全 文 ：Received 27 May 2003 Accepted 21 Jul. 2003
Supported by the State Key Basic Research and Development Plan of China (G1998010100) and the National Natural Science Foundation of
China (39870533, 30270794).
* Author for correspondence. E-mail: .
Abbreviations: CA, carbonic anhydrase; Fv/Fm , PSⅡelectron transport efficiency; MA, malate; NADP-MDH, NADP-malate dehydrogenase;
NADP-ME, NADP-malic enzyme; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; PK, pyruvate,
orthophosphate dikinase; PFD, photon flux density; Pn , net photosynthetic CO2 uptake rate; qN, non-photochemical quenching; qP ,
photochemical quenching; RuBPC, ribulose-1,5-biphosphate carboxylase; WT, wild type.
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (5): 542-551
A Limited Photosynthetic C4-Microcycle and Its Physiological Function
in Transgenic Rice Plant Expressing the Maize PEPC Gene
JI Ben-Hua1, 2, ZHU Su-Qin1, JIAO De-Mao2*
(1. Department of Life Sciences and Technology, Nantong Normal College, Nantong 226007, China;
2. Institute of Agrobiological Genetics and Physiology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China)
Abstract : Photosynthetic C4-microcycle and its function in different genotype rices were explored
comparatively using PEPC transgenic rice and homozygous wild genotype (WT) rice (Oryza sativa L.
subsp. japonica Kitaake) as experimental material. In untransformed WT, there existed an intact C4
photosynthetic enzyme system detected by the activities of enzymes of photosynthetic C4 pathway, i.e.
phosphoenolpyruvate carboxylase (PEPC), NADP+-malic enzyme (NADP+-ME), NADP+-malate dehydrogenase
(NADP+-MDH), pyruvate orthophosphate dikinase (PPDK), and indicating that there is a primitive photosynthetic
C4-pathway with increased photosynthetic rate in leaf discs or chloroplasts fed with exogenous oxaloacetate
(OAA) or malate (MA). Furthermore, photosynthetic C4 microcycle was promoted in a great range in transgenic
rice introduced a maize specific PEPC gene. Enhancement of photosynthetic C4-microcycle further
played some role in raising the net photosynthetic rates (Pn) and debasing the ratio of Pr/Pn by comparing
the CO2 gas exchange rates in different genotype rices, WT rice and PEPC transgenic rice. Analyzing the
chlorophyll a fluorescence characteristics showed that increase of photosynthetic C4-microcycle companied
with the raising PSⅡmaximum photochemical efficiency (Fv/Fm) and photochemical quenching (qP), and
the lowering of non-photochemical quenching (qN). These results will provide scientific evidence for
genetic breeding to improve photosynthetic efficiency in crops by gene engineering.
Key words: transgenic rice; photosynthet ic C4-microcycle; chlorophyll a fluorescence; phospho-
enolpyruvate carboxylase (PEPC); C4-bicarboxylate
In the last decade, more attention has been paid to the
introduct ion of C4 photosynthetic gene in to C3 plan ts to
raise their photosynthetic capacity (Matsuoka et al., 2001).
Due to the development of recombinant DNA technology,
PEPC- (Ku et al., 1999), PPDK- (Fukayama et al., 1999),
NADP+-ME- (Tsuchida et al., 2001), and PEPC+PPDK- (Ku
et al., 2000) transgen ic rice p lants have been ob tained.
Relative to unt ransformed wild type (W T) rice, PEPC
transgenic rice exhibits higher photosynthetic rate and char-
acteristics of tolerance to pho to-oxidation under strong
light and higher temperature (Jiao et al., 2001). This raised
some con jecture about it s mechanism. For example, rice
plants obtained exogenous PEPC gene from C4 maize plant
enhance photosynthetic capacity either by increase of sto-
matal conductance in one opinion (Ku et al., 2001) o r by
raising a CO2-concentrated capacity in photosynthetic cell
in another opinion (Huang et al., 2002). Recent experimental
result (Jiao et al., 2003) showed that there are no relation-
ships between the increase of pho tosynthetic rates and
the enhancement of stomatal conductance of leaves in PEPC
transgenic rice. Chen et a l. (2001) had validated the en-
hancement of photosynthetic capacity by feeding heter-
ogenous C4 bicarboxylate into spinach leaves and made
sure that there was photosynthetic C4-microcycle pathway
in leaves of C3-plant. On the basis of former research work,
whether photosynthetic C4-microcycle pathway existed in
C3-rice plants and its physiological function behaved were
explored comparat ively us ing untransformed WT rice
Kitaake and PEPC t ransgen ic rice as research material in
the present study, by determining the photosynthetic C4
pathway related key enzymes , analyzing the changes of
fluorescence characteristics o f chlorophyll in leaves after
being with C4-bicarboxylate such as OAA or MA, as to
offer the scient ific ev idence for theoretical research and
JI Ben-Hua et al.: A Limited Photosynthetic C4-Microcycle and Its Physiological Function in Transgenic Rice Plant Express-
ing the Maize PEPC Gene 543
practical application to the enhancement of C4-photosyn-
thetic characteristics in C3-plant.
1 Materials and Methods
1.1 Plant materials
Untrans formed W T rice (Oryza sat iva L. subsp .
japonica Kitaake) and PEPC transgenic rice were used in
this s tudy. Homozygous transgenic rice over-expressing
the maize s pecific PEPC was the 7th stable generation
germplasm detected and derived by Jiao et al. (2000) from
the 3rd generation transgenic germplasm which was trans-
fo rmed with WT Kitaake as gene receptor by Ku et a l.
(1999). The seeds were germinated directly in soil and plants
were grown in 4-L pots and maintained in a naturally illumi-
nated out-door net room at Jiangsu Academy of Agricul-
tural Sciences (Nanjing). There were five hills per pot and
one s eedling for each h ill. The whole growth duration of
the transgenic rice sown in the first ten-day period of May
was about 90 d. Average temperature varied from 21 ℃ to
27 ℃ and the diurnal temperatu re difference was from 7.1
℃ to 8.7 ℃. Chemical fertilizer was applied at the tillering
and booting stages as a combination of 2.0 g N, P2O5, and
K2O per pot as basal dressing, and 0.5 g N and K2O per pot
as top dressing. The maximum photosynthetically active
radiation (PAR) at noon on a sunny day in Nanjing during
July-August was approximately 1 800 mmol .m-2.s-1. Rice
shoot was used in the assay of photos ynthetic oxygen-
evolution of chloroplasts and leaf discs, CO2 exchanging
rates and chlorophyll fluorescence parameters of rice leaves.
1.2 Photoinhibitory treatment
During ear stage detached functional leaves were put in
leaf chambers illuminated by a white light (quartz halogen
lamp) which passed a layer of 12 cm water as a heat filter for
3 h at 30 ℃. There were two groups replicated five times.
The first group (moderate conditions) was illuminated un-
der a PFD of 350 mmol .m-2.s-1 and flushed with 21% O2
and 350 mL/L CO2 (relative humidity of 78%-82%). The
second group (photoinhibitory conditions) was illuminated
under a PFD of 1 000 mmol .m-2.s-1 and flushed with 2% O2
and 60 mL/L CO2 (relative humidity of 78%-82%). Then,
the rice leaves were used for obtaining crude enzyme prepa-
ration and measurement of enzyme activities.
1.3 Assay method
1.3.1 Preparation of intact chloroplast in vitro and deter-
mination of photosynthetic oxygen-evolution rates Ma-
ture leaves of rice were harvested from each p lant in the
light and quickly ground in 3-fold volume and pre-cooled
buffer A (330 mmol/L of sorbito l, 50 mmol/L o f MES, 2
mmol/L of EDTA, 2 mmol/L of MgCl2, 2 mmol/L of ascorbic
sodium, pH 6.1). The homogenate was filtered through 8-
layer of gauze cloth, and the filtrate was centrifuged for 30
s at 2 500 g and 4 ℃. Supernatant was abandoned and
chloroplast debris was scoured out. Intact chloroplast was
co llected and re-sus pended in buffer B (330 mmol/L of
sorbitol, 50 mmol/L of Hepes, 2 mmol/L of EDTA, 2 mmol/L
of MgCl2, pH 6.7), with chlo rophyll concent rat ion of 2
mg/mL and kept for next experiment. More than 70% of
intact chlo roplasts was obtained and identified by Clark
electrode accord ing to the method of Ye and Mi (1999).
Such intact chloroplasts were suspended in buffer C (330
mmol/L of sorbitol, 50 mmol/L of Hepes, 2 mmol/L of EDTA,
2 mmol/L of MgCl2, pＨ 7.5 ) containing 0.4 mmol/L of
NaHCO3. Then photosynthetic oxygen-evolution rates of
chloroplas ts were determined in the presence of different
concentrations of OAA, MA or PEP (Sigma Company) by
Oxylab system (liquid phase, Hansatech UK).
1.3.2 Determination of photosynthetic oxygen-evolution
rates of leaf discs Leaf discs of about 1.5 mm× 1.5 mm
were obtained from the leaves of rice grown in outdoor net
room, and submerged in to STN buffer (400 mmol/L of
sucrose, 50 mmol/L of Tris-HCl, pH 7.5, 10 mmol/L of NaCl)
containing 0.4 mmol/L of NaHCO3. Photosynthetic oxygen-
evolution rates of leaf discs were determined with different
concentrations of OAA, PEP or MA (Sigma Company) by
Oxylab system (liquid phase, Hansatech, UK).
1.3 .3 Determination of CO2 exchanging rates of rice
leaves Rice leaves were cut and immediately inserted into
OAA or MA solution (200 mmol/L), respectively, as treat-
ment group, and other rice leaves were inserted into dis-
tilled water, as control group, under the photons flux den-
sity (PFD) of 1 000 mmol .m-2.s-1 for 30 min. Then net
photosynthetic rates (Pn) of rice leaves were measured by
a portable photosynthetic gas analysis system（model
TPS-1, PP Systems Company, UK）under 350 mmol/L of
CO2 and a PFD of 1 000 mmol .m-2.s-1. Photorespirat ion
rates (Pr) were calculated according to the following formula:
Pr = Pn(2%) - Pn (21%). Where Pn (2%) and Pn (21%) are the net
photosynthetic rates under 2% O2 and 21% O2, respectively.
1.3.4 Preparation of crude enzyme and measurement of
enzyme activities About 1 g leaf tissue was collected from
photo inh ibit ion-treated leaves in the light and qu ickly
ground in 6 mL buffer containing 50 mmol/L of Tris-HCl, pH
7.5, 10 mmol/L of MgCl2, 5 mmol/L of dithiothreitol (DTT),
2% (W/V) insoluble polyvinylpolypyrrolidone (PVP) and
10% glycero l. After mixing the filtrate thoroughly, 40 mL
aliquots were used for determination of chlorophyll
contents , the crude extract was centrifuged at 13 000g for
10 min at 4 ℃ and the supernatant was used immediately
Acta Botanica Sinica 植物学报 Vol.46 No.5 2004544
for assay of various C4 enzymes.
PEPC activity was ass ayed spectrophotometrically at
room temperature in a mixtu re con tain ing 50 mmol/L of
Hepes-KOH (pH 8.0), 10 mmol/L of NaHCO3, 5 mmol/L of
MgCl2, 1.5 units NAD+-malate dehydrogenase (MDH), 0.2
mmol/L of NADH and 20-50 mL enzyme extract according
to the method of Gonzeles et al. (1984) and Ku et al. (1999).
The reaction was started by adding PEP to a final concen-
tration of 2 mmol/L. The change in NADH absorbance was
monitored at 340 nm.
PPDK activity was assayed by the method of Hatch and
Slack (1975). The ass ay medium contained 25 mmol/L of
Hepes-KOH (pH 8.0), 8 mmol/L of MgSO4, 10 mmol/L of
DTT, 10 mmol/L of NaHCO3, 2 mmol/L of sodium pyruvate,
5 mmol/L of (NH4)2SO4, 1 mmol/L of glucose-6-phosphate,
2.5 mmol/L of K2HPO4, 0.2 mmol/L o f NADH, 0.5 units
PEPC and 2 units NAD-MDH. The reaction was started by
adding 20 mL 50 mmol/L of ATP into 1 mL assay medium to
give a final concent ration o f 1 mmol/L. The reaction was
performed at 30 ℃.
NADP+-ME activity was assayed as described by Chen
et al. (1981) and the assay medium contained 50 mmol/L of
Tris-HCl (pH 8.0), 1 mmol/L of MgCl2, 1 mmol/L of MnCl2,
1 mmol/L of EDTA, and 0.33 mmol/L of NADP+. The reac-
tion was started by adding 50 mL of 100 mmol/L of malate to
give a final concent ration o f 5 mmol/L. The reaction was
performed at 30 ℃ and the reduction of NADP+ was moni-
tored by absorbance at 340 nm.
NADP+-MDH activity was determined by the method
of Li et a l. (1987). For determining the maximal potent ial
activity of NADP+-MDH, the crude enzyme preparation was
supplemented with 0.1 mmol/L of DTT and 0.2 mol/L of
MgCl2 and incubated at 30 ℃ for 1.5 h prior to assay. The
assay medium contained 100 mmol/L of Tris-HCl (pH 7.5),
1 mmol/L of EDTA, and 0.18 mmol/L of NADPH. Reaction
was started by adding oxaloacetic acid to a final concentra-
tion of 0.5 mmol/L. The reaction was performed at 30 ℃ and
the oxidation of NADPH was monitored by the absorbance
at 340 nm.
RuBPC was prepared and its activ ity was assayed ac-
cording to Wei et a l. (1994) and Kung et a l. (1980).
Photo inhibition-treated leaves (0.5 g) were cut into small
pieces, and immediately homogenized in a prechilled pestle
and mortar with acid-washed quartz sand in 2.5 mL extrac-
tion medium containing 50 mmol/L of Tris-HCl (pH 7.5), 1
mmol/L of MgCl2, 5 mmol/L of DDT, and 2% insoluble PVP.
After mixing the filtrate thoroughly, 40 mL aliquots were
used for determination of chlorophyll contents. The homo-
genate was clarified by centrifugation at 10 000g for 10 min;
the clear supernatant was used for enzyme activity assay.
All these steps were carried out at 4 ℃.
Carbonic anhydrase (CA) activ ity was determined by
the method of Guo et al. (1988). About 5 g leaf tissue was
quickly ground in 30 mL extraction medium (5 mmol/L of
DTT, 10 mmol/L of barbital buffer, pH 8.2) in a p re-chilled
mortar and pestle, and the homogenate was filtered through
eight layers of gauze cloth and centrifuged at 5 000g fo r 5
min. The supernatant was used for CA ass ay in a vessel
after injected with pure CO2 for a few minutes. The activity
of CA was calculated based on the rate of change in pH in
the assay medium.
Enzyme activities were expressed on chlorophyll basis.
Chlorophyll concentration was determined after extraction
with 96% ethanol.
1.3.5 Measurement of chlorophyll fluorescence param-
eters of rice leaves Chlorophyll fluorescence parameters
of rice leaves were determined by PAM-2000 fluorescence
meter with data collection soft DA-2000 at 30 ℃ after dark
adaptation for 10 min. The fluorescence kinetics param-
eters were measured by the method of Schreiber et al. (1986)
and Genty et a l. (1989) and calculated by the fo llowing
formula: PSⅡ photochemical efficiency Fv /Fm = (Fm–
Fo)/Fm; photochemical quenching qP = (Fm＇- Fs)/(Fm＇-
Fo); non-photochemical quenching qN = (Fm – Fm＇)/( Fm
-Fo).
2 Results
2.1 Changes in the activities of photosynthetic enzymes
in the leaves of PEPC transgenic rice and wild type (WT)
rice under photoinhibitory conditions
Only phosphoenolpyruvate carboxylase (PEPC) activ-
ity was obviously higher in PEPC transgenic rice than in
WT rice, the activities of other C4-enzyme, pyruvate phos-
phate dikinase (PPDK), NADP+-ME, NADP+-MDH, CA and
C3 photosynthetic enzyme, RuBPC, in PEPC transgenic rice
were the same as in WT rice under moderate conditions (a
PFD of 350 mmol .m-2.s-1, 21% O2 and 350 mL/L CO2, at 30
℃) for 3 h (Fig.1A, B). However, under photoinhibitory
conditions (a PFD of 1 000 mmol .m-2.s-1, 2% O2 and 60
mL/L CO2 at 30 ℃) for 3 h, all photosyntheticＣ 4-enzyme
activities increased obviously . For example, the activities
of PEPC, CA, NADP+-MDH, NADP+-ME and PPDK in-
creased by 113.7%, 178.9%, 123.7%, 135.6% and 115.0%
(Fig .1A, B), res pect ively, in W T rice, and by 364.7%,
342.3%, 304.1%, 294.2% and 398.1%, respectively, in PEPC
trans genic rice. While the activity of pho tosynthet ic C3 -
enzyme, RuBPC, did not change obviously (increased by
about 4%), and exhibited no differences between the two




JI Ben-Hua et al.: A Limited Photosynthetic C4-Microcycle and Its Physiological Function in Transgenic Rice Plant Express-
ing the Maize PEPC Gene 549
mmol/L, then it increases slowly and keeps in a higher and
stable level as the concentrations of OAA or MA are be-
tween 30 and 200 mmol/L (Fig.2). This indicated that C3 rice
plant leaves possess photosynthetic C4 poly-enzyme sys-
tem (Fig.1) to catalyze the production of C4 acids and higher
concentration of OAA or MA may accelerate the operation
of C4 cycle, and photosynthetic capacity may further rise
by raising the activit ies of PEPC and catalyzing more C4
acids formed. Fifty percent and 100% increment of photo-
synthetic capacity in leaf discs or chloroplasts in WT rice
Kitaake and PEPC transgenic rice, respectively, after feed-
ing with OAA or MA (30-200 mmol/L), indicate that PEPC
is the velocity-limited enzyme of C4 micro-cycle in C3 rice
plant, and photosynthetic potential may further increase if
PEPC activity can be improved by DNA recombinant tech-
nology made in existent high-yield rice cultivars.
There is some argumentation about the role of extrane-
ous PEPC gene in PEPC transgenic rice at present, for dif-
feren t res u lt s were go t under varied experimental
conditions. For example, Japanese scientists have not ob-
served the improvement of photosynthetic capacity when
PEPC transgenic rice was grown indoors under moderate
ligh t intensity (Osaki et a l., 2000). In con tras t, PEPC
transgenic rice cultivated under high light intensity and
high temperature conditions of summer in Nanjing, China,
exhibited the characteristics of enhanced photosynthetic
efficiency and alleviated pho toinhibition/photooxidation,
and its grain y ield per plan t increased by 10%-30% as
compared with WT rice Kitaake (Jiao et al., 2001). Results
in this investigation show that additional primary photo-
synthetic C4-products such as OAA or MA may improve
the photosynthetic trait s of PEPC transgenic rice, by de-
creas ing photoresp iration, raising net photosynthet ic
capacity, and thereby, raising photosynthetic efficiency
(Fig.3). Increase of CO2-assimilation capacity needs more
light energy consumed and alleviates the photo-inhibitive
damage, namely, more light energy abs orbed is used in
photochemical react ion. As a result, qN, which d issipates
exces sive light energy, decreases, and PSⅡ Fv/Fm and
qP increase correspondingly (Fig.4). Above biochemical
and biophysics process may be the physiological base of
enhancing pho tos ynthetic efficiency, higher yield and
stress-resistance in PEPC transgenic rice.
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