Plant Cell Physiol.
46(7): 1036–1045 (2005)
doi:10.1093/pcp/pci113, available online at www.pcp.oupjournals.org
JSPP © 2005
Photosynthetic Acclimation in Rice Leaves to Free-air CO2 Enrichment
Related to Both Ribulose-1,5-bisphosphate Carboxylation Limitation and
Ribulose-1,5-bisphosphate Regeneration Limitation
Gen-Yun Chen 1, Zhen-Hua Yong 1, Yi Liao 1, Dao-Yun Zhang 1, Yue Chen 1, Hai-Bo Zhang 1, Juan Chen 1,
Jian-Guo Zhu 2 and Da-Quan Xu 1, *
1
Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, PR China
2
State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, PR China
Net photosynthetic rates (Pns) in leaves were com-
pared between rice plants grown in ambient air control and
free-air CO2 enrichment (FACE, about 200 µmol mol–1
above ambient) treatment rings. When measured at the
same CO2 concentration, the Pn of FACE leaves decreased
significantly, indicating that photosynthetic acclimation to
high CO2 occurs. Although stomatal conductance (Gs) in
FACE leaves was markedly decreased, intercellular CO2
concentrations (Ci) were almost the same in FACE and
ambient leaves, indicating that the photosynthetic accli-
mation is not caused by the decreased Gs. Furthermore,
carboxylation efficiency and maximal Pn, both light and
CO2-saturated Pn, were decreased in FACE leaves, as shown
by the Pn–Ci curves. In addition, the soluble protein,
Rubisco (ribulose-1,5-bisphosphate caboxylase/oxygenase),
and its activase contents as well as the sucrose-phosphate
synthase activity decreased significantly, while some soluble
sugar, inorganic phosphate, chlorophyll and light-harvest-
ing complex II (LHC II) contents increased in FACE leaves.
It appears that the photosynthetic acclimation in rice leaves
is related to both ribulose-1,5-bisphosphate (RuBP) carb-
oxylation limitation and RuBP regeneration limitation.
Keywords: CO2 enrichment — Photosynthetic acclimation —
Rice — RuBP carboxylation limitation — RuBP regeneration
limitation.
Abbreviations: Ca, atmospheric CO2 concentration; CE, carb-
oxylation efficiency; Ci, intercellular CO2 concentration; FACE, free-
air CO2 enrichment; Fru, fructose; Glc, glucose; Gs, stomatal conduct-
ance; LHC II, light-harvesting complex II; Pi, inorganic phosphate; Pn,
net photosynthetic rate; PPFD, photosynthetic photon flux density;
Rubisco, ribulose-1,5-bisphosphate caboxylase/oxygenase; RuBP,
ribulose-1,5-bisphosphate; Φc, apparent quantum yield of carbon
assimilation; SPS, sucrose-phosphate synthase; Suc, sucrose; WUE,
water use efficiency.
Introduction
For the past several decades there have been numerous
reports about the response and acclimation of plant photosyn-
*
Corresponding author: E-mail, dqxu@sippe.ac.cn; Fax, +86-21-54924015.
1036
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;
thesis to an elevated atmospheric CO2 concentration. In gen-
eral, when C3 plants are exposed to a high CO2 concentration,
the net photosynthetic rate (Pn) of their leaves is accelerated
due to both enrichment of substrate CO2 and inhibition of pho-
torespiration by high CO2 concentration. The stimulatory effect
of high CO2 concentration on photosynthesis, however,
decreases gradually as the time of the exposure is prolonged.
After long-term exposure to a high CO2 concentration, the Pn
in leaves is significantly lower than that in ambient leaves
when measured at the same CO2 concentration (DeLucia et al.
1985, Spencer and Bowes 1986, Rowland-Bamford et al. 1991,
Xu et al. 1994a, Xu et al. 1994b). The phenomenon is gener-
ally termed acclimation or down-regulation of photosynthesis.
Of course, there are some reports that the acclimation does
not occur in plants grown at elevated CO2 concentration (e.g.
Radin et al. 1987, Herrick and Thomas 2001). Since the accli-
mation occurs often in potted but not in field-grown plants, the
root growth restriction or the imbalance in the source–sink
relationship of photosynthates has been considered as a major
factor causing the acclimation (Arp 1991). However, this
hypothesis has been challenged because the acclimation is also
observed in field-grown plants under free-air CO2 enrichment
(FACE) conditions where no root growth restriction exists
(Seneweera et al. 2002, Ainsworth et al. 2003).
Additionally, some studies indicate that the down-regula-
tion of photosynthesis in plants with a low nitrogen (N) level is
more obvious than in plants with a high N level (Wong 1979)
and no photosynthetic acclimation occurs when the N supply is
adequate (Stitt and Krapp 1999). Therefore, the N supply level
may be another possible determinant of the acclimation. Never-
theless, a study has shown that the acclimation is an indirect
effect of N and is dependent on the sink–source balance of
plant (Rogers et al. 1998). Moreover, when N is supplied in
direct proportion to plant growth, elevated CO2 does not induce
the acclimation regardless of whether the N supply is strongly
limiting growth or is optimal (Farage et al. 1998).
The mechanism leading to the acclimation is as yet
unclear, although some explanations or hypotheses have been
proposed.
Based on photosynthetic inhibition in cotton leaves after
long-term exposure to elevated CO2, DeLucia et al. (1985) sug-
 Photosynthetic acclimation in rice to FACE 1037

Fig. 1 Pn (upper), Gs (middle) and Ci/Ca (lower) measured at their growth CO2 concentrations in rice leaves. Ambient, leaves grown at the nor-
mal atmospheric CO2 concentration (380 µmol mol–1); FACE, leaves grown at elevated atmospheric CO2 concentration (580 µmol mol–1); T, till-
ering stage; H, heading stage; F, filling stage; F(1), early filling stage; F(2), late filling stage. Each value in this figure is the mean of 30 leaves in
three rings with the SE expressed as a bar. *P < 0.05 and **P < 0.01 for differences between FACE and ambient leaves.

gested that carbohydrate accumulation causes a decline in
photosynthesis by feedback inhibition and/or physical damage
at the chloroplast level. However, there is no convincing evi-
dence for a direct end-product limitation of photosynthesis
when sugars accumulate in leaves during prolonged exposure
to elevated CO2 (Stitt 1991). Moreover, photosynthetic accli-
mation is not always associated with increased total non-struc-
tural carbohydrate level when plants grow under elevated CO2
(Lee et al. 2001), so the explanation needs stronger evidence.
Stitt (1991) proposed that accumulating carbohydrate
could lead to a direct inhibition of photosynthesis, through
mechanical damage by large starch grains or inorganic phos-
phorus (Pi) limitation due to inhibition of sucrose synthesis, but
indirect or ‘adaptive’ regulation is probably more important,
involving decreases in the amounts of key photosynthetic
enzymes, including ribulose-1,5-bisphosphate caboxylase/
oxygenase (Rubisco). Photosynthetic acclimation to high CO2
has been attributed to decreased Rubisco concentration (Rogers
et al. 1996). Moore et al. (1999) considered the reduction in
Rubisco content as a central response of photosynthetic accli-
mation to elevated CO2, and presented a model describing the
molecular control of Rubisco accumulation, including the role
of hexose signal, hexokinase and Rubisco subunit mRNA
translation. However, there were reports that the tobacco and
rice plants with decreased Rubisco content due to an antisense
gene of the Rubisco small subunit (RbcS) could grow to a size
similar to those of the respective wild-type plants under ele-
vated CO2 conditions (Malse et al. 1993, Makino et al. 2000).
These facts seem to imply that decreased Rubisco does not
limit CO2-enriched photosynthesis.
Bowes (1993) predicted that a rise in atmospheric CO2
should exacerbate ribulose-1,5-bisphosphate (RuBP) limitation
unless Rubisco was down-regulated and/or RuBP regeneration
was up-regulated. There are some reports suggesting up-regula-
tion of RuBP and Pi regeneration capacity concomitant with
down-regulation of Rubisco (Ziska and Teramura 1992, Hus-
sain et al. 1999), but the up-regulation is not universal.
Whether the down-regulation of Rubisco and/or up-regulation
of RuBP regeneration are enough to avoid limitation of RuBP
regeneration to photosynthesis during photosynthetic accli-
mation to elevated CO2 is not clear at present (Bowes 1993,
Mitchell et al. 2000).

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 1038 Photosynthetic acclimation in rice to FACE
Fig. 2 Effects of FACE on WUE (upper) and Φc (lower) in rice leaves. The measurements were made at their growth CO2 concentrations. Val-
ues in this figure are the means of 30 (for WUE) or five leaves (for Φc), with the SE expressed as a bar.
Most of the previous studies on photosynthetic accli-
mation were carried out with plants grown in the greenhouse,
phytotron and open-top chambers where an increase in
atmospheric CO2 concentration was always accompanied by
significant alteration of microclimate conditions such as air
temperature, humidity and radiation (Kimball et al. 1997).
Thus, the results obtained in chamber-based facilities may be
different from those of field experiments to a certain extent.
Fortunately, the FACE technique developed in recent years
allows the study of high CO2 effects on plants grown in the
field without direct modification of the microclimate. Kimball
et al. (2002) have examined the responses of many agricultural
crops to FACE in detail, including increases in photosynthesis
and radiation use efficiency, but have not involved photo-
synthetic acclimation. The photosynthetic acclimation of rice
plants to FACE has been reported (Kim et al. 2001), and
Seneweera et al. (2002) have suggested that under FACE con-
ditions, photosynthetic acclimation in rice flag leaves is due to
a large demand for N at the reproductive stage.
In the present study, we attempted to explore the role of
RuBP carboxylation limitation and RuBP regeneration limita-
tion in photosynthetic acclimation to high CO2 through exam-
ining the changes in Rubisco, Rubisco activase, light-
harvesting complex II (LHC II) and Pi contents in order to
understand the mechanism of the acclimation.
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Results
Response and acclimation of photosynthesis in rice leaves to
FACE
When measured at their growth CO2 concentrations, the
leaf Pn of rice plants grown in FACE rings was significantly
higher than that of those grown in ambient rings at the tillering
and heading stages, but not at the filling stage (Fig. 1, upper).
In most cases, stomatal conductance (Gs) was lower in FACE
leaves than that in ambient leaves, but only the difference at the
tillering stage was significant (Fig. 1, middle). Although the
ratio of intercellular to air CO2 concentration (Ci/Ca) was
always higher in FACE leaves than that in ambient leaves, the
difference was not always remarkable (Fig. 1, lower). Water
use efficiency (WUE) and apparent quantum yield of carbon
assimilation (Φc) in FACE leaves were significantly higher
than those in ambient leaves in almost all cases (Fig. 2).
When measured at the same CO2 concentration of
580 µmol CO2 mol–1, the Pn in FACE leaves was significantly
decreased after heading (Fig. 3, upper), indicating that accli-
mation or down-regulation of photosynthesis occurs under
FACE conditions. Although Gs in FACE leaves decreased sig-
nificantly in most cases (Fig. 3, middle), no significant differ-
ence in Ci was observed between FACE and ambient leaves
(Fig. 3, lower), meaning that photosynthetic acclimation in
FACE leaves is not due to decreased Gs.
In consonance with decreased Pn, the CE (initial slope of
the Pn–Ci curve at low CO2 concentrations <380 µmol CO2
mol–1) of FACE leaves declined at the heading and filling
 Photosynthetic acclimation in rice to FACE 1039

Fig. 3 Pn (upper), Gs (middle), and Ci (lower) measured at the same CO2 concentration in rice leaves. Measurements were made at a CO2 con-
centration of 580 µmol mol–1 in 2002, 2003 and 2004, but 380 µmol mol–1 in 2001. Each value in this figure is the mean of 30 leaves in three
rings, with the SE expressed as a bar.

Fig. 4 Effects of FACE on CE in rice flag leaves. Each value in this figure is the mean of five leaves, with the SE expressed as a bar. H, heading
stage; F, filling stage.

stages (Fig. 4), implying that the capacity for RuBP car-
boxylation is decreased in FACE leaves. In order to explore the
relationship of the photosynthetic acclimation with RuBP car-
boxylation limitation and RuBP regeneration limitation, the
complete Pn–Ci curves were made (Fig. 5). From Fig. 5, it is
seen that the Pn–Ci curves for FACE and ambient leaves are
composed of three parts: (A) the oblique line at low CO2 con-
centration; (B) the horizontal at high or saturating CO2 concen-
tration; and (C) the curve at medium CO2 concentration
between A and B. Obviously, there are RuBP carboxylation
limitation, RuBP regeneration limitation and co-limitation of
them, respectively, in parts A, B and C. Under FACE condi-
tions, the RuBP carboxylation limitation still exists in FACE
leaves although it is unlikely to be a predominant limiting fac-
tor to photosynthesis.
Soluble protein, Rubisco and Rubisco activase
Similar to most of the previous experimental results under
elevated CO2 (Bowes 1993), the soluble protein content was
significantly decreased in FACE rice flag leaves (Fig. 6 upper).
Moreover, the contents of Rubisco and its activase, expressed
as a percentage of those in ambient leaves, were also remarka-

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 1040 Photosynthetic acclimation in rice to FACE

bly decreased at the heading and filling stages (Fig. 6 middle

and lower).

Sucrose, fructose and glucose contents and sucrose-phosphate

Fig. 5 The responses of light-saturated Pn to intercellular CO2 con-
centration (Ci) in the flag leaves of rice plants grown in ambient (open
circles) and FACE rings (filled circles). In the figure, A, B and C are
the RuBP carboxylation limitation region, RuBP regeneration limita-
tion region, and carboxylation and regeneration co-limitation region of
the Pn–Ci curve, respectively. Also shown are the operating points and
the supply functions (dotted line) of photosynthesis at growth CO2
concentration. Measurements were made at the heading and filling
stage at the Xiaoji site of Chinese rice FACE in 2004, and results
obtained at both stages were identical. The values shown were meas-
ured at the heading stage.
synthase activity
Compared with those in ambient leaves, the contents of
soluble sugars such as sucrose (Suc), fructose (Fru) and glu-
cose (Glc) in FACE leaves were significantly increased in most
cases (Fig. 7), similar to those in many other reports (Xu et al.
1994a, Curtis 1996). On the contrary, sucrose-phosphate
synthase (SPS) activity in FACE leaves was significantly
decreased (Fig. 8). Seneweera et al. (2002) reported that there
was no significant difference in SPS activity between FACE
and ambient rice leaves, but their results were expressed on the
basis of leaf N instead of leaf area. Actually, their SPS activity
in FACE leaves was also decreased when expressed in unit leaf
area because of the decrease in the leaf N of FACE leaves.
Pi, LHC II and chlorophyll contents
In order to reveal the relationship between the photo-
synthetic acclimation and RuBP regeneration, Pi and LHC II

Fig. 6 Effects of FACE on the soluble protein (upper), Rubisco (middle) and Rubisco activase (lower) contents in rice flag leaves. The amounts
of Rubisco in FACE leaves are expressed as a percentage of those of ambient leaves at the heading stage, while the amounts of Rubisco activase
in FACE leaves are expressed as a percentages of those of ambient leaves at each growth stage. Rubisco and its activase amounts were expressed
on a leaf area basis. Each value in this figure is the mean of eight leaves, with the SE expressed as a bar.

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 Photosynthetic acclimation in rice to FACE
Fig. 7 Effects of FACE on the Suc (upper), Fru (middle) and Glc
(lower) contents in rice flag leaves. Each value in this figure is the
mean of six leaves, with the SE expressed as a bar.
contents were measured at the early filling stage in 2003 and
2004 (LHC II only). Pi and LHC II contents were increased in
FACE leaves, compared with those in ambient leaves (Table 1).
An increase in LHC II content means that the LHC II /Rubisco
ratio was also increased because of the decrease in Rubisco
content. Contrary to the many results reported previously in
other plants, Chl contents in FACE leaves were significantly
increased, compared with those in ambient leaves (Fig. 9).
Discussion
Photosynthetic acclimation and root volume or N supply
Photosynthetic acclimation to elevated atmospheric CO2
concentration is not an inevitable phenomenon. Since most of
the phenomena are observed in chamber-based experiments
and potted plants where the root volume is limited, the limita-
tion of root growth and sink has been considered as one impor-
tant determinant of photosynthetic acclimation (Arp 1991).
However, in our FACE experiments, photosynthetic acclimation
of rice leaves still occurred under field conditions without root
growth limitation and with ample N supply (Fig. 3). Our results
indicate that the root volume and N supply level are not the
determinants for occurrence of photosynthetic acclimation.
Photosynthetic acclimation and decreased stomatal conductance
Stomata on the leaf surface, as the ultimate gates of CO2
entrance, exert a substantial effect on photosynthesis, and
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1041
Fig. 8 Effects of FACE on SPS activity in rice flag leaves. Each
value in this figure is the mean of eight leaves, with the SE expressed
as a bar.
photosynthetic acclimation is often accompanied by a decline
in Gs. However, the decreased conductance is not considered
an important cause of the acclimation, because the Ci/Ca ratios
are nearly identical for plants grown at elevated and normal Ca
(Xu et al. 1994a, Drake et al. 1997, Lee et al. 2001, Liao et al.
2003).
In the rice experiment reported here, although Gs in FACE
leaves was remarkably lower than that in ambient leaves in
most cases, no significant difference in Ci was observed
between FACE and ambient leaves when measured at the same
CO2 concentration (Fig. 3). Therefore, the partial closure of
stomata is not a reason for the photosynthetic acclimation to
FACE in rice leaves. Nevertheless, whether decreased Gs lim-
its photosynthesis is probably species-dependent. Chen et al.
(2002) observed that both Gs and Ci were significantly
decreased when Pn was markedly decreased in the leaves of
Echinochloa crusgalli (C4 plant) grown under FACE condi-
tions, implying that the partial closure of stomata is an impor-
tant factor leading to photosynthetic acclimation.
Photosynthetic acclimation and RuBP carboxylation limitation
Rubisco is widely accepted as the ultimate rate-limiting
step in photosynthetic carbon fixation at saturating light and
ambient CO2 concentration (Jensen 2000). Furthermore, its loss
or RuBP carboxylation limitation has been considered as the
main cause of the photosynthetic acclimation to elevated CO2
Table 1 Effects of FACE on Pi and LHC II contents in rice
flag leaves
Ambient FACE F/A (%)
–9 –2
Pi (10 mol cm ) 301 ± 20 378 ± 22 125**
LHC II (%) 100 ± 14 157 ± 24 157* (2003)
100 ± 2.0 112 ± 4.2 112* (2004)
The LHC II content in FACE leaves was expressed as a percentage of
that in ambient leaves (100%). Each value is the mean of eight leaves
with (±) SE, and the data for LHC II content are expressed on a leaf
area basis. The measurement of Pi content was made at the filling stage
in 2003, and the measurements of LHC II content were made at the fill-
ing stage in 2003 (Anzhen site) and 2004 (Xiaoji site). *P<0.05 and
**P<0.01 for differences between FACE and ambient leaves.
 1042 Photosynthetic acclimation in rice to FACE
Fig. 9 Effects of FACE on Chl content in rice flag leaves. Each value in this figure is the mean of five leaves, with the SE expressed as a bar.
concentration (Yelle et al. 1989, Besford et al. 1990, Bowes
1991, Stitt 1991, Rogers et al. 1996, Moore et al. 1999, Rogers
and Humphries 2000). In our rice FACE experiments, RuBP
carboxylation limitation may be a cause of the photosynthetic
acclimation, as shown by decreased CE, Rubisco and its acti-
vase contents (Fig. 4, 6), but not the main cause, as shown by
the Pn–Ci curves (Fig. 5, part C). Under FACE conditions,
photosynthesis is in a way transferring RuBP carboxylation
limitation into RuBP regeneration limitation. In other words,
photosynthesis in FACE rice leaves is being limited by both
RuBP carboxylation and RuBP regeneration. Although the con-
siderable decreases in Rubisco content did not lead to signifi-
cant decreases in biomass of transgenic tobacco and rice plants
(Malse et al. 1993, Makino et al. 2000), it cannot be concluded
that Rubisco never limits CO2-enriched photosynthesis. It
should be pointed out that their experiments were carried out
under CO2-saturated conditions (930 µbar and 100 pa, respec-
tively), while our experiments were conducted under CO2-non-
saturated conditions (580 µmol mol–1). Therefore, only under
CO2-saturated conditions may it be safely said that Rubisco
does not limit CO2-enriched photosynthesis.
The decrease in Rubisco and its activase contents may be
attributed to more accumulation of soluble sugars such as Suc,
Fru and Glc in FACE leaves (Fig. 7). It has been proposed that
increased hexose levels lead, via hexokinase-related signaling,
to repression of expression of RbcS and other genes, resulting
in the decreases of Rubisco and other proteins (Jang and Sheen
1994, van Oosten et al. 1994). The fact that Rubisco content
was decreased much more when additional Glc was present in
the culture at elevated CO2 concentration (de la Vina et al.
1999) strongly supports this proposition.
Photosynthetic acclimation and RuBP regeneration limitation
The fact that the stimulatory effect of elevated CO2 con-
centration on photosynthesis could not persist in the long term,
and disappeared finally at the late filling stage (Fig. 1), indi-
cates that besides RuBP carboxylation limitation, RuBP regen-
eration limitation exists in FACE leaves. Furthermore,
compared with that in ambient leaves, the substantially
declined Pn in part B of the Pn–Ci curve (Fig. 5) shows a pre-
dominant limitation of RuBP regeneration to photosynthesis in
FACE rice flag leaves. At high CO2 and high light, a limited
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capacity for triose phosphate use in starch and/or sucrose syn-
thesis may result in a shortage of both Pi and ATP (Bowes
1993). In our study, the RuBP regeneration limitation is proba-
bly not due to Pi shortage because Pi content is increased in
FACE leaves (Table 1).
Under CO2 enrichment conditions, the up-regulation of
RuBP regeneration predicted by Bowes (1993) has been
reported, for example, an increased SPS activity (Hussain et al.
1999) and an increased LHC II/Rubisco ratio (Adam et al.
2000). The significant increases in the Chl content and LHC II
/Rubisco ratio in FACE leaves observed by us (Fig. 9, Table 1)
may be considered as an indication of the up-regulation of
RuBP regeneration. Thus, RuBP regeneration in FACE
leaves may be up-regulated through more light absorption by
increased Chl and LHC II. Nevertheless, if the decrease in
photosynthetic electron transport capacity is due to a decrease
in the content of cytochrome f and/or the number of plastocy-
anins, the two key components of the photosynthetic electron
transport chain, the increased Chl content and LHC II/Rubisco
ratio cannot resolve the problem of RuBP regeneration limita-
tion in FACE leaves. There have been some reports showing
that the plastocyanin number is correlated with the light-
saturated photosynthetic electron transport rate in tobacco
thylakoids (Schöttler et al. 2004), and the gene encoding plas-
tocynin is repressed in the high-sugar state (Oswald et al.
2001). Also the cytochrome f content is highly correlated with
CO2-saturated photosynthesis (Sudo et al. 2003). Whether
plastocyanin and/or cytochrome f contents are decreased in
FACE leaves will be worth studying.
Conclusion
Our results demonstrate that under FACE conditions with-
out both root volume limitation and N supply limitation, photo-
synthetic acclimation in rice leaves occurs, and it is related to
both RuBP caboxylation limitation and RuBP regeneration
limitation.
Materials and Methods
FACE site and rice growth
The Chinese rice FACE facilities were located at Anzhen village
(120°27′ 51′′ E, 31°37′ 24′′ N), Wuxi city in 2001–2003 and were
 Photosynthetic acclimation in rice to FACE
transferred to Xiaoji village (119° 42′ 0′′ E, 32°35′ 5′′ N), Yangzhou
city in 2004, in Jiangsu Province, East China. Both sites are in a typi-
cal region for rice production in China. The running and controlling
systems of the facilities were transferred from a Japanese rice FACE
site (Okada et al. 2001). A full description of Chinese rice FACE facil-
ities has been provided by Liu et al. (2002). Briefly, in the experimen-
tal field, there were eight (Anzhen site, but six in the Xiaoji site) rings
of 12 m diameter. Among them, three rings were sprayed with pure
CO2 as FACE treatment, and the others were in the normal atmos-
phere as ambient control. The distances between FACE and ambient
rings were >90 m. Target CO2 concentration in the center of FACE
rings was 200 µmol mol–1 above the ambient air CO2 concentration.
CO2 enrichment of rice plants in FACE rings was commenced immedi-
ately after transplanting, and applied continuously day and night until
harvesting.
Rice cultivar Japonica 9915 used in this study is a new cultivar
planted commonly in this region. Its growth duration (from transplant-
ing to harvesting) is about 130 d (from mid June to mid October). Its
cultivation was performed with typical agronomic management tech-
niques for this region. Seeds of Japonica 9915 were germinated in a
seedbed without a layer of water in ambient air, and the seedlings were
transplanted into the plots of the experimental field on June 13. The
planting density was 17×25 cm. N was supplied as urea (NH2CONH2)
(85%) and (NH4)2HPO4 (15%) at 250 kg N ha–1 (normal N supply for
local rice fields), with 40% of N supplied as a basal dressing, 20% on
the fifth day after transplanting and 40% at the panicle initiation stage.
Phosphorus was applied at 75 kg P2O5–ha–1. The soil was flooded
before transplanting, and the water layer of 5 cm above soil level was
maintained except when the field was drained several times.
Gas exchange measurement
Gas exchange measurements were made in situ using a portable
gas analysis system LI-COR 6400 (LI-COR Inc. Lincoln, NE, USA)
with 10–12 fully expanded leaves in each ring between 10 : 00 and
14 : 30 h (Beijing time) in early July (tillering stage), mid August
(heading stage), early September (early filling stage) and early October
(late filling stage). These measurements were performed alternately in
FACE and ambient rings. At the tillering stage, the second fully
expanded leaves counted from the top of plant were used, while the
fully expanded flag leaves were used at other stages. The Pn of leaf
was measured at 580 µmol CO2 mol–1 in FACE rings, and at 580 and
380 µmol CO2 mol–1 in ambient rings. During the measurements, CO2
concentration was controlled with the Li-Cor CO2 injection system,
and a saturating photosynthetic photon flux density (PPFD) of
1,200 µmol m–2 s–1 from a Li-Cor LED light source was supplied. Air
temperature of the leaf chamber was maintained at about 30°C. Before
recording data, the measured leaves were kept in the leaf chamber for
2 min to reach a steady state of photosynthesis. Then, some of these
leaves were used to measure Φc (Xu et al. 1987) and CE (Farquhar et
al. 1980, von Caemmerer and Farquhar 1981). During Φc measure-
ment, CO2 concentrations were kept at 580 and 380 µmol mol–1,
respectively, for FACE and ambient leaves, and the PPFD was set at
160, 135, 110, 85, 60 and 35 µmol m–2 s–1 in turn. For CE measure-
ment, the PPFD was kept at 1,200 µmol m–2 s–1, and the CO2 concen-
tration controlled with LI-COR CO2 injection system was set at 250,
200, 150, 100, 50 and 25 µmol mol–1 in turn. To produce the Pn–Ci
curve, the CO2 concentration was set at 580 (for FACE leaves) or 380
(for ambient leaves), 250, 200, 150, 100, 50, 350, 450, 550, 650 and
750 µmol mol–1 in turn, and the PPFD was kept at 1200 µmol m–2 s–1.
Leaf samples
For biochemical analysis, all leaf samples were collected
between 10: 00 and 13: 00 h in the light. The detached leaf samples
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1043
were excised to a 5 cm long segment (excluding the tip and base) and
their areas were measured with a portable area meter LI-3000A (Li-
Cor Inc, Lincoln, NE, USA). Then, the leaf segments were immedi-
ately dropped into liquid N2, taken back to the laboratory with dry ice,
and preserved at –80 °C until biochemical analysis.
Enzyme content and activity assay
Each eight-leaf segments from one FACE ring and one ambient
ring were separately ground to a fine powder in liquid N2. The soluble
protein was extracted from the fine powder with an extraction buffer
containing Tris–HCl (100 mM, pH 7.6), EDTA (5 mM), insoluble
polyvinylpyrrolidone (1%), NaCl (50 mM) and β-mercaptoethanol
(0.2%). The extract was centrifuged at 15,000×g at 4°C for 10 min,
and the soluble protein content of the obtained supernatant was deter-
mined according to Bradford (1976).
For analysis of Rubisco and its activase contents, about 50–
100 ng of protein of the supernatant was immobilized on a 96-well cell
culture cluster for 2 h, then the immobilized mixture was removed, and
the cell culture cluster was incubated with pre-hybrid buffer contain-
ing bovine serum albumin (BSA, 2%). The contents were measured
with a protein detector enzyme-linked immunosorbent assay (ELISA)
kit, ABTS system (KPL Inc., Gaithersburg, MD, USA; Fleming and
Pen 1988). The procedure to quantify the membrane protein LHC II
was the same as that for Rubisco and its activase, but their extraction
procedure was different: the extraction buffer mentioned above con-
tained SDS (1%) and the crude extract was incubated at 100°C for
5 min before centrifugation.
Antibodies of Rubisco, Rubisco activase and LHC II were pre-
pared from the antisera of rabbits immunized respectively with the
purified Rubisco, Rubisco activase of tobacco leaves, and soybean
LHC II b subunit expressed in Escherichia coli in our laboratory. The
working curve for Rubisco quantification was prepared with purified
tobacco Rubisco, and the purification of Rubisco from tobacco leaf
was performed according to the method described by Li (1997). The
working curves for the quantification of Rubisco activase and LHC II
were prepared with the crude extracts of rice leaves from the ambient
ring. The amounts of Rubisco at both heading and filling stage were
expressed as a percentages of those of ambient leaves at the heading
stage, while Rubisco activase and LHC II in FACE leaves were
expressed as the percentages of those in ambient leaves at each growth
stage. All relative amounts here were on the leaf area basis.
SPS activity measurement was based on the method described by
Seneweera et al. (1995) with some modifications. Namely, 0.05 ml of
the soluble protein extract supernatant mentioned above was added to
0.15 ml of assay buffer including Tris–HCl (50 mM, pH 7.0), MgCl2
(10 mM), Fru-6-phosphate (10 mM) and UDP-Glc (3 mM), and the
mixture was incubated at 30°C for 10 min. Then, 0.05 ml of NaOH
(2 M) was added, and incubated at 100°C for 10 min. After cooling
with flowing water, 0.7 ml of 30% HCl and 0.2 ml of 0.1% resorcinol
(w/v, dissolved in 95% ethanol) were added, then the mixture was
incubated at 80°C for 10 min. Finally, the optical density of this mix-
ture was measured at 480 nm after cooling. For blanks, the first incu-
bation was performed on ice.
Pi and soluble sugar content measurement
For soluble sugar and Pi measurement, the leaf segments col-
lected from the experimental plots were baked at 80°C and ground into
a fine powder, then preserved in a vacuum at 4°C.
Pi was extracted from this fine powder with boiling water for
30 min. Then, the extract was centrifuged. The residue was re-
extracted twice. Pi extraction and measurement were performed
according to Fiske and Subbarrow (1925).
 1044 Photosynthetic acclimation in rice to FACE
The soluble sugars were extracted from the fine powder with
95% ethanol, and the extract was centrifuged. The residue obtained
after centrifugation was re-extracted with 95% ethanol twice. The
supernatants were combined. Suc, Glc and Fru contents were meas-
ured according to Cardini et al. (1955).
Chl content measurement
Chl was extracted separately from 3–4 fresh leaves in each ring
with 80% acetone and determined according to Arnon (1949).
Statistical analyses
Statistical analysis of all data, including mean, SE and t-tests,
was carried out using Sigma Plot 4.0 software (SPSS Inc., Chicago, IL,
USA).
Acknowledgments
The Chinese Rice/Wheat FACE Project was a research program
involved in the China–Japan Science and Technology Cooperation
Agreement. The main instruments and apparatus of the system were
supplied by Japan National Institute for Agro-Environmental Sciences
(NIAES) and Japan Agricultural Research Center for Tohoku Region
(NARCT). The project was funded by the Chinese Academy of Sci-
ences (CAS, KZCX2-408 and KSCX2-SW-133), National Natural Sci-
ence Foundation of China (NSFC, 40231003 and 40120140817),
Ministry of Science and Technology of China (2002cb71403), and
State Key Laboratory of Soil and Sustainable Agriculture, Institute of
Soil Science, Chinese Academy of Sciences (035104).
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