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. 2001 Nov;127(3):1204–1211.

Growth in Elevated CO2 Can Both Increase and Decrease Photochemistry and Photoinhibition of Photosynthesis in a Predictable Manner. Dactylis glomerata Grown in Two Levels of Nitrogen Nutrition1

Graham J Hymus 1,2, Neil R Baker 1, Stephen P Long 1,3,*
PMCID: PMC129288  PMID: 11706199

Abstract

Biochemically based models of C3 photosynthesis can be used to predict that when photosynthesis is limited by the amount of Rubisco, increasing atmospheric CO2 partial pressure (pCO2) will increase light-saturated linear electron flow through photosystem II (Jt). This is because the stimulation of electron flow to the photosynthetic carbon reduction cycle (Jc) will be greater than the competitive suppression of electron flow to the photorespiratory carbon oxidation cycle (Jo). Where elevated pCO2 increases Jt, then the ratio of absorbed energy dissipated photochemically to that dissipated non-photochemically will rise. These predictions were tested on Dactylis glomerata grown in fully controlled environments, at either ambient (35 Pa) or elevated (65 Pa) pCO2, and at two levels of nitrogen nutrition. As was predicted, for D. glomerata grown in high nitrogen, Jt was significantly higher in plants grown and measured at elevated pCO2 than for plants grown and measured at ambient pCO2. This was due to a significant increase in Jc exceeding any suppression of Jo. This increase in photochemistry at elevated pCO2 protected against photoinhibition at high light. For plants grown at low nitrogen, Jt was significantly lower in plants grown and measured at elevated pCO2 than for plants grown and measured at ambient pCO2. Elevated pCO2 again suppressed Jo; however growth in elevated pCO2 resulted in an acclimatory decrease in leaf Rubisco content that removed any stimulation of Jc. Consistent with decreased photochemistry, for leaves grown at low nitrogen, the recovery from a 3-h photoinhibitory treatment was slower at elevated pCO2.

The majority of experimental evidence points to a stimulation of light-saturated photosynthesis (Asat) for C3 plants, grown in the atmospheric partial pressure of CO2 (pCO2) predicted for the end of this century (for review, see Drake et al., 1997). In the field, increased photosynthesis in elevated pCO2 has been shown to both increase and decrease photochemical requirements for light-saturated electron flow through photosystem (PS) II (Jt; Scarascia-Mugnozza et al., 1996; Hymus et al., 1999). What basis might there be for a variable response in electron transport when assimilation is consistently increased?

Elevated pCO2 will stimulate the photosynthetic carbon reduction cycle and the electron flow that drives it (Jc). However, elevated pCO2 will also competitively suppress the photorespiratory carbon oxidation (PCO) cycle and the electron flow that drives it (Jo). Whether or not there is an increase in the demand of carbon metabolism for Jt will depend on the net effect of these changes in Jc and Jo. The mechanistic understanding of C3 photosynthesis proposed by Farquhar et al. (1980) predicts that, when pCO2 is increased, if Asat is limited by the amount of Rubisco the stimulation of Jc will be greater than the suppression of Jo, and an increase in Jt will result. This predicted increase in Jt may not be observed where growth in elevated pCO2 results in acclimation of either: (a) the amount of Rubisco in the leaf, or (b) sinks for Jt other than the photosynthetic carbon reduction and PCO cycles. Decreased leaf Rubisco content in elevated pCO2 will decrease both Jc and Jo. In addition, Jo will be competitively suppressed by increasing pCO2. Many studies have shown no effect of growth in elevated pCO2 on sinks for Jt, other than carbon metabolism (Epron et al., 1994; Habash et al., 1995; Bartak et al., 1996; Hymus et al., 1999). However, there is some evidence that growth in elevated pCO2 decreases antioxidant activity (Polle et al., 1997), suggesting a possible change in potential electron flux to a Mehler reaction.

Where elevated pCO2 changes photochemical quenching of absorbed photosynthetically active photon flux density (PPFD), changes in non-photochemical quenching of absorbed PPFD will result. Non-photochemical quenching constitutes a dynamic form of photoinhibition (Baker and Ort, 1992; Long et al., 1994; Osmond, 1994). At high light, if elevated pCO2 increases photochemistry, a decrease in non-photochemical quenching and protection against photoinhibiton would be expected.

In this study, Dactylis glomerata was grown under controlled environment conditions, at two levels of nitrogen nutrition. A previous study of D. glomerata showed decreased leaf Rubisco content with growth in elevated pCO2, but only when nitrogen supply was limiting (Davey, 1998). Given this potential to change leaf Rubisco content of plants growing in elevated pCO2 in controlled environments, the following two hypotheses were tested: (a) In the absence of acclimation, elevated pCO2 will result in a net increase in Jt because the stimulation of Jc will be greater than the inhibition of Jo, decreasing photoinhibition; and (b) an acclimatory decrease in leaf Rubisco content in elevated pCO2 will offset the stimulation of Jc, Jo will be suppressed, Jt will decrease, and photoinhibition will increase in elevated pCO2.

RESULTS

Light-Saturated Photosynthesis

Growth in elevated pCO2 did not affect Vc,max or Jmax in the high-nitrogen treatment (Fig. 1a; Table I), where Vc,max is the maximal Rubisco catalyzed carboxylation rate and Jmax the maximal whole-chain electron transport rate. In the low-nitrogen treatment, Vc,max was significantly decreased, by 42%, in elevated pCO2, the reduction in Jmax was not significant (Fig. 2a; Table I). In high nitrogen, Asat was Rubisco limited under ambient pCO2. As a consequence, Asat, Jc, and Jt were significantly increased, by 86%, 73%, and 55%, respectively, for plants grown and measured at elevated pCO2, relative to those grown and measured at ambient pCO2 (Fig. 1a; Table I). In the low-nitrogen treatment, acclimation to elevated pCO2 resulted in no stimulation of Asat and a significant decrease in both Jc and Jt of 11% and 20%, respectively, when the comparisons at respective growth pCO2 were made (Fig. 2a; Table I). In high nitrogen, the suppression of photorespiration by elevated pCO2 reduced Jo by an apparent 16%. In low nitrogen, Jo was decreased by 57% due to suppression of photorespiration by elevated pCO2 and decreased Rubisco (Table I). Neither decrease in Jo was statistically significant.

Figure 1.

Figure 1

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Light-saturated photosynthesis: high nitrogen. a, The responses of light-saturated CO2 uptake (Asat) against intercellular CO2 concentration (Ci) for leaves of D. glomerata grown in high nitrogen and at either elevated (black symbols and black lines) or current ambient (white symbols and dotted lines) pCO2. Values of Vc,max and Jmax, calculated using the equations and constants in von Caemmerer (2000) and Bernacchi et al. (2001), were used to fit a nonlinear regression to observed values above (Jmax) and below (Vc, max) the inflection of the curves. Also shown are the supply functions for each curve (dashed line) that indicate the operating point of photosynthesis at the growth pCO2 for each treatment. Data points shown are the means (±1 se) for five replicate leaves. Measurements were made in 21 kPa O2 and at a PPFD of 1,300 μmol m−2 s−1. b, Jt, Jc, and Jo for ambient (white bar) and elevated (black bar) pCO2 treatments were calculated for measurements at the respective growth pCO2 for each group of leaves using the equations of Valentini et al. (1995). Values shown are the means (±1 se) for five replicate leaves.

Table I.

Light-saturated photosynthetic characteristics

Measure Low Nitrogen
High Nitrogen
pCO2 × N
35 Pa 65 Pa 35 Pa 65 Pa
Asat 9.6 (0.6)a 9.4 (0.4)a 9.8 (0.6)a 18.3 (0.9)b F1,16  = 45
φPSII 0.14 (0.01)ab 0.11 (0.01)a 0.16 (0.01)b 0.27 (0.01)c F1,16  = 41
qP 0.44 (0.02)ab 0.39 (0.02)a 0.42 (0.02)a 0.52 (0.03)b F1,16  = 11
F′v/F′m 0.32 (0.01)b 0.28 (0.01)a 0.39 (0.01)c 0.51 (0.02)d F1,16  = 29
Vc,max 44.3 (4.4)b 25.8 (3.7)a 62.7 (2.4)c 62.4 (4.0)c F1,16  = 6
Jmax 81.4 (6.3)ab 62.7 (5.5)a 101.0 (5.3)bc 118.8 (6.3)c F1,16  = 8
Jt 59.7 (4)b 46.5 (2)a 62.0 (3)b 95.2 (5)c F1,16  = 38
Jc 47.8 (2)b 42.7 (1)a 49.1 (2)b 84.8 (4)c F1,16  = 30
Jo 11.3 (3) 4.9 (2) 12.3 (1) 10.4 (2) F1,16  = 2
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Asat, the quantum efficiency of PSII (φPSII), photochemical quenching coefficient (qP), and F′v/F′m measured simultaneously at the growth pCO2 for ambient (35 Pa) and elevated (65 Pa) treatments. Vc,max and Jmax were calculated using the equations and constants in von Caemmerer (2000) and Bernacchi et al. (2001). Jt, Jc, and Jo were estimated using the model of Valentini et al. (1995). All values are the means (±1 se) for five replicate plants. A significant interaction between growth pCO2 and nitrogen was found for each parameter measured, except Jo. As a consequence, differences between individual means were tested using a post hoc Tukey's test. Different superscript letters identify means that are significantly different within that row (P < 0.05). Asat, Vc,max, Jmax, Jt, Jc, and Jo are expressed in μmol m−2 s−1. Units for φPSII, qP, and F′v/F′m are dimensionless.

Figure 2.

Figure 2

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Light-saturated photosynthesis: low nitrogen. a, Plot of light-saturated A against Ci for leaves of D. glomerata grown in low nitrogen. As described previously for Figure 1. b, Measurements of Jt, Jc, and Jo made at the respective growth pCO2 for D. glomerata grown in low nitrogen. As described previously for Figure 1.

In the high-nitrogen treatment, elevated pCO2 significantly increased φPSII when measurements made at the two growth pCO2 were compared, as a result of increases in both Fv'/Fm' and photochemical quenching coefficient (qP; Fig. 1b; Table I), where Fv′/Fm′ is the probability of an absorbed photon reaching an open PSII reaction center. In low nitrogen, Fv'/Fm' was significantly lower in elevated pCO2; the decreases in φPSII and qP were not statistically significant (Fig. 2b; Table I).

Ratio of Electron Transport to CO2 Fixation

For all nitrogen and pCO2 treatments, φPSII and φCO2 were highly correlated (r2 = 0.74–0.82; P < 0.05) and linearly related with an intercept that was not significantly different from zero (Table II), where φCO2 and φPSII are the quantum efficiencies of CO2 uptake and of linear electron transport through PSII, respectively. It is important that growth pCO2 had no statistically significant effect on the φPSII/φCO2 relationship for plants grown in either high or low nitrogen (Table II). These relationships indicated that growth in elevated pCO2 had not resulted in additional sinks for Jt, such as to a Mehler reaction.

Table II.

Ratio of electron transport to CO2 fixation

Regression Coefficients Low Nitrogen
High Nitrogen
35 Pa 65 Pa 35 Pa 65 Pa
k 13.9 14.1 17.0 14.9
b 0.035 −0.030 −0.040 −0.018
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The gradient (k) of the relationship φPSII/φCO2 was unaffected by elevated pCO2 in low nitrogen (F1.64 = 2.1; P > 0.05) and high nitrogen (F1.62 = 2.79; P > 0.05). The intercept (b) was not significantly different from zero for each plot. Low nitrogen ambient, t1.29 = 0.02, P = 0.98; low nitrogen elevated, t1.31 = 0.95, P = 0.34; high nitrogen ambient, t1.34 = 1.14, P = 0.26; high nitrogen elevated, t1.28 = 0.34, P = 0.73.

Photoinhibition and Recovery

Fv/Fm measured prior to the beginning of the photoperiod was unaffected by growth pCO2 for both nitrogen treatments (F1,16 = 0.1; P > 0.1). For all treatments, Fv/Fm declined during the 3-h high-light treatment at a PPFD of 2,000 μmol m−2 s−1 (Figs. 3a and 4a). After 3 h, Fv/Fm was significantly higher by 7% in elevated pCO2 for the high-nitrogen plants (t18 = 2.3; P < 0.05; Fig. 4a). In low nitrogen, Fv/Fm measured after 3 h was unaffected by pCO2 (Fig. 4a).

Figure 3.

Figure 3

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Photoinhibition and recovery: high nitrogen. a, Photo-inhibitory reduction in Fv/Fm. b, Changes in Fo during a 3-h exposure to a PPFD of 2,000 μmol m−2 s−1. c, Recovery of Fv/Fm measured after 10 min dark adaption (black lines), and Fv'/Fm' measured under growth PPFD (dashed lines), for D. glomerata grown in high nitrogen. Plants were grown, photoinhibited, then allowed to recover in their growth pCO2 either ambient (white symbols) or elevated (black symbols). Each symbol represents the mean (±1 se) for 10 replicate plants.

Figure 4.

Figure 4

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Photoinhibition and recovery: low nitrogen. a, Photo-inhibitory reduction in Fv/Fm. b, Changes in Fo during a 3-h exposure to a PPFD of 2,000 μmol m−2 s−1. c, Recovery of Fv/Fm measured after 10 min of dark adaption (black line), and Fv'/Fm' measured under growth PPFD (dashed line), for D. glomerata grown in low nitrogen. As described previously for Figure 3.

In high nitrogen with elevated pCO2, 60% of the reduction in Fv/Fm had recovered after 10 min of dark adaptation and after 1 h, Fv/Fm had returned to dark-adapted levels. Although the ambient pCO2 treatment similarly recovered after 1 h, Fv/Fm and Fv'/Fm' after 10 min were lower than in elevated pCO2. This difference in Fv/Fm was statistically significant (t18 = 3.5; P < 0.05; Fig. 3c).

For plants grown in low nitrogen, the recovery of Fv/Fm took between 3 and 4 h (Fig. 4c). Values of Fo measured before and at the end of the 3-h treatment were not significantly different in either the ambient (t18 = 1.5; P > 0.1) or elevated (t18 = 0.01; P > 0.1) pCO2 treatments (Fig. 4b). Although there was no effect of pCO2 on the recovery of Fv'/Fm' in the low-nitrogen treatment, the initial recovery of Fv/Fm was significantly reduced by elevated pCO2 during the first (t18 = 3.9; P < 0.05) and second (t18 = 3.0; P < 0.05) hours of the recovery. Given that Fv'/Fm' was unaffected by pCO2, the slower recovery of Fv/Fm should reflect an affect of pCO2 on the dark-adapted relaxation of non-photochemical quenching.

DISCUSSION

This controlled-environment study confirmed the hypothesis that elevated pCO2 decreases photoinhibition in high nitrogen, but increases photoinhibition in low nitrogen, at a level assumed to restrict growth. This is explained by a greater demand for electrons in photosynthetic carbon metabolism in the absence of limitations, and a decreased demand by both photosynthetic and photorespiratory carbon metabolism, relative to ambient pCO2, when resources other than carbon restrict production.

For D. glomerata, light-saturated photosynthesis at ambient pCO2 was limited by the amount of Rubisco, regardless of nitrogen treatment (Fig. 1a). At high nitrogen, elevated pCO2 had no effect on Vc,max. Therefore, in keeping with the theory of Farquhar et al. (1980), elevated pCO2 stimulated Jc to a greater extent than it suppressed Jo, increasing Jt (Fig. 1b; Table I). As a consequence, when exposed to a high PPFD, the proportion of absorbed photons dissipated photochemically was increased in the elevated pCO2 treatment, reducing photoinhibition (Fig. 3).

For D. glomerata grown under low nitrogen supply, acclimation of the photosynthetic apparatus significantly reduced Vc,max (Fig. 2). The magnitude of the acclimation was sufficient to totally remove the short-term stimulation of Jc by elevated pCO2 (Fig. 2). Because photorespiration was suppressed, a similar Asat at elevated pCO2 to that in ambient pCO2, was achieved with about 20% lower Jt (Table I). If triose-phosphate utilization (TPU) had been limiting photosynthesis, we would have expected a similar decrease in Jt. Under conditions of TPU limitation, Asat and Jc will be insensitive to increasing pCO2 and Jo will be suppressed, and decreased Jt and increased non-photochemical quenching can result (Sharkey et al., 1988; Pammenter et al., 1993). In this study, Asat of leaves grown in low nitrogen and elevated pCO2 increased with an increase in Ci to 150 Pa. As a consequence, decreased Vc,max, not TPU limitation of photosynthesis, was responsible for decreased Jt. The nitrogen dependence of the pCO2-dependent decrease in carboxylation capacity observed here is consistent with other studies of the interactive effects of growth at elevated pCO2 and nitrogen supply (Tissue et al., 1993; Thomas et al., 1994; Curtis, 1996; Rogers et al., 1996). Rogers et al. (1998) showed, with a related herbage grass, Lolium perenne, that when nitrogen supply was limited there was a loss of carboxylation capacity and Rubisco at elevated pCO2, but not when nitrogen supply was adequate. Partial defoliation removed this acclimatory response. This showed that low nitrogen affected acclimation by limiting sink size relative to source because when source size was decreased acclimation was removed. For D. glomerata, in low nitrogen, the deceased demand for photochemical energy was reflected in a significant depression in Fv'/Fm' in elevated pCO2 and therefore increased the probability of absorbed photons being dissipated as radiation-less decay from the antenna of PS II (Table I, Fig. 2). The gradient of the straight line describing the dependence of φPSII on φCO2 was not significantly different in elevated pCO2 for either nitrogen treatment. This suggested that growth in elevated pCO2 did not produce significant sinks for photochemical energy other than the photosynthetic carbon reduction or PCO cycles, in agreement with published findings (Epron et al., 1994; Habash et al., 1995; Bartak et al., 1996; Hymus et al., 1999). However, there was no evidence of any significant sink beyond photosynthetic and photorespiratory carbon metabolism at the current ambient pCO2 in D. glomerata. In other species, notably those that bare leaves throughout long periods of environmental stress restricting carbon metabolism, alternative sinks for electron flow, primarily to oxygen, are suggested to be very significant (Lovelock and Winter, 1996; Cheeseman et al., 1997). Responses of species with these stress tolerance strategies might be very different.

After the 3-h photoinhibitory treatment, the recovery of Fv/Fm in low nitrogen was slower for the elevated pCO2 treatment. Because Fo was unaffected (Fig. 3), the decreased Fv/Fm was almost certainly associated with zeaxanthin-dependent quenching (Demmig-Adams and Adams, 1992; Owens, 1994; Horton et al., 1996). Under stress conditions, this recovery can require many hours (Demmig-Adams and Adams, 1992; Fryer et al., 1995). The results suggest that in addition to increasing the potential for photoinhibition, elevated pCO2 may also decrease capacity for recovery. Hymus et al. (1999) showed that loblolly pine (Pinus taeda) photoinhibited during winter low temperature stress recovered more slowly when growing at elevated pCO2. Together, these results indicate that elevated pCO2, may decrease the capacity of the plant to recover from stress-induced photoinhibition. This may be part of a wider pattern of decreased stress tolerance in leaves growing at elevated pCO2 (Lutze et al., 1998; Terry et al., 2001).

In a study on wheat (Triticum aestivum) grown for 6 weeks under optimal conditions, without an acclimatory loss of Rubisco, both A and total non-cyclic electron flow through PSII were enhanced by elevated pCO2 at high light (Habash et al., 1995). A similar study on ryegrass (Lolium perenne) showed that A and φPSII were increased by instantaneous elevation of pCO2 for plants grown at current pCO2, but longer term acclimation completely reduced the stimulation of both A and φPSII (Bartak et al., 1996). For natural vegetation growing in the field, studies show seasonal decreases in photochemistry, and increased photoinhibition in elevated pCO2 (Scarascia-Mugnozza et al., 1996; Hymus et al., 1999).

Here, we have extended these findings by providing quantitative evidence that elevated pCO2 can either increase or decrease photochemistry and photoinhibition, depending on whether down-regulation of photosynthetic capacity has occurred. Plants were grown at 400 μmol m−2 s−1, but exposed to higher light for the photoinhibitory treatment. This is not unrealistic of many areas of the globe where a series of cloudy days may be followed by clear sky days with higher photon flux or where grazing exposes lower canopy leaves to high light. This finding has important ecological implications. Although under optimal conditions, elevated pCO2 increases photochemical energy use and decreases the probability of photoinhibition, the reverse is true of limiting nitrogen conditions. Most of the natural terrestrial biosphere and much subsistence agriculture is nitrogen limited. It has been widely appreciated that the response of photosynthesis to rising pCO2 may be diminished by acclimation in these conditions. Here, we show that not only is capacity for carbon assimilation decreased, but the probability of photoinhibition, due to increased non-photochemical quenching, is increased. Such non-photochemical quenching would serve to protect the reaction centers from photo-inactivation and damage when the rate of excitation of PSII is in excess of the rate of photochemistry. The cost of this protection is that when a leaf is in low light after photoinhibition the efficiency of photosynthesis remains low for minutes to hours, resulting in a significant loss of potential carbon fixation (Long et al., 1994). Elevated pCO2 will increase this loss, both by increasing the potential for photoinhibition and by slowing the rate of recovery.

MATERIALS AND METHODS

Growth Conditions

Dactylis glomerata (IGER, Aberystwyth, UK) was grown from seed for 56 to 57 d in a washed silver sand media (William Sinclair Horticulture, Lincoln, UK), in 0.62-L pots. Four seeds were sown in each pot. These were then divided between two controlled environments (PG660, Sanyo, Loughborough, UK); one was maintained at 35 Pa pCO2 (ambient), the other at 65 Pa pCO2 (elevated). An infrared gas analyzer integrated with a feedback control system (WMA-2, PP Systems, Hitchin, UK), which controlled the injection of scrubbed, pure CO2 gas (Linde Gas Ltd, Stoke on Trent, UK), maintained the pCO2 within the controlled environment cabinets. Plants were grown in a day/night temperature regime of 16°C/12°C and a relative humidity of 80%, giving a daytime water vapor pressure deficit of 0.5 kPa. The photoperiod was 14 h long at a PPFD of 400 μmol m−2 s−1 at pot height, providing a total photon flux of 20 mol m−2 d−1 over the photoperiod and similar to that which D. glomerata would receive in the field during summer in Western Europe.

From planting to 1 week after emergence, the sand media was fully saturated with deionized water. At this point, the plants were divided into two nutrient regimes in each cabinet. They were supplied with either high (12 mm) or low (4 mm) nitrogen by a Long Ashton (nitrate type) solution (Hewitt, 1966). Throughout the growth period, the sand media was flushed twice weekly with deionized water to prevent accumulation of salts.

To minimize undetected inter-cabinet environmental differences, pCO2 treatments and their plants were swapped between cabinets each week. To minimize the effects of intra-cabinet environmental gradients, the plants were randomly repositioned within the cabinets each week. Between 56 and 57 d into growth, five plants of the eight grown for each treatment were randomly selected for simultaneous measurements of leaf gas exchange and chlorophyll a fluorescence. Measurements were made on the youngest leaf with an emerged ligule on the main stem.

Leaf Gas Exchange

Leaf net CO2 uptake (A) and water vapor efflux were measured in an open gas exchange system. A combined CO2 and water vapor analyzer (LI-6262, LI-COR, Lincoln, NE), calibrated against a water vapor generator (WD600, ADC Ltd., Hoddesdon UK) and a standard CO2 concentration of 50 Pa (Linde Gas Ltd) was used. Inlet CO2 concentration was controlled by a gas dilutor (GD-600, ADC Ltd.), and inlet humidity was controlled by passing the dry airflow through a temperature-controlled ferrous-sulfate crystal column (WG-600, ADC Ltd.). The temperature-controlled leaf section chamber used (LSC, ADC Ltd.) allowed for rapid mixing of gases, and a small response time to changes in pCO2 and PPFD. Chamber cooling was by circulating coolant through each half of the chamber. All measurements were made at a leaf temperature of 16.0°C (±0.3°C) and a water vapor pressure deficit of 1.2 (±0.04) kPa.

The light-saturated response of A to Ci was made at a PPFD of 1,300 μmol m−2 s−1. Photosynthetic induction was performed at the growth pCO2. Calculations of A and Ci followed von Caemmerer and Farquhar (1981). Vc,max and Jmax were estimated for each individual leaf by fitting maximum likelihood regressions to the initial slope and plateau of the A/Ci response curves, respectively, using the calculations of von Caemmerer (2000) and Bernacchi et al. (2001).

Leaf Chlorophyll a Fluorescence

A modulated chlorophyll fluorimeter and leaf clip (PAM 2000, H Walz, Effeltrich, Germany) were used to measure minimum (Fo'), maximum (Fm'), and steady-state (Fs) levels of fluorescence simultaneously with the gas exchange measurements. These values were used to calculate the efficiency of excitation energy capture by open PSII reaction centers (Fv'/Fm'), the qP, and φPSII (Genty et al., 1989).

The response of A and φPSII to PPFD was determined over a range of light levels from 0 to 1,420 μmol m−2 s−1 under non-photorespiratory conditions (1 kPa O2; Linde Gas Ltd). Leaf absorptance (α) was measured with a Taylor integrating sphere attached to a quantum sensor (SKP 215, Skye Instruments Ltd, Llandrindod Wells, UK) following the method of Rackham and Wilson (1968). From these measurements, φCO2 was then calculated as:

graphic file with name M1.gif

where Q is PPFD. When measured in 1% (v/v) O2, the relationship of φPSII to φCO2 is linear (Genty et al., 1989). The model and assumptions of Valentini et al. (1995) use the φPSII/φCO2 relationship in 1% (v/v) O2, to calculate light-saturated linear electron flow through PSII (Jt) and partition it between electron flow to the photosynthetic carbon reduction (Jc) and PCO (Jo) cycles. In this model, the linear relationship between φPSII and φCO2 is assumed to describe the apparent quantum efficiency of photosynthetic linear electron flow (φe−) using the equation:

graphic file with name M2.gif

where 4 is the number of electrons needed per CO2 molecule fixed, k is the gradient, and b is the y intercept. The model assumes that this relationship holds in both photorespiratory and non-photorespiratory conditions, enabling Jt to be calculated as:

graphic file with name M3.gif

By assuming only the photosynthetic carbon reduction and PCO cycles are sinks for linear electron flow, the model calculates the partitioning of Jt between Jc and Jo as:

graphic file with name M4.gif
graphic file with name M5.gif
graphic file with name M6.gif

where 4 is the number of electrons required to fix one molecule of CO2 and Rl is the rate of CO2 production by photorespiration. Jc and Jo are then solved as:

graphic file with name M7.gif
graphic file with name M8.gif

Photoinhibitory Treatment

Between 54 and 58 d into growth, leaves of D. glomerata grown in the nitrogen and pCO2 treatments described previously were selected for a photoinhibitory, high-light treatment. The criteria used for leaf selection were as for the previous measurements. Leaves were exposed to a PPFD of 2,000 μmol m−2 s−1 for 3 h, within a custom-built controlled environment in which the pCO2 was maintained at growth levels, air temperature was maintained at 16°C to 18°C, and relative humidity was maintained at 75% to 82%, approximating growth conditions. Eight plants, two from each treatment, were randomly selected and photoinhibited on each of 5 consecutive d. In total, 10 plants from each treatment were photoinhibited.

Measurements of Fo and Fm were made in the dark prior to the beginning of the photoperiod to determine the maximum quantum yield of PSII (Fv/Fm), then every 45 min during the photoinhibitory treatment, using a modulated chlorophyll fluorometer (PAM 2000). The photoinhibitory treatment was begun 1 h into the photoperiod. After 3 h, the plants were returned to their respective growth environments to recover. The recovery of Fv/Fm measured after 10 min of dark adaptation, and Fv'/Fm' measured under growth light levels, was followed. Measurements were made immediately following the photoinhibitory treatment and then at hourly intervals until the recovery was complete, using the equipment and protocols for measurement described previously.

Statistical Analysis

The effects of growth pCO2 and nitrogen supply on Asat, Vc,max, Jmax, φPSII, qP, Fv'/Fm', Jt, Jc, Jo, and α were tested using ANOVA. Where a significant interaction between pCO2 and nitrogen was found, post hoc pair-wise comparisons using Tukey's test were performed to identify differences between individual means. The effect of pCO2 treatment on the recovery of Fv/Fm was tested using a two-tailed Student's t test. All ANOVA and Student's t tests were performed using statistical software (Systat 7.0, Systat Inc, Evanston, IL). The effect of pCO2 treatment on the relationship between φPSII and φCO2 was examined by regression analysis of variance (Zar, 1999). Fluorescence yields were arcsine transformed to generate a normal distribution for statistical analysis (Zar, 1999). An effect was described as significant where P < 0.05.

ACKNOWLEDGMENTS

We thank Mr. Paul Beckwith and Mrs. Sue Corbett for their skilled technical support.

Footnotes

1

This work was supported by the Natural Environment Research Council of the United Kingdom (research studentship to G.J.H.).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.010248.

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