Tree Physiology 34, 944–954
doi:10.1093/treephys/tpu066
Research paper
High CO2 concentration increases relative leaf carbon gain
under dynamic light in Dipterocarpus sublamellatus seedlings
in a tropical rain forest, Malaysia
Hajime Tomimatsu1,5, Atsuhiro Iio2, Minaco Adachi3, Leng-Guan Saw4, Christine Fletcher4
and Yanhong Tang1
1Center
for Environmental Biology and Ecosystem Studies, National Institute for Environmental Studies, Tsukuba, Japan; 2Center for Education and Research in Field Science,
Agricultural Faculty, Shizuoka University, Ohya, Shizuoka, Japan; 3Center for Global Environmental Research, National Institute for Environmental Studies, Tsukuba, Japan;
4Forest Research Institute Malaysia, Kepong, Selangor, Malaysia; 5Corresponding author ([email protected])
Received February 7, 2014; accepted July 10, 2014; published online September 2, 2014; handling Editor Ülo Niinemets
Understory plants in tropical forests often experience a low-light environment combined with high CO2 concentration. We
hypothesized that the high CO2 concentration may compensate for leaf carbon loss caused by the low light, through increasing light-use efficiency of both steady-state and dynamic photosynthetic properties. To test the hypothesis, we examined CO2
gas exchange in response to an artificial lightfleck in Dipterocarpus sublamellatus Foxw. seedlings under contrasting CO2
conditions: 350 and 700 μmol CO2 mol−1 air in a tropical rain forest, Pasoh, Malaysia. Total photosynthetic carbon gain from
the lightfleck was about double when subjected to the high CO2 when compared with the low CO2 concentration. The increase
of light-use efficiency in dynamic photosynthesis contributed 7% of the increased carbon gain, most of which was due to
reduction of photosynthetic induction to light increase under the high CO2. The light compensation point of photosynthesis
decreased by 58% and the apparent quantum yield increased by 26% at the high CO2 compared with those at the low CO2.
The study suggests that high CO2 increases photosynthetic light-use efficiency under both steady-state and fluctuating light
conditions, which should be considered in assessing the leaf carbon gain of understory plants in low-light environments.
Keywords: dynamic photosynthesis, elevated CO2, induction efficiency, lightfleck, tropical trees.
Introduction
Photosynthetic photon flux density (PFD) is a crucial resource
for green plants, but it is often limited under forest canopy, in
particular under tropical forest canopies (Chazdon and Pearcy
1991, Pearcy 2007). For example, in the Pasoh forest, Malaysia,
PFD was reported to be <16 μmol m−2 s−1 for >70% of the daytime at understory sites (Tang et al. 1999). Photon flux density
can become even lower under some particular sky conditions,
such as the hazy conditions associated with forest fires and/or
burning (Tang et al. 1996). Because PFD is often very low in
tropical forest floor, sunflecks, which have brief high PFD due
to direct sunlight penetrating through the forest canopy, have
been considered as an important light resource for understory
plants (Chazdon 1988, Pearcy 1990, 2007, Timm et al. 2002).
Carbon dioxide is one of the substrates of plant photosynthesis. In contrast to the limited light resource, CO2 is relatively
sufficient for understory plants. Within a closed tropical rainforest, CO2 concentration is often higher on the forest floor than
above the canopy during late night and early morning because
of soil respiration. For example, several studies reported much
higher CO2 concentrations near the forest floor within the
Pasoh tropical rainforest, Malaysia, when compared with the
atmospheric CO2 concentration above the canopy (Aoki et al.
© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Lightfleck utilization in tropical seedlings under enhanced CO2­ 1978, Ohkubo et al. 2008) and compared with the CO2 concentration of a gap area (Tang et al. 2003). The recent rapid
increase of atmospheric CO2 concentration has further added
to our interest in the effects of environmental CO2 concentrations on photosynthetic light utilization in understory plants.
However, studies have produced contradictory results regarding the combined effects of low-light levels and high CO2
concentrations on photosynthetic light utilization, although
some studies suggest that increased CO2 concentrations can
enhance photosynthetic light utilization (Wurth et al. 1998,
DeLucia and Thomas 2000, Naumburg and Ellsworth 2000).
Naumburg and Ellsworth (2000) reported that photosynthesis
can be greatly enhanced by elevated CO2 concentrations in
shade-tolerant species. At very low PFD, CO2 enhancement of
seedling biomass was reported to be related to a decrease in
dark respiration and the light compensation point in Fagus and
Taxus species (Hättenschwiler 2001). In situ measurements in
tropical forest understory plants support the hypothesis that
tropical plants growing near the photosynthetic light compensation point are responsive to elevated CO2 concentrations
(Wurth et al. 1998). The authors suggest that an improved
plant carbon balance in deep shade is likely to influence understory plant recruitment and competition as atmospheric CO2
concentrations continue to rise. However, DeLucia and Thomas
(2000) reported that there were no differences in the responsiveness to CO2 concentrations among species with varying
shade tolerance. Moreover, the highly temporal variation of
PFD within a plant canopy adds to the complexity of the combined effects of CO2 concentration and PFD on photosynthesis
and shade tolerance of understory plants in tropical rain forests. The relative enhancement of photosynthesis by high CO2
concentrations may be higher in lightflecks than under steadystate light conditions (Leakey et al. 2002, 2005).
However, very limited information is available from field
observations regarding how instantaneous high CO2 concentrations may potentially affect the photosynthetic light utilization
of tropical understory plants. At the tropical forest floor where
CO2 concentration is often high, it is reasonable to expect that
high CO2 concentrations may contribute to the carbon balance
of understory plants. Previous experimental studies also suggest that high CO2 concentrations can increase leaf carbon gain
through increasing photosynthetic efficiency under dynamic
light conditions (Leakey et al. 2002, Košvancová et al. 2009,
Holišová et al. 2012, Tomimatsu and Tang 2012). We hypothesize that high CO2 at the tropical forest floor may enhance the
photosynthetic light-use efficiency of understory plants under
both steady-state and dynamic light conditions. The possible
mechanisms may include the increase of quantum-use efficiency under steady-state light and/or the increase of light-use
efficiency under dynamic light conditions, which may include
the decrease of photosynthetic induction limitation in responding to a light increase and/or the increase of post-illumination
945
CO2 fixation responding to a light decrease. We tested the
hypothesis by examining the photosynthetic characteristics of
one of the most common tree species in the tropical rain forest in Malaysia, Dipterocarpus sublamellatus Foxw., in relation to
lightflecks at both controlled low and high CO2 concentrations.
Materials and methods
Study site
The Pasoh Forest Reserve (2°59′N, 102°08′E) consists of a
core of virgin dipterocarp forest (600 ha) surrounded by a buffer zone that was selectively logged from 1955 to 1959 (Wong
1983). Meteorological data collected here from 1971 to 1974
(Soepadmo 1978) indicated that the mean monthly temperature
at ground level is 24 °C, ranging from 21 to 26 °C. At the top
of a 52-m tower in the Pasoh forest, the mean temperature is
22 °C, ranging between 17 and 32 °C. Relative humidity measured 2.5 m above the ground is invariably >96% during the
wet months (April, May, November and December) and ranges
from 60 to 93% during the dry months (February, March, July
and August). Ambient CO2 concentrations are higher in the
early morning, reaching 520 μmol CO2 mol−1 air at ∼08:00 h in
the closed understory (Tang et al. 2003). The CO2 concentrations decrease rapidly thereafter and reach their lowest levels
around noon, reaching ∼400 μmol CO2 mol−1 air. In the afternoon, ambient CO2 concentration levels increase again.
Carbon dioxide gas exchange measurements
Fully expanded younger leaves of D. sublamellatus from six
individual seedlings, which were in general 2 or 3 years old,
were used for CO2 gas exchange measurements using a portable gas exchange system (LI-6400; Li-Cor, Inc., Lincoln,
NE, USA). The species is widespread in the subcanopy and
understory of Pasoh forest. We selected seedlings along a gap
edge in the forest with an intermittent light regime on 13 April
2012. The average leaf area was 68.5 ± 5.1 cm2 (mean ± SE,
hereafter) with a leaf mass area of 6.8 ± 0.5 mg cm−2 for the
six samples. The selected leaves for photosynthetic measurements had a height of 1.1 ± 0.2 m aboveground.
To exclude the effects of light and soil water conditions
on photosynthesis measurements from individual seedlings,
we covered the leaves with an opaque cloth and provided an
ample water supply in the afternoon of the day before the gas
exchange measurement. We performed the same measurement
twice for the same leaf at 350 and 700 μmol CO2 mol−1 air.
Leaf photosynthetic CO2 gas exchange was measured inside
a chamber measuring 2 × 3 cm with a 6400-02B LED lamp
as the light source from 13 to 21 April 2012. Measurements
were made at 28 °C and 75% relative humidity. The air stream
entering the chamber was first scrubbed of CO2 using soda
lime, and the subsequent CO2 concentration was controlled by
injecting 100% CO2 gas. The flow rate was kept constant at
Tree Physiology Online at http://www.treephys.oxfordjournals.org
946 Tomimatsu et al.
500 µmol s−1 for all measurements, which is essential for the
measurement of dynamic photosynthesis. To measure the photosynthetic response under dynamic PFD (Figure 1), leaves were
kept at 0 µmol photons m−2 s−1 for >30 min, gas exchange rates,
i.e., dark respiration rates, were recorded, and the PFD was
then changed to 50 µmol photons m−2 s−1 (PFD50). In addition,
we established a program where the PFD intensity was set to
sequentially change to 50 µmol photons m−2 s−1 (PFD50; 30-min
period), 500 µmol photons m−2 s−1 (PFD500; 30-min period) and
20 µmol photons m−2 s−1 (PFD20; 10-min period). The irradiation
period at each PFD was sufficient for steady-state photosynthesis. The auto-logging program, Timed-Lamp, provided by the
instrument was used to send data from the LI-6400 system to a
computer, with data-logging intervals set at 5 s.
When examining rapid photosynthetic responses, time lags in
the gas analysis system must be considered. Variations in time
lags depend primarily on the flow rate, chamber volume and
tubing volume. To examine the effect of these volumes on the
instantaneous photosynthetic response, Tang and Liang (2000)
calculated the equilibrium concentration based on Model 1 of
Pearcy et al. (1985). They found that even if the effective chamber
volume was five times the actual chamber volume, the corrected
assimilation rate was <1.1% of the measured assimilation rate if
the time interval was 2.08 s at the flow rate of 500 cm3 min−1.
Niinemets (2012) has also shown that the time for the steady
state in a LI-6400 standard 2 × 3-cm cuvette is 2.12 s. The actual
time delay may differ due to leaf surface properties, temperature
and moisture conditions. In this study what we focused on was
mainly the difference in photosynthesis between the two contrast
CO2 conditions. The absolute time delay may not be a major
issue. In addition, using LI-6400 perhaps was the best choice for
the field study of CO2 exchange in our study because no similar
instrument for the purpose was then available.
After finishing the measurement of dynamic photosynthesis, we measured the CO2 response curve at a PFD of
500 µmol m−2 s−1. The relationship between net photosynthesis (A) and the calculated intercellular CO2 concentration (Ci)
was determined based on the photosynthetic rates measured
over eight external concentrations of CO2 ranging from 50 to
1500 μmol CO2 mol−1 air. The A–Ci curve was fitted based on
equations described by von Caemmerer and Farquhar (1981).
Calculations
Steady-state photosynthesis Photosynthetic light-response
curves were plotted for steady-state photosynthesis at 0, 20,
50 and 500 µmol photons m−2 s−1 based on the dynamic PFD
program described above, and the apparent quantum yield (φ)
and light compensation (Qlcp) of steady-state photosynthesis
were calculated.
Induction state From the photosynthetic induction curves,
we calculated the time to reach 50 and 90% of full induction
(IT50 and IT90, respectively). The photosynthetic induction state
(ISt) was calculated based on the equation of Chazdon and
Pearcy (1986):
ISt = ( At − A50 ) / ( A500 − A50 )
t +1
where At = (1/3) ∫ t −1 A(t )dt , which is the instantaneous assim­
ilation rate at time t after the increase in PFD at Time 0, and
is averaged from three readings to reduce the signal noise in the
measurement system. A50 and A500 are steady-state assimilation
rates before (PFD = 50 μmol photons m−2 s−1) and after the PFD
increase (PFD = 500 μmol photons m−2 s−1), respectively.
Post-illumination effect To evaluate the post-illumination effect
on photosynthetic carbon gain, the post-illumination carbon gain
(PICG) and post-illumination CO2 burst (PICB) were calculated:
Figure 1. The time course of the measured and expected net assimilation rate (A) at time (t) over 40 min (T40). T0 and T30 indicate the start
and end of a lightfleck, respectively. The upward arrow indicates that
the PFD was suddenly increased from 50 to 500 μmol photons m−2 s−1
(induction response). The downward arrow indicates that the PFD was
suddenly decreased from 500 to 20 μmol photons m−2 s−1 (post-illumination effect). The leaves used for measurement were exposed to a flux
of 50 μmol photons m−2 s−1 for 60 min before the PFD was increased.
Gray and hatched areas indicate the post-illumination carbon gain (PICG)
and post-illumination CO2 burst (PICB), respectively.
Tree Physiology Volume 34, 2014
PICG =
∫
T40
PICB =
∫
T40
T40
T30
T30
( A(t ) − A20 )dt , when A(t )> A20
( A(t ) − A20 )dt , when A(t )< A20 where ∫T ( A(t ) − A20 )dt is the cumulative value of the mea30
sured assimilation rate A(t) − A20 from the time when PFD suddenly decreased from 500 to 20 μmol photons m−2 s−1 (T30) until
the time 10 min after the steady state was reached (T40). A20 is
the steady-state assimilation rates at the PFD of 20 μmol m−2 s−1
(see Figure 1).
Lightfleck utilization in tropical seedlings under enhanced CO2­ Induction efficiency Photosynthetic induction efficiencies for
various induction times (IEt) were calculated according to Tang
et al. (1994):
IEt =
∫
t
T0
A(t )dt − A50(t − T0 )
(A500 − A50 ) (t − T0 )
,
t
where ∫T A(t )dt is the integration of the measured ­assimilation
0
rate A(t) from the time when the light changed (T0) to any
given time (t). Leaves experiencing a high CO2 concentration
may show a higher IE than leaves at ambient CO2 concentration
levels if they respond more rapidly to transient PFDs.
To evaluate the influence of lightflecks on the photosynthetic
carbon gain, the lightfleck utilization efficiency (LUE) for single
lightflecks was calculated based on the equation of Chazdon
and Pearcy (1986):
LUE =
∫
T40
T0
A(t )dt − { A50(T30 − T0 ) + A20(T40 − T30 )}
A500(T30 − T0 ) + A20(T40 − T30 ) − { A50(T30 − T0 ) + A20(T40 − T30 )}
∫
=
T40
∫
T30
∫
T30
T0
=
=
A(t )dt − {A50(T30 − T0 ) + A20(T40 − T30 )}
( A500 − A50 )(T30 − T0 )
T0
A(t )dt − A50(T30 − T0 ) +
∫
T40
T30
A(t )dt − A20(T40 − T30 )
( A500 − A50 )(T30 − T0 )
T0
A(t )dt − A50(T30 − T0 ) + PICG + PICB
( A500 − A50 )(T30 − T0 )
,
PICG + PICB
∫
T40
T0
=
∫
∫
T30
T0
=
A(t )dt − { A50(T30 − T0 ) + A20(T40 − T30 )}
PICG + PICB
T30
A(t )dt − A50(T30 − T0 ) +
∫
T40
T30
A(t )dt − A20(T40 − T30 )
PICG + PICB
T0
where PICG + PICB is the post-illumination effect during
T40 – T30 (Figure 1).
Statistical analysis
All measurements were replicated six times using leaves from
different seedlings, although the same leaves were selected
for the measurements at two different CO2 concentrations.
The results are presented as means and standard errors of
the mean. One- or two-way analysis of variance (ANOVA) was
used to compare the means. In all analyses, P < 0.05 was taken
to indicate statistical significance.
Results
Steady-state gas exchange
The steady-state photosynthetic rate (A) was higher at the higher
CO2 concentration at all PFDs (Table 1). The relative enhancement at the higher CO2 concentration was >50% at all PFDs.
Steady-state stomatal conductance (gs) and transpiration
rates (E) were decreased at the higher CO2 concentration
in the ranges of 22.8–26.4% and 18.9–24.4%, respectively.
A relative decrease at the higher CO2 concentration of both gs
and E was not apparent among different PFDs.
The values of the apparent quantum yield (φ) of leaves at the
higher CO2 concentration were significantly greater than the
values for leaves at the lower CO2 concentration (Table 1). This
is one of the reasons why the photosynthetic rates at steady
state in low PFDs increase under elevated CO2 concentrations.
The light compensation point (Qlcp) and dark respiration (Rd)
were significantly lower at the higher CO2 concentration.
Dynamic gas exchange
where T30 – T0 is the effective lightfleck duration and T40 – T30 is
the post-illumination duration (Figure 1).
The photosynthetic carbon gain due to a lightfleck consists
of two components: carbon gained during the lightfleck and
that achieved after the lightfleck, i.e., the post-illumination CO2
fixation. The contribution of post-illumination CO2 fixation to the
total carbon gain due to a lightfleck is described by the ratio of
post-illumination CO2 carbon gain to the total carbon gain during the lightfleck (RPC):
RPC =
947
A(t )dt − A50(T30 − T0 ) + PICG + PICB
,
After a sudden increase in PFD from 50 to 500 μmol photons m−2 s−1, the photosynthetic CO2 uptake rate increased rapidly within the first 2–5 min and increased gradually thereafter,
reaching a steady-state under high PFD in leaves at both low
and high CO2 concentrations (Figure 2).
The dynamic photosynthetic response to Ci was plotted to
separate the stomatal and biochemical limitations during photosynthetic induction and post-illumination (Figure 3). The dynamic
relations of A–Ci in photosynthesis at induction and post-­
illumination showed a virtually straight line. These straight lines in
the dynamic relations of A–Ci indicate that the stomatal limitation
is small. The time taken to reach a steady-state photosynthetic
rate was very short (∼30 s), after a sudden decrease in PFD.
The time required for the CO2 uptake rate to reach 50%
(IT50) and 90% (IT90) of the steady-state assimilation rate after
an increase in light differed between the two CO2 concentrations (Figure 4). Both IT50 and IT90 were smaller at the higher
CO2 concentration.
The temporal variation of the induction state was compared
with IS30, IS120 and IS300 (Figure 5). Leaves exposed to both
Tree Physiology Online at http://www.treephys.oxfordjournals.org
948 Tomimatsu et al.
Table 1. Net assimilation rate (A), stomatal conductance (gs) and transpiration rate (E) obtained under constant PFD of 20, 50 and 500 μmol photons m−2 s−1 (indicated by subscripts under each parameter), dark respiration (Rd), light compensation point (Qlcp) and apparent quantum yield (φ) in
D. sublamellatus at CO2 concentrations of 350 and 700 μmol CO2 mol−1 air, respectively. Values are means (±SE) for six fully expanded leaves. The
effects of high CO2 concentrations are indicated by (X700 − X350)/X350, where X is A, gs, E, Rd, Qlcp and φ obtained under 350 and 700 μmol CO2 mol−1
air, respectively. The significance of differences in each parameter between the two CO2 concentrations was determined by one-way ANOVA
(*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Values are means (±SE) for six leaves each from a different tree. ns, not significant.
Parameter
PFD
Measurement CO2 treatments
350 μmol CO2
mol−1
air
Net assimilation rate (μmol CO2 m−2 s−1)
A20
20
0.50 ± 0.05
A50
50
1.86 ± 0.04
A500
500
4.60 ± 0.30
Stomatal conductance (mol H2O m−2 s−1)
gs20
20
0.088 ± 0.008
gs50
50
0.074 ± 0.004
gs500
500
0.103 ± 0.010
Transpiration rate (mmol H2O m−2 s−1)
E20
20
0.87 ± 0.11
E50
50
0.81 ± 0.05
E500
500
1.10 ± 0.10
Apparent quantum yield (mol CO2 mol−1 photons)
φ
0.052 ± 0.001
Light compensation point (μmol photons m−2 s−1)
Qlcp
12.9 ± 0.72
Dark respiration(μmol CO2 m−2 s−1)
Rd
0.75 ± 0.04
of the CO2 concentrations exhibited an increased state of
induction with induction time. The difference in induction state
between the high and low CO2 concentrations decreased with
induction time.
The photosynthetic carbon gain during the induction response
was evaluated by the induction efficiency (IE) (Figure 5). In
contrast to the IS, the difference in IE between the high and low
CO2 concentrations increased with induction time.
Total carbon gain and LUE
Total carbon gain in leaves during a single lightfleck increased
significantly from ∼4824 to 9290 μmol CO2 m−2 (increasing
by ∼92.6%) under the higher CO2 concentration (Table 2).
Lightfleck utilization efficiency was significantly larger in leaves
at the higher CO2 concentration than at the lower CO2 concentration. The contribution of LUE to the total carbon gain with
a single lightfleck increased by ∼3.3% at the higher CO2 concentration. Thus, the carbon gain under the higher CO2 concentration increased by ∼307 μmol CO2 m−2 (9290 × 0.033)
by the LUE.
Contribution of post-illumination CO2 fixation
to total carbon gain
To evaluate the post-illumination effect on the photosynthetic carbon gain, the PICG and PICB were calculated. The
Tree Physiology Volume 34, 2014
High CO2 effect (%)
P
0.85 ± 0.08
2.94 ± 0.12
8.47 ± 0.60
+70.6
+58.1
+84.1
**
***
***
0.068 ± 0.001
0.054 ± 0.007
0.080 ± 0.003
−23.1
−26.4
−22.8
ns
*
ns
0.71 ± 0.06
0.61 ± 0.10
0.86 ± 0.06
−18.9
−24.4
−21.8
ns
ns
ns
0.065 ± 0.001
+26.3
****
5.41 ± 2.01
−58.0
****
0.31 ± 0.17
−58.9
*
700 μmol CO2
mol−1
air
PICG was higher at the higher CO2 concentration but the PICB
was similar at both of the CO2 concentrations. Thus, the
|PICG/PICB| of leaves at elevated CO2 concentrations was
significantly greater than for leaves at low CO2 concentrations
(Table 3). These observations suggested that the contribution of post-­illumination CO2 fixation to the total carbon gain
is large in the presence of high CO2 concentrations. The RPC
increased by ∼0.65% at the higher CO2 concentration. Thus,
the carbon gain under the higher CO2 concentration increased
∼60 μmol CO2 m−2 (9290 × 0.0065) by the RPC.
Discussion
Steady-state gas exchange
In D. sublamellatus seedlings, the increase in photosynthetic
rates at high CO2 concentrations was very large. This may benefit the carbon fixation of plants growing on the tropical forest floor where CO2 concentration is often high, in particular
during early morning (Tang et al. 2003). Photosynthetic rates
under the higher CO2 concentration were often limited by ribulose-1,5-bisphosphate (RuBP) (Farquhar and von Caemmerer
1982). The steady-state A–Ci relationship suggests that high
CO2 enhanced RuBP regeneration, which should contribute to
more positive net carbon for D. sublamellatus seedlings in lowlight environment (Figure 3).
Lightfleck utilization in tropical seedlings under enhanced CO2­ 949
Figure 2. Representative time courses of net assimilation rate (A), stomatal conductance (gs), transpiration rate (E) and intercellular CO2 concentration (Ci) in response to a lightfleck in leaves of D. sublamellatus seedlings
measured under 350 (Low CO2) and 700 μmol CO2 mol−1 air (High CO2).
The PFD was initially increased from 20 to 500 μmol photons m−2 s−1 for
30 min and decreased to 50 μmol photons m−2 s−1 for 10 min.
Plants growing in deep shade, such as in the understory of
tropical forests, may be expected to benefit from the increase of
CO2 due to the decrease of photorespiration, increase of quantum yield and thus decrease the light compensation point of
photosynthesis (e.g., Valle et al. 1985, Wong and Dunin 1987,
Long and Drake 1991). Quantum yield for terrestrial vascular
plants varies often between 0.05 and 0.12 (Larcher 2003),
though the apparent quantum yield for some shade-­tolerant
species seems to be much lower (Kubiske and Pregitzer 1994).
Quantum yield is, in general, higher at higher CO2 (Ehleringer
and Bjorkman 1977, Ogren and Evans 1993, Herrich and
Thomas 1999). The apparent quantum yield observed for
D. sublamellatus was consistent with previous studies. The
increase of the initial slope of light reaction curve was perhaps
mainly caused by the decrease of ­
photorespiration, though
other mechanisms might also be involved (e.g., Ogren and
Figure 3. Mean net CO2 assimilation rate (A) plotted against intercellular
CO2 concentration (Ci) in leaves of D. sublamellatus seedlings (n = 6) during the induction process (top) after the PFD was increased from 50 to
500 μmol photons m−2 s−1, and during the post-illumination period (bottom) after the PFD was decreased from 500 to 20 μmol photons m−2 s−1.
The CO2 concentration was 350 μmol CO2 mol−1 air (Low CO2) and
700 μmol CO2 mol−1 air (High CO2). The dashed line (with open circles)
is the steady-state relationship between A and Ci measured at the PFD of
500 μmol photons m−2 s−1 for the same leaves (n = 6). Note that the photosynthetic induction state is shown at 0, 5, 10, 30, 60, 120, 300, 600
and 1800 s, and the photosynthetic rate in the post-illumination period
is shown at 1800, 1805, 1810, 1830, 1860, 1920, 2100 and 2400 s.
Figure 4. Photosynthetic induction time required for the CO2 uptake
rate to reach 50% (IT50) and 90% (IT90) of the steady-state photosynthetic rate following an increase in the PFD from 50 to
500 μmol photons m−2 s−1 in D. sublamellatus leaves measured
under conditions of low (350 μmol CO2 mol−1 air, black boxes) and
high CO2 (700 μmol CO2 mol−1 air, open boxes) concentrations. Bars
and vertical lines indicate the means and standard error (n = 6) for
gas exchange measurements, respectively. The differences between
results for the two CO2 concentrations were examined for significance
by one-way ANOVA.
Tree Physiology Online at http://www.treephys.oxfordjournals.org
950 Tomimatsu et al.
Table 3. Parameters of the post-illumination effects (PICG, PICB,
PICG + PICB and |PICG/PICB|) and the ratio of post-illumination CO2 carbon
gain to the total carbon gain during the lightfleck (RPC): for programmed
single lightflecks (see Figure 1) for D. sublamellatus under conditions of
low and high CO2 concentrations (350 and 700 μmol CO2 mol−1 air,
respectively). Values are means for six leaves each from a different
tree ±SE. Significant differences between means indicated by (oneway ANOVA) *P < 0.05, **P < 0.01. ns, not significant.
Parameter
Measurement CO2 treatments
350 μmol CO2 mol−1 air
RPC (%)
−0.27 ± 0.13
Post-illumination parameters
PICG
24.4 ± 7.7
PICB
−37.1 ± 9.8
PICG + PICB
−12.7 ± 6.4
|PICG/PICB|
0.65 ± 0.2
P
700 μmol CO2 mol−1 air
0.38 ± 0.08
61.1 ± 4.9
−29.9 ± 4.1
31.2 ± 6.2
2.15 ± 0.3
**
**
ns
**
*
the dark respiration rate at enhanced CO2 concentrations may
contribute to an increase in the net carbon gain of plants.
Dynamic gas exchange
Figure 5. Photosynthetic induction state (top) and photosynthetic
induction efficiency (bottom) at 30, 120 and 300 s following an
increase in the PFD from 50 to 500 μmol photons m−2 s−1 in D. sublamellatus leaves measured under conditions of low (350 μmol CO2 mol−1
air, black boxes) and high CO2 (700 μmol CO2 mol−1 air, open boxes)
concentrations. Bars and vertical lines extending from the bars indicate the means and standard error (n = 6) for gas exchange measurements, respectively. The differences between results for the two CO2
concentrations were examined for significance by Student's t test.
Table 2. Total carbon gain and LUE for a single lightfleck (see
Figure 1) in D. sublamellatus under conditions of low and high CO2
concentrations (350 and 700 μmol CO2 mol−1 air, respectively). Values
are means for six leaves each from a different tree ±SE. Significant
differences between means indicated by (one-way ANOVA) *P < 0.05,
**P < 0.01. ns, not significant.
Parameter
LUE (%)
Total carbon gain
Total water loss
Measurement CO2 treatments
350 μmol CO2 mol−1 air
700 μmol CO2 mol−1 air
91.9 ± 1.1
4824.3 ± 528.0
436.8 ± 95.0
95.2 ± 1.2
9290.2 ± 1043.0
271.6 ± 51.4
P
*
**
ns
Evans 1993). Tropical plants growing near the photosynthetic
light compensation point are responsive to elevated levels of
CO2 concentration (Wurth et al. 1998). In our study, the compensation point was much lower under the high than under
the low experimental CO2 concentration. This indicates that an
equivalent leaf carbon gain under a high CO2 can be achieved
under a low PFD, which is expected to compensate the loss of
leaf carbon from the deep shade. Furthermore, the decrease in
Tree Physiology Volume 34, 2014
Photosynthetic induction response Previous studies have
suggested that photosynthetic induction response is enhanced
at high CO2 concentrations. This is caused by an increase in the
speed of the stomatal opening (Tinoco-Ojanguren and Pearcy
1993, Košvancová et al. 2009) or decreases of both stomatal
and biochemical limitations (e.g., Tomimatsu and Tang 2012).
In this study, the photosynthetic induction response in the first
phase (i.e., IS30 and IT50) was also enhanced by the high CO2
concentrations, and contributed to an increase in plant carbon
gain (Figures 4 and 5).
However, in the longer second phase of photosynthetic
induction (i.e., IS300 and IT90), the photosynthetic induction did
not differ significantly between leaves exposed to high and
low CO2 concentrations (Figures 4 and 5). These results suggest that the initial induction response was mainly due to the
accelerated regeneration of RuBP and/or a buildup of Calvin
cycle metabolites (e.g., Kirschbaum and Pearcy 1988, Pearcy
1990, Sassenrath-Cole and Pearcy 1992), rather than due to
the change in stomatal conductance.
The linear trajectory of A versus Ci suggests that most of
the limitations were biochemical in nature (Figure 3). The relationship between A and Ci during the induction response provides insights into the limitations of stomatal conductance and
biochemical capacity on photosynthetic induction (Chazdon
and Pearcy 1986, Pearcy et al. 1996). If the increase in A is
caused solely by the increase of gs, then A and Ci should follow
the steady-state A–Ci curve. If the increase in A is also limited
by biochemical capacity, then A should fall below the steadystate A–Ci curve. In a Populus species with only minor stomatal response to light change and with large stomatal opening,
Lightfleck utilization in tropical seedlings under enhanced CO2­ 951
A increased almost linearly with a decrease in Ci during the
induction response (Tang and Liang 2000). In this study,
the linear dependency of A on Ci also may indicate that the
increase in assimilation rate was limited mainly by biochemical
capacity but not by gs. Sufficient water supply before experimental measurement and high air humidity in the tropical forest may contribute to the small limitation of gs.
The change of apparent Ci during photosynthetic induction
is, in general, a result of the changes in gs and A. Both A
and gs are continuously increased after the sudden increase
of PFD, but their time courses are different (Figure 2). By
a quick activation of photosynthetic enzyme reaction, A
increased rapidly, which was combined with a fast decrease
of Ci. On the other hand, gs and E showed a very slow change
in the induction response, which may be contributed to by
the high air humidity and sufficient soil water experimentally
available in this forest floor (Figure 2). The difference in the
time courses of A, or Ci between the high and low CO2 concentrations therefore was mainly due to the differences in the
activation rate of photosynthetic enzymes, but almost not in
the stomatal limitation between the two CO2 concentrations
in this study (Figure 3).
These results suggest that a short-term high CO2 environment on the tropical forest floor increases the photosynthetic
induction response by decreasing the effects of biochemical
limitations, which may also imply the importance of short sunflecks to leaf carbon gain under a high CO2 environment.
Post-illumination effect The use of intermediates after light
irradiation is important for carbon gain in the leaves of seedlings growing in the understory and/or a dark habitat (e.g.,
Tang et al. 1994). Therefore, the RPC, which is significantly
higher at elevated CO2 concentrations (Table 3), is important
for dynamic carbon gain, especially in high-frequency lightfleck
environments, in which post-irradiance metabolism contributes
a greater proportion of the net carbon gain (Pearcy 1990). It
has been reported that short-term treatments with high CO2
concentrations or low O2 concentrations lead to an increased
PICG and a decreased PICB (Doehlert et al. 1979, Peterson
1983, Vines et al. 1983, Laisk et al. 1984). This is likely to be
due to changes in the flux of intermediates in the photosynthetic and photorespiratory pathways, respectively (Sharkey
et al. 1986, Rawsthorne and Hylton 1991).
In this study, the PICG (+36.6) increased significantly at the
high CO2 concentration, but the PICB (+7.3) was unaffected
(Table 3). An increase in the PICG at high CO2 concentration
levels may be affected by an increased maximum photosynthetic rate in the steady state, because the decreasing rate
of photosynthesis did not change between high and low CO2
concentrations when the PFD decreased (Figure 3). However,
the PICB did not change with elevated CO2 concentration,
despite the high maximum photosynthetic rate, and therefore
it may be limited by the decrease in photorespiration at high
CO2 concentrations. As a result, short-term CO2 treatments
increased the |PICG/PICB| and contributed to an increased
post-illumination effect (PICG+PICB). The relative contribution
of the RPC of leaves at elevated CO2 concentration levels was
significantly greater than that at low CO2 concentration levels
(Table 3). These results suggest that an enhanced post-illumination effect with elevated CO2 concentration levels contributes to the increase in total carbon gain under dynamic PFD
conditions.
The post-illumination burst is considered to be related to the
metabolism of glycollate in photorespiration, and CO2 assimilation resulting from the buildup of RuBP and its high energy
precursors. Since these biochemical processes are very quick,
it is difficult to record a detailed time course using an LI-6400
analyzer. Instead of examining the detailed time course, we
focused on the accumulated values of CO2 uptake under the
two contrast CO2 concentrations. This result clearly indicates
that high CO2 concentration decreased the accumulated CO2
during the post-illumination burst. Further studies are required
to clarify the biochemical and stomatal involvements during the
post-illumination CO2 fixation.
Lightfleck utilization efficiency In this study, the LUE of
D. sublamellatus leaves increased by ∼3.2% at the high CO2
concentration compared with the lower CO2 concentration
(Table 2). The enhancement of light-use efficiency at elevated
CO2 concentration under both steady-state and dynamic light
such as sunflecks is expected to benefit leaf carbon gain, and
thus should have positive consequences for seedling growth
and regeneration, and even to have an effect on forest structure and composition (e.g., Leakey et al. 2002, Holišová et al.
2012). Lightfleck utilization efficiency is also mainly determined by the IE and post-illumination CO2 fixation (e.g., Pearcy
1988, Chazdon and Pearcy 1991). The increase in LUE was
mainly due to the IE rather than post-illumination CO2 fixation
(Table 3). The relative contribution of induction response and
post-illumination CO2 fixation should differ with different durations of lightflecks. In general, LUE tends to decrease with
increasing lightfleck duration (Chazdon and Pearcy 1986,
Pons and Pearcy 1992, Tang et al. 1994). This is primarily
due to the relatively small contribution of post-illumination
RPC during long-duration lightflecks (Pons and Pearcy 1992).
However, when the lightfleck duration increases (to over
100 s), the LUE again rises due to increases in the induction
gain during the lightfleck (Tang et al. 1994). In this study,
the lightfleck duration was set at 30 min (which is relatively
long) as this was sufficient for the photosynthetic rate to stabilize. As a result, the contribution of the increased level of
CO2 concentration to LUE, i.e., the difference in LUE between
the low and high CO2 concentrations, is low at only 3.2%.
If the LUE were to be measured during a shorter lightfleck
Tree Physiology Online at http://www.treephys.oxfordjournals.org
952 Tomimatsu et al.
duration, the contribution of the increased level of CO2 concentration to the LUE may be due to the reduced induction
gain during the lightfleck. Further research into the relationship between lightfleck duration and LUE under different CO2
conditions is required to understand the effects of short-term
high CO2 concentrations on dynamic photosynthetic carbon
gain in tropical forest species.
Total carbon gain for a single lightfleck
Short-term high CO2 concentrations in a closed tropical forest
understory increased the dynamic carbon gain in leaves of
D. sublamellatus seedlings. The results support our hypothesis that high CO2 concentrations can enhance the photosynthetic carbon gain through increasing the light-use efficiency
under both steady-state and dynamic PFD conditions. In
this study, when gas exchange measurements were conducted under a long single lightfleck (30 min) in the same
leaves exposed to 350 and 700 μmol CO2 mol−1 air, the
total carbon gain increased by about twofold (from 4824 to
9290 μmol CO2 m−2) (Figure 6). The enhanced carbon gain at
an instantaneous high CO2 concentration was almost entirely
due to the CO2 supply effect as photosynthetic substrate
under steady-state conditions (∼93.1%). Some of the carbon
gain was due to LUE (∼6.9%) that could be further broken
down into an induction effect (∼5.5%) and a post-­illumination
effect (∼1.3%). However, the relative contribution of the
increased photosynthetic response at high CO2 concentrations
may be large under a shorter sun-fleck environment, because
the photosynthetic induction response is promoted at high
CO2 concentrations in the early induction phase (Figures 4
and 5). Further studies are necessary to determine the relationship between lightfleck duration and LUE under changing
CO2 conditions.
Conclusion
Our results suggest that a short-term high CO2 concentration can increase leaf carbon gain by both LUE and changing
steady-state photosynthetic characteristics. The contribution of
high CO2 concentration to leaf carbon gain may vary with light
fluctuating patterns and other environmental conditions, for
which further studies are needed. This study provides insights
into our understanding of photosynthesis in tree seedlings in
response to the CO2 environment, and the ecological consequences of high CO2 for the tropical forest floor in relation to
forest regeneration as well as forest carbon budgets under
future environments including high atmospheric CO2.
Acknowledgments
The authors thank Mr Quah Eng Seng for identifying the plant
species in the field, and thank the Forest Research Institute
Malaysia and the Forestry Department of Negeri Sembilan for
using research facilities.
Conflict of interest
None declared.
Funding
This study was supported by a Grant-in-Aid for Scientific
Research on Innovative Areas ‘Comprehensive studies of plant
responses to high CO2 world by an innovative consortium of
ecologists and molecular biologists’ (No. 22114513) and the
project on tropical forest ecology and environment funded by
National Institute for Environmental Studies.
References
Figure 6. Total carbon gain for a single lightfleck (see Figure 1) in D.
sublamellatus leaves measured at 350 and 700 μmol CO2 mol−1 air.
Black and gray boxes indicate the increase of carbon gain contributed by the increase of lightfleck utilization efficiency (LUE), and by
other effects of high CO2, respectively. The carbon gain contributed
by the increase of LUE is calculated as 9290 × (95.2 − 91.9 (%)) = 307
(i.e., contribution is 6.9%), in which induction response contributed
∼5.5% and post-illumination CO2 fixation contributed ∼1.3% (see also
Tables 2 and 3).
Tree Physiology Volume 34, 2014
Aoki M, Yabuki K, Koyama H (1978) Micrometerology of Pasoh Forest.
Malay Nat J 30:149–159.
Chazdon RL (1988) Sunflecks and their importance to forest understorey plants. Adv Ecol Res 18:1–63.
Chazdon RL, Pearcy RW (1986) Photosynthetic responses to light variation in rainforest species. I. Induction under constant and fluctuating light conditions. Oecologia 69:517–523.
Chazdon RL, Pearcy RW (1991) The importance of sunflecks for forest
understory plants—photosynthetic machinery appears adapted to
brief, unpredictable periods of radiation. Bioscience 41:760–766.
DeLucia EH, Thomas RB (2000) Photosynthetic responses to CO2
enrichment of four hardwood species in a forest understory.
Oecologia 122:11–19.
Lightfleck utilization in tropical seedlings under enhanced CO2­ 953
Doehlert DC, Ku MSB, Edwards GE (1979) Dependence of the post
illumination burst of CO2 on temperature, light, CO2, and O2 concentration in wheat (Triticum aestivum L.). Physiol Plant 46:299–306.
Ehleringer J, Bjorkman O (1977) Quantum yields for CO2 uptake in C3
and C4 plants. Plant Physiol 59:86–90.
Farquhar GD, von Caemmerer S (1982) Modeling of photosynthetic responses to environmental conditions. In: Lange OL, Nobel
PS, Osmond CB, Ziegler H (eds) Physiological plant ecology II.
Encyclopedia of plant physiology. New Series, Vol. 12b. Springer,
Heidelberg, pp 550–587.
Hättenschwiler S (2001) Tree seedling growth in natural deep shade:
functional traits related to interspecific variation in response to
­elevated CO2. Oecologia 129:31–42.
Herrich JD, Thomas RB (1999) Effects of CO2 enrichment on the photosynthetic light response of sun and shade leaves of canopy sweetgum
trees (Liquidambar styraciflua) in a forest ecosystem. Tree Physiol
19:779–786.
Holišová P, Zitová M, Klem K, Urban O (2012) Effect of elevated carbon dioxide concentration on carbon assimilation under fluctuating
light. J Environ Qual 41:1931–1938.
Kirschbaum MUF, Pearcy RW (1988) Gas exchange analysis of
the relative importance of stomatal and biochemical factors in
photosynthetic induction in Alocasia macrorrhiza. Plant Physiol
86:782–785.
Košvancová M, Urban O, Šprtová M, Hrstka M, Kalina J, Tomášková
I, Špunda V, Marek MV (2009) Photosynthetic induction in broadleaved Fagus sylvatica and coniferous Picea abies cultivated under
ambient and elevated CO2 concentrations. Plant Sci 177:123–130.
Kubiske ME, Pregitzer KS (1994) Effects of elevated CO2 and light
availability on the photosynthetic light response of trees of contrasting shade tolerance. Tree Physiol 16:351–358.
Laisk A, Kiirats O, Oja V (1984) Assimilatory power (postillumination
CO2 uptake) in leaves—measurement, environmental dependencies, and kinetic-properties. Plant Physiol 76:723–729.
Larcher W (2003) Carbon utilization and dry matter production. In:
Larcher W (ed) Physiological plant ecology. 4th edn. Springer,
Berlin, pp 69–184.
Leakey ADB, Press MC, Scholes JD, Watling JR (2002) Relative
enhancement of photosynthesis and growth at elevated CO2 is
greater under sunflecks than uniform irradiance in a tropical rain
forest tree seedling. Plant Cell Environ 25:1701–1714.
Leakey ADB, Scholes JD, Press MC (2005) Physiological and ecological significance of sunflecks for dipterocarp seedlings. J Exp Bot
56:469–482.
Long SP, Drake BG (1991) Effect of the long-term elevation of CO2
concentration in the field on the quantum yield of photosynthesis of
the C3 sedge, Scirpus olneyi. Plant Physiol 96:221–226.
Naumburg E, Ellsworth DS (2000) Photosynthesis sunfleck utilization potential of understory saplings growing under elevated CO2 in
FACE. Oecologia 122:163–174.
Niinemets Ü (2012) Whole-plant photosynthesis: potentials, limitations and physiological and structural controls. In: Flexax J, Loreto F,
Medrano H (eds) Terrestrial photosynthesis in a changing environment. Cambridge University Press, Cambridge, UK, pp 399–423.
Ogren E, Evans JR (1993) Photosynthetic light-response curves: I.
The influence of CO2 partial pressure and leaf inversion. Planta
189:182–190.
Ohkubo S, Kosugi Y, Takanashi S, Matsuo N, Tani M, Nik AR (2008)
Vertical profiles and storage fluxes of CO2, heat and water in
a tropical rainforest at Pasoh, Peninsular Malaysia. Tellus 60B:
569–582.
Pearcy RW (1988) Photosynthetic utilisation of lightflecks by understory plants. Aust J Plant Physiol 15:223–238.
Pearcy RW (1990) Sunflecks and photosynthesis in plant canopies.
Annu Rev Plant Phys 41:421–453.
Pearcy RW (2007) Responses of plants to heterogeneous light environments. In: Valladares F, Pugnaire FI (eds) Functional plant ecology.
CRC Press, London, pp 213–258.
Pearcy RW, Katherine O, Calkin HW (1985) Photosynthetic responses
to dynamic light environments by Hawaiian trees. Plant Physiol
79:896–902.
Pearcy RW, Krall JP, Sassenrath-Cole GF (1996) Photosynthesis in
fluctuating light environments. In: Barber NR (ed) Photosynthesis
and the environment. Kluwer Academic Publishers, Dordrecht, The
Netherlands, pp 321–346.
Peterson RB (1983) Estimation of photo-respiration based on the initial rate of postillumination CO2 release. 2. Effects of O2, CO2, and
temperature. Plant Physiol 73:983–988.
Pons TL, Pearcy RW (1992) Photosynthesis in flashing light in soybean leaves grown in different conditions. II. Lightfleck utilization
efficiency. Plant Cell Environ 15:577–584.
Rawsthorne S, Hylton CM (1991) The relationship between the postillumination CO2 burst and glycine metabolism in leaves of C3 and
C3–C4 intermediate species of Moricandia. Planta 186:122–126.
Sassenrath-Cole GF, Pearcy RW (1992) Regulation of photosynthetic
induction state by magnitude and duration of low light exposure.
Plant Physiol 105:1115–1123.
Sharkey TD, Seemann JR, Pearcy RW (1986) Contribution of metabolites of photosynthesis to postillumination CO2 assimilation in
response to lightflecks. Plant Physiol 82:1063–1068.
Soepadmo E (1978) Introduction to the Malaysian IBP synthesis meeting. Malay Nat J 30:119–124.
Tang Y, Liang N (2000) Characterization of the photosynthetic induction response in a Populus species with stomata barely responding
to light changes. Tree Physiol 20:969–976.
Tang Y, Koizumi H, Satoh M, Washitani I (1994) Characteristics of transient photosynthesis in Quercus serrata seedlings grown under lightfleck and constant light regimes. Oecologia 100:463–469.
Tang Y, Naoki K, Akio F, Awang M (1996) Light reduction by regional
haze and its effect on simulated leaf photosynthesis in a tropical forest of Malaysia. For Ecol Manag 89:205–211.
Tang Y, Kachi N, Furukawa A, Awang MB (1999) Heterogeneity of light
availability and its effects on simulated carbon gain of tree leaves in
a small gap and the understory in a tropical rain forest. Biotropica
31:268–278.
Tang Y, Okuda T, Awang M, Rahim Nik A, Tani M (2003) Sunfleck
contribution to leaf carbon gain in tree seedlings from gap and the
understory. In: Okuda T (ed) Pasoh—ecology of a lowland rain forest in Southeast Asia. Springer, Tokyo, Japan, pp 251–260.
Timm HC, Stegemann J, Küppers M (2002) Photosynthetic induction strongly affects the light compensation point of net photosynthesis and coincidentally the apparent quantum yield. Trees
16:47–62.
Tinoco-Ojanguren C, Pearcy RW (1993) Stomatal dynamics and its
importance to carbon gain in 2 rain-forest piper species. 2. Stomatal
versus biochemical limitations during photosynthetic induction.
Oecologia 94:395–402.
Tomimatsu H, Tang Y (2012) Elevated CO2 differentially affects photosynthetic induction response in two Populus species with different
stomatal behavior. Oecologia 169:869–878.
Valle R, Mishoe JW, Campbell WJ, Jones JW, Allen LH Jr (1985)
Photosynthetic responses of ‘bragg’ soybean leaves adapted to different CO2 environments. Crop Sci 25:333–339.
Vines HM, Tu ZP, Armitage AM, Chen SS, Black CC (1983)
Environmental responses of the post-lower illumination CO2 burst as
related to leaf photo-respiration. Plant Physiol 73:25–30.
Tree Physiology Online at http://www.treephys.oxfordjournals.org
954 Tomimatsu et al.
von Caemmerer S, Farquhar GD (1981) Some relationships between
the biochemistry of photosynthesis and the gas exchange of leaves.
Planta 153:376–387.
Wong M (1983) Understory phenology of the virgin and regenerating
habitats in Pasoh Forest Reserve, Negeri Sembilan, West Malaysia.
Malay For 46:197–223.
Tree Physiology Volume 34, 2014
Wong SC, Dunin FX (1987) Photosynthesis and transpiration of trees
in a eucalypt forest stand: CO2, light and humidity responses. Aust J
Plant Physiol 14:619–632.
Wurth MKR, Winter K, Korner C (1998) In situ responses to elevated CO2 in tropical forest understorey plants. Funct Ecol
12:886–895.