全 文 ：Received 22 Oct. 2003 Accepted 15 Jan. 2004
Supported by the State Key Basic Research and Development Plan of China (G1998010100).
* Current address: Research Center of Freshwater Fishery, National Institutes of Aquatic Products Research, Wuxi 214081, China.
** Author for correspondence. Tel: +86 (0)10 82610114; E-mail:.
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (7): 873-882
Photosynthetic Features of Transgenic Rice Expressing Sorghum
C4 Type NADP-ME
CHI Wei, ZHOU Jing-Song*, ZHANG Fang, WU Nai-Hu**
(Institute of Genetics and Developmental Biology, The Chinese Academy of Sciences, Beijing 100080, China)
Abstract: The gene encoding sorghum NADP malic enzyme, which plays a key role in C4 photosynthetic
pathway, was isolated by RT-PCR and cDNA library screening. The 2 139 bp cDNA sequence obtained
includes a 1 911-bp open reading frame that encodes 636 amino acids and a terminating codon (GenBank
accession number: AY274836). It was then introduced into Nongken 58, a rice variety, using an Agrobacterium-
mediated system. Southern hybridization, Northern hybridization and enzyme activity determination all
confirmed the effective expression of sorghum (Sorghum vulgare L.) C4 type NADP-ME in rice, with the
enzyme activity being elevated 1–7 folds. However, no appreciable change was demonstrated in carbon
assimilation of the transgenic rice though increased photoinhibition was noted under high light intensity.
Key words: NADP malic enzyme (NADP-ME); sorghum; transgenic rice; physiology of photosynthesis
According to differences in carbon assimilation path-
ways in photosynthesis, three photosynthetic types may
be distinguished among land plants: C3, C4, and CAM.
Compared with C3 plants, the unique Kranz structure and
C4 pathway endow C4 plants and CAM plants with higher
efficiency in photosynthesis, water utilization and nutrient
utilization (Hatch, 1987). These advantages of C4 plants are
especially valuable during stresses caused by high
temperature, high light intensity and drought. It has there-
fore long been a much sought for objective to improve the
photosynthetic properties of C3 plants by introducing the
photosynthetic traits of C4 plants (CAM plants) into them.
NADP-ME, a key enzyme in the C4 pathway in the NADP-
ME subtype of C4 plants, was located in the chloroplasts
of the bundle sheath cells. In the C4 pathway, ambient CO2
is first fixed by PEPCase in the form of malic acid in meso-
phyll cells of leaves, which is then transported to bundle
sheath cells where it is decarboxylated by NADP-ME, re-
leasing CO2 again. The released CO2 is further fixed by
RuBisco into the C3 pathway. Evidently, it is the decar-
boxylation of NADP-ME that helps enrich the CO2 immers-
ing RuBisco, thus lowering photorespiration and improv-
ing the photosynthetic efficiency. As pointed out by Ku et
al. (1991), the activity of NAPD-ME shows negative corre-
lation with the activity of photorespiration. It thus appears
that transfer of NADP-ME gene into C3 plants might be an
effective way of lowering photorespiration and improving
photosynthetic efficiency of C3 plants.
There are many reports on the successful transfer of
photosynthetic enzymes of C4 plants into C3 plants and
their high level expression in the latter by means of gene
engineering techniques (Ku et al., 1999, Takeuchi et al.,
2000, Furayama et al., 2001, Zhang et al., 2003). Yet incon-
sistent results have been obtained in studies aimed at elu-
cidating the underlying physiological mechanisms.
Recently, Takeuchi et al. (2000) tried to account for some of
the physiological characteristics of transgenic rice express-
ing high level maize NADP-ME in terms of chloroplast de-
velopment (Takeuchi et al., 2000). So far, however, no re-
port has been published on systematic study of the photo-
synthetic physiology such as CO2 exchange in transgenic
plants expressing NADP-ME of the C4 plant. In this work of
ours, sorghum NADP-ME was cloned and introduced into
rice using the transformation system mediated by
Agrobacterium, and then systematic study on the photo-
synthetic physiology involved was carried out. It is hoped
that better understanding of the physiology might prove
helpful to the genetic approach for improving photosyn-
thesis in C3 plants.
1 Materials and Methods
1.1 Experimental materials
Jinzhong 405, a variety of sorghum (Sorghum vulgare
L.), was cultivated in greenhouse and, after 15 d of growth,
seedlings were harvested for further experiments.
The variety of rice (Oryza sativa L.) used for
Acta Botanica Sinica 植物学报 Vol.46 No.7 2004874
transformation experiment was Nongken 58N. Both the
transgenic rice (ME) and the untransformed controls (WT)
were cultivated under controlled conditions until the boot-
ing stage when specimens were collected for determination
of the physiological indices.
1.2 Cloning and sequencing of sorghum NADP-ME
Based on the published conserved regions of the se-
quences encoding C4 type NADP-ME, two primers were
synthesized: primer 5: 5-GAAGGTTTGGCTIGTGGACTC -
3; primer 3: 5-GATGCTGGTGAAIGGTGGGAA-3,where I
stands for hypoxanthine. Using cDNA taken from green
leaves of sorghum as templates and the above two primers,
PCR amplification was performed in a reaction system of 20
mL. After initial denaturing at 94 ℃ for 5 min, the sample
underwent the following steps for 30 cycles: denaturing at
94 ℃ for 1 min, annealing at 61 ℃ for 1 min, then extension
at 72 ℃ for 1 min. After final extension at 72 ℃ for 10 min,
the amplification product was cloned into the vector pGEM-
T-easy. It was then sent to Bo Ya Company (Shanghai,
China) for sequencing. As shown by homology study, it
was identified as a fragment of NADP-ME gene.
mRNA was extracted from 1 g of green leaves of
sorghum, using the Quickprep Micro mRNA isolation kit
(Phamacia, Piscataway, New Jersy, USA). Then cDNA was
synthesized from the mRNA obtained, using the TimeSaver
cDNA synthesis kit (Phamacia Piscataway, New Jersy, USA).
Next, the cDNA was ligated to the vector, l ZAP
(Stratagene, La Jolla, California, USA), and packed with the
packing protein of Statagene to form a cDNA library.
The cDNA library was screened, using a gene fragment
of sorghum NADP-ME as probe. Eight positive clones were
obtained from about 1× 105 plagues. The recombinant
DNA isolated therefrom was then digested with EcoRⅠ to
determine the length of the insert and clones with inserts
longer than 2 kb were sequenced.
Analysis of the nucleotide sequence obtained and its
homology study were performed using GENTYX-WIN 3.1.
The sequence of the chloroplast transfer peptide was ana-
lyzed using Chlop V1, a program downloaded from Internet.
1.3 Amplification of CAB promoter, construction of ex-
pression vector, and genetic transformation of rice
Two primers were designed based on the sequence of
the promoter of chloroplast a/b binding protein gene (CAB)
of rice (GenBank accession number: E03236): Cab5: 5-
ACTCTAGAGATTGGGATTAAGGTAATG-3; Cab3: 5-
ACGATATCGATGCAGTGAGCTGTGAGAG-3.
Next, PCR amplification was performed in a 20-mL reac-
tion system using rice genomic DNA as template. After
initial denaturing at 94 ℃ for 5 min, the sample underwent
the following steps for 25 cycles: denaturing at 94 ℃ for 1
min, annealing at 55 ℃ for 1 min, then extension at 72 ℃ for
1 min. After final extension at 72 ℃ for 10 min; the product
was cloned into pBluescript. Sequencing showed it to be
basically the same as the CAB promoter as recorded in
GenBank, with a similarity of 99%.
A transgenesis vector was constructed on the basis of
pCAMBIA1301: at the polycloning sites were inserted CAB
promoter, ME cDNA and NOS terminator. Designated as
p1301-ME, the structure is shown in Fig.1.
p1301-ME was then introduced into the Agrobacterium
tumefaciens strain EHA105 by freezing-thawing. The trans-
formation of rice was performed by the method of Hiei et al.
(1994).
1.4 Southern and Northern hybridization of transgenic
rice
Genomic DNA was extracted from rice using the method
of CTAB. Fifteen mg of the extracted DNA, digested with
BstXⅠ, were separated by electrophoresis in 0.7% agarose
gel before being transferred to Hybond membrane for South-
ern hybridization. All the procedures, including
prehybridization, were performed in routine manner.
Total RNA was extracted from the leaves using
guannidine isothiocyanate and was separated with elec-
trophoresis in 1.2% denaturing gel. It was then transferred
to Hybond nylon membrane for Northern hybridization.
1.5 Extraction of soluble proteins and determination of
its enzyme activity
Naught point two gram of rice leaves that had been
exposed to full light (at 9:00-11:00, with an intensity of
about 1 400 mmol×m–2×s–1) for 2 h was rapidly placed in a
pre-cooled mortar and ground with 1.5 mL extracting buffer
in ice bath. The buffer was composed of 50 mmol/L Tris-
HCl (pH 7.0), 10 mmol/L MgCl2, 1 mmol/L EDTA, 5 mmol/L
DTT and 5% insoluble PVP. After full maceration, the
Fig.1. The schematic diagram of transgenesis vector of p1301-ME. 35s, promoter of cauliflower mosaic virus; GUS, b-glucuronidase
gene； Tnos, terminator of the nopine synthase gene; PCAB, promoter of chloroplast a/b binding protein gene; ME, cDNA of sorghum
NADP-ME, HPTII, hygromycin phosphotransferase gene; LB, left T-DNA border; RB, right T-DNA border.
CHI Wei et al.: Photosynthetic Features of Transgenic Rice Expressing Sorghum C4 Type NADP-ME 875
extract was centrifuged at 13 000g and 4 ℃ for 10 min and
the supernatant was gathered for activity determination.
Activities of the enzymes were determined respectively
by the following methods: PEPCase by that of Gonzalez
et al. (1984); NADP-ME and NADP-MDH by those of Li
et al. (1987) and Chen et al. (1981) ; PPDK by that of Hatch
et al. (1975); and Rubisco by Kung et al. (1980).
1.6 Determination of substrate contents for photosynthe-
sis
The contents of malate, pyruvate and phospho-
enolpyruvate (PEP) were determined by the method of Sakae
et al. (2002).
1.7 Measurement of CO2 exchange and chlorophyll fluo-
rescence
Photosynthetic rates of rice leaves in booting stage
under various light intensities were measured using a por-
table photosyntometer, LI-6400 (LI-COR Co., USA). After
adaptation to darkness for 30 min, parameters of chloro-
phyll fluorescence were measured using a portable
fluorometer, FMS2 (Hansatech Co., UK).
2 Results
2.1 Amplification of fragments of sorghum NADP-ME
and cloning of full-length cDNA
A pair of primers were designed based on a relatively
conserved region of NADP-ME of C4 plants and each had
an hypoxanthine incorporated. Since hypoxanthine may pair
with anyone of the three nucleotides, T, C, or A, its incor-
poration ensures both the specificity and conservative-
ness of the primers, thus enhancing the efficiency of
amplification. PCR amplification of a single chain of the
cDNA retrotranscribed from leaf RNA using this pair of
primers produced a fragment about 500 bp long, within the
range of the molecular weight as expected.
The amplified fragment was cloned into pGEM-T-easy
vector and subsequent sequence showed it had 532 bp
and a 91% similarity with maize NADP-ME. This confirmed
our result as in the cDNA fragment of sorghum NADP-ME.
Next, screening of cDNA library with the fragment ob-
tained as probe produced a positive clone about 2.1 kb.
The insert of this clone was subcloned into pBluescript SK
and sequencing showed the insert to be 2 139 bp in length.
Analysis using GENETYX Win 3.1 revealed an opening
reading frame of 1 911 bp which encodes 636 amino acids
and a terminator (GenBank accession number: AY274836).
2.2 Structural analysis of sorghum NADP-ME
Using software GENETYX Win 3.1, homology study of
the amino acid sequence encoded by sorghum NADP-ME
as compared with those encoded by NADP-MEs of other
plants revealed a 89% similarity (the highest) to that of the
C4 monocot maize, and a 40% similarity (the lowest) to the
non-C4 type NADP-ME of the C3 plant Arabidopsis. It was
interesting that it was more similar to non-C4 type NADP-
ME of C4 monocot plant (maize) than to C4 type NADP-ME
of C4 dicot plants (Flaveria trinervia). A similar finding
has also been reported in other C4 type photosynthetic
enzymes (Toh et al., 1994). In terms of plant evolution, this
seems to indicate that the divergence of plants into C3 and
C4 photosynthetic types occurred earlier than that into di-
cots and monocots.
At the N terminal of C4 type NADP-ME, there is a transit
peptide sequence that serves to transport the precursor of
ME into chloroplast where it is further processed into the
mature enzyme. Structural prediction of the transit peptide
was performed by importing the amino acid sequence of
sorghum C4 type NADP-ME into Chlop V.1, an analytical
software. A transit sequence was indeed found at the N
terminal and two splicing sites at the 62nd and 80th amino
acids (Fig.2). The predicted molecular weight of the mature
protein is somewhere between 61 and 63 kD, basically the
same as the value determined for C4 type NADP-ME in
other plants.
As a rule, each NADP-ME molecule combines four
NADPs in performing its biochemical function. In addition,
NADP-ME combines NAD as well as NADP so that the
former may work as a competitive inhibitor for the latter. As
may be noted in the amino acid sequence predicted, highly
conserved sequences are found between the positions
183-191 and 239-246 (Fig.2) as compared with those re-
ported for NADP and NAD combining sites (Rothermel
and Nelson, 1989).
2.3 Expression of NADP-ME from sorghum in transgenic
rice plants
Altogether 33 plants of transgenic rice transformed with
sorghum C4 type NADP-ME were obtained using
Agrobacterium tumefaciens as mediator. Determination of
NADP-ME activity in leaves of transgenic rice revealed
elevation to various extents; 1-7 folds increase was noted
in comparison with the control. Examples of transgenic rice
plants ME1.2, ME5.2, ME6.0 and ME6.7 are shown in
Fig.3A, with enzyme activity elevated by 1.2, 5.2, 6.0 and
6.7 folds respectively.
Further molecular-level examination was performed on
the transgenic rice. Southern hybridization of BstXⅠ-di-
gested genomic DNA of the transgenic rice was carried out
using a 1.3-kb BstXⅠ-digested fragment of the cDNA of
sorghum NADP-ME as probe (Fig.3B). As expected, a hy-
bridization band was found at the position of 1.3 kb in each
Acta Botanica Sinica 植物学报 Vol.46 No.7 2004876
Fig.2. Nucleotide sequence and predicted amino acid sequence of cDNA of sorghum C4 type NADP-ME. Underlined sequence, predicted
combining site for NADP; boxed sequence, predicted combining site for NAD; arrows, predicted splicing sites for chloroplast transit
peptide; asterisk, stop codon.
CHI Wei et al.: Photosynthetic Features of Transgenic Rice Expressing Sorghum C4 Type NADP-ME 877
Fig.2. (continued)
Acta Botanica Sinica 植物学报 Vol.46 No.7 2004878
of the transgenic plant while nothing was detected in the
corresponding position of the control. This confirms the
successful integration of sorghum NADP-ME into rice
genome. But two further hybridization bands were found in
both the control and transgenic plants, indicating the pres-
ence of other genes homologous to NADP-ME in rice.
In order to make clear if there is any relation between the
level of expression of ME in rice and the level of
transcription, Northern hybridization was done to analyze
the expression of NADP-ME in rice. And correlation is found
between NADP-ME activity and transcription level as can
be seen from Fig.3C.
2.4 Light regulation of NADP-ME activity in transgenic
rice
In C4 plants, the expression of NADP-ME is under the
regulation of light. It has been demonstrated that rice CAB
promoter can direct the mesophyll-specific and light-regu-
lated expression in leaves of C3 plants (Sakamoto et al.,
1991). This has led us to adopt the CAB promoter as the
promoter for the target gene to ensure the specific expres-
sion of NADP-ME in rice. To make clear if the activity of
NADP-ME in the transgenic rice is light-regulated, the ac-
tivity of NADP-ME under light and in darkness was sepa-
rately measured and compared with each other. As shown
in Fig.4, the activity of NADP-ME in rice rapidly declines
10 h after being transferred into darkness. Yet the activa-
tion levels (the ratio of activity under light as compared
with in darkness) of NADP-ME in sorghum and the
transgenic rice were different. The ratios of activity were
11.25 in sorghum, 1.2 in WT, 6.0 in ME5.2 and 8.5 in ME6.7
respectively. This may reflect regulatory differences be-
tween the CAB promoter in rice and the promoter of the
sorghum NADP-ME itself. On the other hand, NADP-ME is
expressed specifically in bundle sheath cells in C4 plant
while, in the transgenic plants, they are expressed in meso-
phyll cells. Consequently, the differences between the
physiological and biochemical microenvironments of the
bundle sheath cells and those of mesophyll, with regard to,
for example, the concentration of substrates, the pH values,
and the mount of regulatory factors of enzyme activity,
may all contribute to the differences at activation levels.
As it has been demonstrated, changes of activity of
certain enzymes in C4 cycle may exert influences on other
enzymes of C4 cycle through feedback mechanism of the
metabolites (Hausler et al., 2001). We thus measured the
activity of other photosynthetic enzymes in the C4 cycle
and that of Rubisco, the rate-limiting enzyme of C3 cycle, in
transgenic rice. As compared with the control, no appre-
ciable changes were noted in PEPCase, NADP-MDH and
PPDK in the transgenic plant, nor was any found in Rubisco
(data not shown), both of these suggested that the trans-
formation with NADP-ME did not lead to enhanced
Fig.3. Expression analysis of transgenic rice plants. A. Activity
of NADP-ME in transgenic rice. B. Southern Hybridization. C.
Northern Hybridization. 1, transgenic plant ME 1.2; 2, ME 5.2;
3, ME 6.0; 4, ME 6.7; WT, control; S, sorghum; P, position of
cDNA of sorghum NADP-ME as revealed by hybridization.
Fig.4. Effect of light on activity of NADP-ME in transgenic
rice. Normally growing transgenic rice was first exposed to light
for 8 h (light intensity: 600 mmol×m-2×s-1) before growing in dark-
ness for another 10 h. Activity of NADP-ME in rice leaves under
each condition was measured. The data are Mean±SE (n = 5). S,
sorghum; ME5.2, ME 6.7, transgenic rice; WT, control.
CHI Wei et al.: Photosynthetic Features of Transgenic Rice Expressing Sorghum C4 Type NADP-ME 879
operation of C4 cycle and any remarkable effect on carbon
assimilation.
2.5 Determination of the substrates and products of
NADP-ME in transgenic rice
Although determination of the activity of NADP-ME in
transgenic rice has demonstrated the expression of sor-
ghum C4 type NADP-ME in rice, the test was conducted in
vitro and consequently it provides no adequate evidence
for the true nature of the expression product in vivo. As
was found by Furayama et al. (2001), despite the high level
of expression of C4 type PPDK in rice, most of the PPDK
molecules produced were in the form of inactive zymogens.
It thus appeared that the contents of substrate and prod-
ucts of NADP-ME might better reflect the effective expres-
sion of NADP-ME in vivo, which we indeed measured in
leaves of the transgenic rice. As compared with the control,
the malic acid content dropped by about 30% and the pyru-
vic acid content rose by about 20%, thus proved that the
sorghum NADP-ME protein is in active form in the
transgenic rice (Fig.5). No appreciable change was noted in
the content of another key enzyme in C4 cycle, which was
consistent with the observation that no change of activity
of PEPcase and PPDK was detected.
2.6 CO2 exchange and chlorophyll fluorescence in
transgenic rice
The introduction of C4 photosynthetic enzymes into C3
plants was intended to improve the efficiency of solar en-
ergy utilization. What then was the actual result of intro-
duction of sorghum NADP-ME into rice? We measured the
net photosynthetic rates of transgenic rice expressing
NADP-ME under various light conditions. As shown in
Fig. 6A, no appreciable differences of net photosynthetic
rates were found between the transgenic rice and the control.
With the increase of light intensity, light saturation was
reached at 1 000 mmol×m– 2×s–1.
PSⅡ photochemical efficiency (Fv/Fm) is an important
parameter characterizing the status of photochemical
reaction. As shown in Fig.6B, greater decline of Fv /Fm was
found under high light intensity at noon for the transgenic
rice (ME5.2 and ME6.7) than for the control (WT), suggest-
ing greater susceptibility of the former to photoinhibition.
3 Discussion
Because C3 plants do not possess the Kranz anatomy,
for a long time it remains doubtful that an effective C4 cycles
could be established in C3 plants simply by transferring C4
photosynthetic enzyme genes into C3 plants. However, it
was recently discovered that some aquatic angiosperms,
such as Hydrilla verticillate ( Magnin et al., 1997) and
Egeria densa ( Casati et al., 2000), possess a primitive type
of C4 photosynthesis without Kranz anatomy. Moreover, it
was reported lately that the expression of C4 type PEPC in
rice can improve its photosynthetic capacity with enhanced
tolerance to photo-oxidation (Chi et al., 2001; Huang et al.,
2001; Zhang et al., 2003). The primitive CO2 concentration
mechanisms in transgenic rice expressing PEPC gene and
those expressing PCK gene were also detected (Suzuki
et al., 2000; Jiao et al., 2003). These results further strength-
ened the possibility of improving C3 photosynthetic per-
formance by gene engineering. In this study, NADP-ME,
another key enzyme involved in C4 photosynthesis was
introduced into a C3 plants, rice and the photosynthetic
characteristics of transgenic plants were analyzed in detail.
To our surprise, although high level expression of NADP-
ME gene in rice was achieved in our study, no marked
improvement in photosynthetic properties was detected
judged from CO2 assimilation. It may suggest that the in-
troduction of C4 NADP-ME alone into C3 plants could con-
tribute little to the improvement of C3 photosynthetic
performance. Therefore, the introduction of other C4-re-
lated enzymes in addition to NADP-ME, such as PEPCase
Fig.5. Contents of malic acid, pyruvic acid and phospho-
enolpyruvic acid in leaves of transgenic rice. The data are M±
SE (n = 5). * indicates significant difference (P < 0.05) as com-
pared with control.
Acta Botanica Sinica 植物学报 Vol.46 No.7 2004880
and PPDK, may be necessary to drive the C4 pathway in C3
plants. Further studies on this point should also shed light
on the coordination of parameters needed for C4
photosynthesis.
A recent report suggests that NADP malic enzyme could
be detrimental in the development of normal chloroplasts
when expressed at high levels (20-70 folds increases) in a
C3 plant (Takeuchi et al., 2000). For the plants analyzed in
this study, which showed 1–7 folds increased activity, there
was no change in phenotype under normal growth condi-
tion except for a slight reduction of chlorophyll content
(data not shown). This seems to indicate that the physi-
ological function varies with the level of expression of its
gene. However, the photoinhibition was enhanced under
intense light at noon for transgenic rice in our study. In
general, photoinhibition happens whenever the reducing
power and ATP produced by photosynthesis exceed the
total consumption of Calvin cycle, photorespiration and
other physiological processes. In the case of transgenic
rice, therefore, a great amount of NADPH is accumulated in
chloroplasts due to overproduction by the fully activated
NADP-ME under the intense light at noon, thus leading to
photoinhibition. Moreover, the accumulated NADPH in
chloroplasts keeps the components of the photosynthetic
electron transfer chain in a highly reduced state, leading to
production of great amount of singlet oxygen at the termi-
nal of the chain, which may attack the active center of
PSⅡ, further aggravating the inhibition (Vass and Styring,
1993). Besides, malic acid, the substrate for NADP-ME,
plays an important role in maintaining pH within the cell,
regulating Mg2+ concentration, and protecting the integ-
rity of membranes, which are closely related to
photoprotection mechanisms including xanthophyll cycle
(Edwards and Andreo, 1992; Casati et al., 1997). Thus the
down-regulation of photoprotection mechanisms caused
by lowering of malic acid concentration might be another
reason for the enhanced photoinhibition in transgenic rice
expressing NADP-ME gene. Further physiological data are
required to support those hypotheses.
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