in year-old needles in elevated pCO2 compared with ambient pCO2 trees (Figure 1). In the summer, current-year needles had significantly higher Asat than year-old needles in both pCO2 treatments (P = 0.0005). In the winter, Asat was not significantly affected by growth pCO2 in current-year needles (Figure 1); year-old needles senesced in the fall, so they were not available for measurement. In the winter, Asat was significantly lower than in the summer for trees at both ambient and elevated pCO2 (P < 0.001; Figure 1).

Figure 1.

Light-saturated rates of photosynthetic CO2 assimilation (Asat) of current-year and year-old needles of loblolly pine (P. taeda) grown at ambient (open bars) or elevated (ambient + 20 Pa; shaded bars) pCO2 using FACE. Current-year needles were measured in July (2004) and January (2005); year-old needles were available for measurement in July only. Measurements were performed at ambient pCO2 (38 Pa; A) or growth pCO2 (i.e., 38 Pa for needles from trees grown at ambient pCO2 and 58 Pa for trees grown at elevated pCO2; B). Data are mean ± SE, n = 3.

Figure 1.

Light-saturated rates of photosynthetic CO2 assimilation (Asat) of current-year and year-old needles of loblolly pine (P. taeda) grown at ambient (open bars) or elevated (ambient + 20 Pa; shaded bars) pCO2 using FACE. Current-year needles were measured in July (2004) and January (2005); year-old needles were available for measurement in July only. Measurements were performed at ambient pCO2 (38 Pa; A) or growth pCO2 (i.e., 38 Pa for needles from trees grown at ambient pCO2 and 58 Pa for trees grown at elevated pCO2; B). Data are mean ± SE, n = 3.

When measured at a common pCO2 (38 Pa) in the summer, Asat was similar in current-year needles of trees grown at ambient pCO2 and elevated pCO2 (Figure 1). However, year-old needles exhibited significantly lower Asat in elevated pCO2 trees compared with ambient pCO2 trees when measured at a common pCO2 (38 Pa), indicating photosynthetic down-regulation in elevated pCO2. In the winter, Asat was similar in current-year needles of ambient pCO2 and elevated pCO2 trees when measured at ambient pCO2 (38 Pa; Figure 1).

Photosynthetic O2 evolution

We determined the photosynthetic capacity as the rate of O2 evolution from detached needles measured during exposure to saturating light intensities and pCO2 at 25 °C (Figure 2). In the summer, there was no significant effect of needle age or of growth pCO2 on the capacity for photosynthetic O2 evolution, nor was there a significant growth pCO2 * age cohort interaction. In the winter, there was no significant effect of growth pCO2 on the capacity for O2 evolution in current-year needles. In the winter, the capacity for photosynthetic O2 evolution was reduced 20% compared to summer (P = 0.017).

Figure 2.

Capacities for photosynthetic O2 evolution of current-year and year-old needles of loblolly pine (P. taeda) grown at ambient (open bars) or elevated (ambient + 20 Pa; shaded bars) pCO2 using FACE. Current-year needles were measured in July and January; year-old needles were available for measurement in July only. Data are mean ± SE, n = 3.

Figure 2.

Capacities for photosynthetic O2 evolution of current-year and year-old needles of loblolly pine (P. taeda) grown at ambient (open bars) or elevated (ambient + 20 Pa; shaded bars) pCO2 using FACE. Current-year needles were measured in July and January; year-old needles were available for measurement in July only. Data are mean ± SE, n = 3.

Chlorophyll fluorescence emission

In the summer, there were no significant effects of needle age or of growth pCO2 on ΦPSII at 1500 μmol photons m−2 s−1 ((Fm′ − Fs)/Fm′; Figure 3A) or on the efficiency of PSII in the fully-oxidized state (Fv′/Fm′; Figure 3B). There were no significant growth pCO2 * age cohort interactions for these parameters. Both parameters were significantly lower in the winter compared to summer in current-year needles (P < 0.001 for both). In the winter, the decrease in ΦPSII was greater than that observed in Fv′/Fm′ because of a significant increase in the estimated PSII reduction state ([Fs′ − Fo′]/[Fm′ − Fo′]) in the winter relative to summer (data not shown). There was no effect of growth pCO2 on either fluorescence parameter in the winter.

Figure 3.

The steady-state photosystem II efficiency (ΦPSII) at 1500 μmol photons m−2 s−1 ((Fm′ − Fs)/Fm′; A) and the intrinsic efficiency of PSII in the fully-oxidized state Fv′/Fm′ upon transient darkening after exposure to 1500 μmol photons m−2 s−1 (B) of current-year and year-old needles of loblolly pine (P. taeda) grown at ambient (open bars) or elevated (ambient + 20 Pa; shaded bars) pCO2 using FACE. Measurements were performed during exposure to growth pCO2 (i.e., 38 Pa for needles from trees grown at ambient pCO2 and 58 Pa for trees grown at elevated pCO2). Current-year needles were measured in July and January; year-old needles were available for measurement in July only. Data are mean ± SE, n = 3.

Figure 3.

The steady-state photosystem II efficiency (ΦPSII) at 1500 μmol photons m−2 s−1 ((Fm′ − Fs)/Fm′; A) and the intrinsic efficiency of PSII in the fully-oxidized state Fv′/Fm′ upon transient darkening after exposure to 1500 μmol photons m−2 s−1 (B) of current-year and year-old needles of loblolly pine (P. taeda) grown at ambient (open bars) or elevated (ambient + 20 Pa; shaded bars) pCO2 using FACE. Measurements were performed during exposure to growth pCO2 (i.e., 38 Pa for needles from trees grown at ambient pCO2 and 58 Pa for trees grown at elevated pCO2). Current-year needles were measured in July and January; year-old needles were available for measurement in July only. Data are mean ± SE, n = 3.

Needle pigment composition

The conversion state of the xanthophyll cycle to antheraxanthin and zeaxanthin, as a proportion of the overall xanthophyll cycle pool ([A + Z][V + A + Z]−1), was not significantly affected by growth pCO2, needle age or season (Figure 4A). No significant effects of growth pCO2, needle age or season were observed for the total chlorophyll content or the xanthophyll cycle pool size (Figure 4B and C).

Figure 4.

The conversion state of the xanthophyll cycle (A), xanthophyll cycle pool size (B) and total chlorophyll content (C) of current-year and year-old needles of loblolly pine (P. taeda) grown at ambient (open bars) or elevated (ambient + 20 Pa; shaded bars) pCO2 using FACE. Needles were collected during exposure to growth pCO2 (i.e., 38 Pa for needles from trees grown at ambient pCO2 and 58 Pa for trees grown at elevated pCO2) at 1500 μmol photons m−2 s−1. Current-year needles were collected in July and January; year-old needles were available for collection in July only. Data are mean ± SE, n = 3.

Figure 4.

The conversion state of the xanthophyll cycle (A), xanthophyll cycle pool size (B) and total chlorophyll content (C) of current-year and year-old needles of loblolly pine (P. taeda) grown at ambient (open bars) or elevated (ambient + 20 Pa; shaded bars) pCO2 using FACE. Needles were collected during exposure to growth pCO2 (i.e., 38 Pa for needles from trees grown at ambient pCO2 and 58 Pa for trees grown at elevated pCO2) at 1500 μmol photons m−2 s−1. Current-year needles were collected in July and January; year-old needles were available for collection in July only. Data are mean ± SE, n = 3.

Discussion

Seasonal differences in photosynthetic performance

In the winter, current-year needles of loblolly pine exhibited reductions in photosynthetic activity (e.g., lower Asat; lower capacity for photosynthetic O2 evolution and lower ΦPSII and PSII efficiency in the fully-oxidized state), which are commonly observed after acclimation to seasonally lower temperatures (Öquist and Huner 2003, Adams et al. 2004, Demmig-Adams and Adams 2006). Decreased photosynthetic activity in the winter can be attributed to the direct inhibitory effects of low temperatures on the enzymes involved in photosynthetic CO2 assimilation. In addition, photosynthetic activity may be reduced by feedback associated with the inhibitory effects of low temperature on the transport and utilization of photoassimilate (Koch 1996, Paul and Foyer 2001). The magnitude of these reductions in photosynthetic performance due to seasonally lower temperatures was relatively small in loblolly pine compared with other conifers experiencing more severe winter conditions (Verhoeven et al. 1999, Adams et al. 2002, Zarter et al. 2005), in part due to the relatively mild winters at the Duke FACE site.

Depressions in PSII efficiency in the fully-oxidized state (Fv′/Fm′) often correlate inversely with the level of energy dissipation, either diurnally regulated or seasonally sustained, in field-grown plants experiencing natural environmental conditions to which they are acclimated (Demmig-Adams et al. 1996, Logan et al. 2007). This suggests that in loblolly pine, the levels of photoprotective energy dissipation were higher in the winter, presumably in response to greater absorption of excess light when photosynthetic light use was lower than in the summer. However, lower Fv′/Fm′ values in the winter were not correlated with increased xanthophyll cycle pool size or conversion to the de-epoxidized carotenoids (e.g., zeaxanthin and antheraxanthin; Demmig-Adams and Adams 1996, Adams et al. 2004), although we observed a nonsignificant trend of slightly higher conversion states in the winter. Recent studies (reviewed in Demmig-Adams and Adams 2006) have revealed that different mechanisms can underlie seasonally-sustained energy dissipation and that these mechanisms may vary in their dependence upon de-epoxidized carotenoids of the xanthophyll cycle. Therefore, in the winter, loblolly pine may engage a form of energy dissipation which does not increase the xanthophyll cycle pool size or conversion state; alternatively, winter-time depressions in Fv′/Fm′ may have been due to photoinhibition.

Elevated pCO2 effects on photosynthetic performance

In the eighth year of elevated pCO2 treatment, the stimulation of Asat in the summer due to elevated pCO2 was greater in current-year needles than in year-old needles, as has been observed throughout the duration of the Duke FACE experiment (Hymus et al. 1999, Myers et al. 1999, Rogers and Ellsworth 2002, Crous and Ellsworth 2004, Springer et al. 2005, Crous et al. 2008). Reduced enhancement of Asat in year-old needles compared with current-year needles in the summer reflected photosynthetic down-regulation in year-old needles (but not current-year needles), which has previously been reported (Rogers and Ellsworth 2002, Crous and Ellsworth 2004, Crous et al. 2008). Most likely, photosynthetic down-regulation in older needles of loblolly pine was due to a combination of factors, including reductions in biochemical efficiency and reductions in local growth generating smaller sinks for carbohydrates (Tissue et al. 1999, 2001, Griffin et al. 2000, Rogers and Ellsworth 2002, Crous and Ellsworth 2004, Springer et al. 2005, Crous et al. 2008). In the winter, cooler temperatures eliminated the elevated pCO2 enhancement of Asat in current-year needles. In previous studies of loblolly pine, elevated pCO2 stimulated Asat year-round, although the magnitude of stimulation was lower in the winter than in the summer (Lewis et al. 1996, Tissue et al. 1997) and not significant during all measurement years (Hymus et al. 1999). Low temperatures inhibit photorespiration, thereby potentially reducing the relative enhancement of Asat at elevated pCO2.

The capacity for O2 evolution, a direct measure of photosynthetic electron transport capacity, was unaffected by growth pCO2 and needle age; hence, we found no evidence that linear photosynthetic electron transport was altered to compensate for the positive effects of elevated pCO2 on Asat in the summer. Our observations are consistent with those of Epron et al. (1996) who reported that growth under elevated CO2 did not affect the capacity for net photosynthetic O2 evolution in Fagus sylvatica L. Our analyses of in vivo chlorophyll fluorescence emission were consistent with our measurements of capacity for O2 evolution since neither elevated pCO2 nor needle age affected ΦPSII. Likewise, neither levels of energy dissipation, estimated as depressions in Fv′/Fm′ (Demmig-Adams et al. 1996), nor the pigment composition of needles indicated that light absorption or light allocation to photochemistry versus energy dissipation was affected by growth pCO2 or needle age.

Hymus et al. (1999) measured chlorophyll fluorescence emission from upper-canopy loblolly pine needles in the first 2 years of CO2 treatment at the Duke FACE experiment. Although Fv′/Fm′ was similar in our study when compared to Hymus et al. (1999), they reported significantly higher ΦPSII at elevated pCO2 in the summer and significantly lower ΦPSII at elevated pCO2 in the winter, whereas we report that elevated pCO2 had no effect on ΦPSII in either season. Myers et al. (1999) reported that elevated pCO2 did not affect photochemical quenching of chlorophyll fluorescence (qP; a parameter that generally correlates positively with ΦPSII) during most of the first growing season of CO2 treatment (i.e., part of the period during which Hymus et al. 1999 conducted their study). The only significant effect on qP reported by Myers et al. (1999) was a depression in trees grown in elevated pCO2 at the end of the first growing season (late September). Differences between the two studies during the first year of CO2 treatment suggest that the impact of elevated pCO2 on chlorophyll fluorescence may be complex, seasonally transient and not easily quantified.

Most commonly, the effects of growth at elevated pCO2 on photosynthetic electron transport are assessed by determinations of Jmax. Given that Jmax is modeled to estimate the maximum rate of RuBP regeneration, which may be limited by ATP synthesis via photosynthetic electron transport, reductions in Jmax may be expected to correlate with reductions in the capacity for photosynthetic O2 evolution, which is a direct measure of O2 production by the O2-evolving complex associated with PSII under CO2- and light-saturated conditions. However, although significant reductions in Jmax have been reported in year-old needles of loblolly pine grown at elevated pCO2 and measured in the summer at the Duke FACE site (Rogers and Ellsworth 2002, Crous and Ellsworth 2004, Crous et al. 2008), we observed no significant effects of needle age or growth pCO2 on capacities for photosynthetic O2 evolution. Taken together, these findings suggest that down-regulation of Jmax at elevated pCO2 was not correlated with changes in the capacity for photosynthetic O2 evolution in year-old needles at the Duke FACE site; however, it should be noted that contemporaneous estimates of Jmax are not available, and therefore direct comparison of the two parameters was not possible.

We offer a potential explanation for the stimulation of Asat in elevated pCO2 without concomitant alterations in photosynthetic electron transport. In elevated pCO2, the competitive inhibition of the oxygenation reaction of Rubisco, which subsequently reduces photorespiration, leads to an increase in Asat (Sharkey 1988, Long 1991). The processes of O2 reduction (i.e., photorespiration) and CO2 reduction (i.e., photosynthesis) share similar requirements for reductant and ATP (Cornic and Fresneau 2002). Thus, although the demand for the products generated by photosynthetic electron transport (i.e., reductant and ATP) to support photosynthesis was larger in elevated pCO2, there may have been a compensatory decrease in the demand to support photorespiration. Subsequently, this reduced or eliminated the difference in the overall demand for the products of photosynthetic electron transport at elevated pCO2 relative to ambient pCO2. In addition, non-assimilatory sinks for reductant, including the water–water cycle (Asada 1999), may represent up to 30% of the total photosynthetic electron flux (Badger 1985, Osmond and Grace 1995, Badger et al. 2000, Logan 2006) and the effect of elevated pCO2 on the magnitude of non-assimilatory sinks in field-grown trees remains unresolved. Perhaps they represent a factor that balances the demand for photo-generated reductant across growth pCO2.

This study was supported by the Office of Science (BER), US Department of Energy, Grant No. DE-FG02-95ER62083, a grant to B.A.L. from the Bowdoin College Faculty Research Fund, US Department of Agriculture Award Nos. MER-2002-04818 and 2005-35101-15338, and National Science Foundation Award No. DUE 0088517. A.C., K.M., R.K. and L.S. were supported by Howard Hughes Medical Institution undergraduate fellowships. The authors thank Robert Nettles for logistical support at the Duke FACE site, Jaret Reblin for assistance with determinations of needle pigment composition and statistical analyses and Dr. Kristine Crous for helpful comments on the manuscript.

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