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. Hymus2, Neil R. Baker, and Stephen P. Long3*
Department of Biological Sciences, John Tabor Laboratories, University of Essex, Wivenhoe Park, Colchester
CO4 3SQ, United Kingdom
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; ScarasciaMugnozza 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 oxi1
This work was supported by the Natural Environment Research Council of the United Kingdom (research studentship to
G.J.H.).
2
Present address: Smithsonian CO2 Site, Mail Code DYN–2,
Kennedy Space Center, FL 32899.
3
Present address: Departments of Crop Science and Plant Biology, University of Illinois, Edward R. Madigan Laboratory 190,
2206 West Gregory Drive, Urbana, IL 61801.
* Corresponding author; e-mail [email protected]; fax 217–
244 –7563.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.010248.
1204
dation (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 pCO 2 changes photochemical
quenching of absorbed photosynthetically active
photon flux density (PPFD), changes in nonphotochemical quenching of absorbed PPFD will
result. Non-photochemical quenching constitutes a
Plant Physiology, November
2001, Vol.
pp. 1204–1211,
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Elevated CO2, Acclimation, and Electron Partitioning
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.
In the high-nitrogen treatment, elevated pCO2 significantly increased ␾PSII 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 ␾PSII and qP were not statistically significant (Fig. 2b; Table I).
Plant Physiol. Vol. 127, 2001
Figure 1. 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 ␮mol
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.
Ratio of Electron Transport to CO2 Fixation
For all nitrogen and pCO2 treatments, ␾PSII and
␾CO2 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 ␾CO2 and ␾PSII 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 ␾PSII/
␾CO2 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.
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1205
Hymus et al.
Table I. Light-saturated photosynthetic characteristics
Asat , the quantum efficiency of PSII (␾PSII), 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 ␮mol m⫺2 s⫺1. Units for ␾PSII, qP, and F⬘v/F⬘m are dimensionless.
Low Nitrogen
Measure
Asat
␾PSII
qP
F⬘v/ F⬘m
Vc,max
Jmax
Jt
Jc
Jo
High Nitrogen
35 Pa
65 Pa
35 Pa
65 Pa
9.6 (0.6)a
0.14 (0.01)a,b
0.44 (0.02)a,b
0.32 (0.01)b
44.3 (4.4)b
81.4 (6.3)a,b
59.7 (4)b
47.8 (2)b
11.3 (3)
9.4 (0.4)a
0.11 (0.01)a
0.39 (0.02)a
0.28 (0.01)a
25.8 (3.7)a
62.7 (5.5)a
46.5 (2)a
42.7 (1)a
4.9 (2)
9.8 (0.6)a
0.16 (0.01)b
0.42 (0.02)a
0.39 (0.01)c
62.7 (2.4)c
101.0 (5.3)b,c
62.0 (3)b
49.1 (2)b
12.3 (1)
18.3 (0.9)b
0.27 (0.01)c
0.52 (0.03)b
0.51 (0.02)d
62.4 (4.0)c
118.8 (6.3)c
95.2 (5)c
84.8 (4)c
10.4 (2)
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 ␮mol 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).
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 darkadapted 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 photoinhi1206
p CO2 ⫻ N
F1,16
F1,16
F1,16
F1,16
F1,16
F1,16
F1,16
F1,16
F1,16
⫽ 45
⫽ 41
⫽ 11
⫽ 29
⫽6
⫽8
⫽ 38
⫽ 30
⫽2
bition 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 triosephosphate 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
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Plant Physiol. Vol. 127, 2001
Elevated CO2, Acclimation, and Electron Partitioning
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
␾PSII on ␾CO2 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
Figure 2. 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.
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
Table II. Ratio of electron transport to CO2 fixation
The gradient (k) of the relationship ␾PSII /␾CO2 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.
Regression
Coefficients
k
b
Plant Physiol. Vol. 127, 2001
Low Nitrogen
High Nitrogen
35 Pa
65 Pa
35 Pa
65 Pa
13.9
0.035
14.1
⫺0.030
17.0
⫺0.040
14.9
⫺0.018
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1207
Hymus et al.
of photosynthetic capacity has occurred. Plants were
grown at 400 ␮mol 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 ex-
Figure 3. Photoinhibition and recovery: high nitrogen. a, Photoinhibitory reduction in Fv /Fm. b, Changes in Fo during a 3-h exposure
to a PPFD of 2,000 ␮mol 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.
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 ␾PSII 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 ␾PSII (Bartak et al., 1996).
For natural vegetation growing in the field, studies
show seasonal decreases in photochemistry, and increased photoinhibition in elevated pCO2 (ScarasciaMugnozza 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
1208
Figure 4. Photoinhibition and recovery: low nitrogen. a, Photoinhibitory reduction in Fv /Fm. b, Changes in Fo during a 3-h exposure
to a PPFD of 2,000 ␮mol 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.
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Plant Physiol. Vol. 127, 2001
Elevated CO2, Acclimation, and Electron Partitioning
citation 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 ␮mol
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 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 ␾PSII (Genty et al., 1989).
The response of A and ␾PSII to PPFD was determined
over a range of light levels from 0 to 1,420 ␮mol m⫺2 s⫺1
under non-photorespiratory conditions (1 kPa O2; Linde
Gas Ltd). Leaf absorptance (␣) 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, ␾CO2 was then calculated as:
␾CO2 ⫽ 共 A ⫺ Rd兲/共␣ Q兲
where Q is PPFD. When measured in 1% (v/v) O2, the
relationship of ␾PSII to ␾CO2 is linear (Genty et al., 1989).
The model and assumptions of Valentini et al. (1995) use
the ␾PSII/␾CO2 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 ␾PSII and ␾CO2 is assumed to
describe the apparent quantum efficiency of photosynthetic linear electron flow (␾e ⫺ ) using the equation:
␾PSII ⫽ k ␾CO2 ⫹ b ⫽ 41 k ␾e ⫺ ⫹ b
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.),
Plant Physiol. Vol. 127, 2001
and inlet humidity was controlled by passing the dry airflow through a temperature-controlled ferrous-sulfate
crystal column (WG-600, ADC Ltd.). The temperaturecontrolled 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 ␮mol 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).
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:
Jt ⫽ Q␾e ⫺
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Copyright © 2001 American Society of Plant Biologists. All rights reserved.
1209
Hymus et al.
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:
Jt ⫽ Jc ⫹ Jo
Jc ⫽ 4共 A ⫹ Rd ⫹ Rl兲
Jo ⫽ 8 Rl
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:
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.
Received March 12, 2001; returned for revision May 8, 2001;
accepted July 17, 2001.
1
Jc ⫽ 3 关 Jt ⫹ 8共A ⫹ Rd兲兴
Jo ⫽ 32 关 Jt ⫺ 4共A ⫹ Rd兲兴
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 ␮mol 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 , ␾PSII , qP, Fv’/Fm’, Jt, Jc, Jo, and ␣ 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 twotailed 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 ␾PSII and ␾CO2 was examined by
1210
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