全 文 ：Received 21 May 2003 Accepted 15 Dec. 2003
Supported by the State Key Basic Research and Development Plan of China (G1998010100, G1999011700), the National Natural Science
Foundation of China (30170088) and the Doctoral Program Foundation of Ministry of Education of China (20020019030).
* Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (3): 294-301
Characteristics of Triose Phosphate/Phosphate Translocator from Wheat
and Its Role in the Distribution of Assimilates
SUN Jin-Yue, WANG Qing-Mei, CHEN Jia*, WANG Xue-Chen
(College of Biological Sciences, China Agricultural University, State Key Laboratory for Plant Physiology
and Biochemistry, Beijing 100094, China)
Abstract: In plants, triose phosphate/phosphate translocator (TPT) is the first regulation point for
partitioning of photosynthate between source and sink. Studies on the characteristic of TPT and its
regulation on the distribution of assimilates are critical for improving the utilization rate of photosynthetic
assimilates. Chloroplasts with intactness of more than 91% and high purity were isolated from wheat
(Triticum aestivum L. cv. Jing 411) leaves. Analysis of SDS-PAGE and labeling with an irreversible specific
inhibitor, [H3]2-DIDS (4, 4-diisothiocyano-2, 2-stilbenedisulfonate, DIDS) demonstrated that wheat TPT
was a chloroplast membrane protein with a 35 kD molecular weight, which comprised about 15% of the total
membrane proteins of chloroplasts. Western blotting analysis showed that wheat TPT is uniquely
distributed in the envelope membrane of chloroplasts, but not detected in the membranes of vacuoles and
mitochondria. The silicone-oil-layer centrifugation system was employed to study the kinetic properties of
TPT. The results showed that the maximal transport activity of TPT was the highest for dihydroxyacetone
phosphate (DHAP)/inorganic phosphate (Pi), then for phosphoenolpyruvate (PEP)/Pi and glucose-6-
phosphate (G6P)/Pi. The Km value of TPT was the lowest for DHAP, followed by Pi, PEP and G6P,
therefore the most preferred substrate of TPT is DHAP. The transport of wheat TPT to DHAP was strongly
inhibited by DIDS with a degree of 95%. Inhibition of TPT transport activity led to an obvious accumulation
of starch in chloroplasts, therefore the TPT protein of wheat controls the export of TP out of chloroplasts
into cytosol. Except for the need of participating in the Calvin cycle, the ratio of TP exported out of
chloroplast to the one used for synthesizing starch was at least 93.6:6.4. The TPT protein from wheat has
much high transport efficiency, which plays an important role in the regulation of the distribution of
assimilates in wheat chloroplasts.
Key words: wheat; triose phosphate/phosphate translocator; characteristic; assimilate; partition;
regulation
Resource allocation between tissues is a fundamental
process in higher plant. Leaves function as the principle
site of resource acquisition by the synthesis of
photoassimilate in chloroplasts, which provide all of the
reduced carbon. Carbon fixed during the day from photo-
synthesis is exported from the chloroplasts in the form of
triose phosphate (TP), which is mostly converted into su-
crose in the cytosol. Sucrose often serves as the predomi-
nant photoassimilate being allocated to sink tissues. The
export of TP from the chloroplasts is mediated by the triose
phosphate/phosphate translocator (TPT) (Häusler et al.,
1998; Häusler et al., 2000; Wang et al., 2001; Schneider et
al., 2002). Rather than being exported, a considerable
amount of the fixed carbon is maintained within the chloro-
plasts and is involved in the biosynthesis of transitory
starch. The export of TP into the cytosol is of central impor-
tance with respect to carbon assimilates partitioning among
various organs and allocation among various pathways
such as sucrose and starch synthesis. Since the TPT gov-
erns the export of photoassimilates from the chloroplast,
one would expect that it is the key regulating site for the
flux of assimilates between source and sink. Revealing the
characteristic of TPT and its regulation on the distribution
of assimilates may be critical for improving the utilization
efficiency of photosynthesis. But the information about
the characteristic of wheat TPT is scarce due to the fact
that the preparation of intact chloroplasts from cereals is
more difficult because of their higher fiber content than
that from spinach and tobacco and other model plants.
The transport by TPT strictly complies with a one-to-
one counter exchange rule. The import of one molecule of
inorganic phosphate (Pi) into chloroplast requires the ex-
port of one molecule of dihydroxyacetone phosphate
(DHAP) or 3-phosphoglyceraldehyde (3-GAP) (Heldt and
SUN Jin-Yue et al.: Characteristics of Triose Phosphate /Phosphate Translocator from Wheat and Its Role in the Distribution of
Assimilates 295
Flügge, 1992). The compound 4, 4-diisothiocyano-2, 2-
stilbenedisulfonate (DIDS) acts as an inhibitor that
covalently, irreversibly and specifically binds with TPT.
The isothiocyano and sulfonate moieties of the inhibitor
covalently combine with Lys or Arg residues, the substrate
attachment site, to permanently inhibit transport activity
(Rumpo et al., 1988; Gross et al., 1990). cDNAs encoding
the complete precursors of TPT from wheat (Wang et al.,
2002) and rice (Wang et al., 2002) have been recently cloned.
In spite of these important advances, however, vanishingly
little is known about transport characteristic and the con-
trol pathways that regulate assimilate partitioning in wheat
and rice. In the results reported here, we described the lo-
calization and transport kinetics of TPT, and its physiologi-
cal role in the distribution of assimilates in wheat.
1 Materials and Methods
1.1 Materials
Seeds of Triticum aestivum L. cv. Jing 411 were germi-
nated in greenhouse set at 24 ℃ (12 h day)/20 ℃ (12 h
night) with a light intensity of 350 mmol.m-2.s-1. The first
and second basal fully expanded leaves of the seedlings
were harvested during the three-leaf-stage (about 18 days
old) after being lightened for 2 h and used for preparation
of chloroplasts.
[3H]2-DIDS was purchased from HSC Research Devel-
opment Corp., USA; [14C]-sorbitol and [32P]-KH2PO4 was
from Sigma, USA.
1.2 Preparation of TPT antibody
A peptide with a length of 13 amino acids (KAK IEE
EKR AKA A) was synthesized (by Genemed Synthesis,
Inc., USA) according to the putative amino acid sequence
at the 3 end of wheat tpt cDNA (39 bp) cloned by us (Wang
et al., 2002). The peptide coupled with bovine serum albu-
min (BSA) was used to inject rabbits to produce the anti-
body against TPT by using a standard immunological
procedure.
1.3 Isolation of intact chloroplasts with photosynthesis
activity
Intact chloroplasts were isolated from wheat leaves and
purified by Percoll density gradient centrifugation based
on the method of Batz et al. (1992), and modified as follows.
The bottom layer of the Percoll discontinuous density gra-
dient centrifugation system consisted of 5 mL 60% Percoll
(V/V) covered with 6 mL 32% Percoll (V/V), on which 4 mL
chloroplast suspension was placed. Chloroplast purity was
estimated by measuring the enzyme activities specific for
cellular compartments in purified chloroplasts (Quick et al.,
1995). Chloroplast intactness was determined by the method
of Sun et al. (1998). Photosynthetic activity of the purified
chloroplasts treated with or without DIDS was measured
by determining the rate of fixing carbon dioxide in light (500
mmol.m-2.s-1) at 20 ℃ (Walker et al., 1968).
1.4 Isolation of chloroplast envelope membrane, tono-
plast and mitochondrial membrane
Chloroplast envelope membranes were isolated from
intact, Percoll-purified wheat leaf chloroplasts by a discon-
tinuous sucrose gradient based on the method of Wiese et
al. (1999), and modified as follows. One hundred and 50 mL
of the purified intact chloroplasts were treated for 15 min in
3.6 mL of swelling medium (10 mmol/L Tricine-NaOH (pH
7.6), 4 mmol/L MgCl2) on ice. Aliquots of 1.8 mL of the
suspension were then layered on the top of a discontinu-
ous sucrose density gradient (1.6 mL of 0.6 mol/L and 1.5
mL of 0.93 mol/L sucrose) in a 5 mL tube and centrifuged at
72 000g for 2 h at 4 ℃. Tonoplast was isolated by the method
of Chen et al. (2002). The purity of the isolated tonoplast
was evaluated by measuring the activity of the marker en-
zyme following the procedure described by Zhu and Chen
(1997). The preparation and purity evaluation of mitochon-
drial membrane were based on the method of Zou et al.
(1999).
1.5 Western blotting analyses of membrane proteins
from different organelles
The membrane proteins from chloroplasts, vacuoles and
mitochondria were solubilized in sample buffer containing
4% SDS, 10% glycerol, 0.25% mercaptoethanol and 60 mmol/
L Tris (pH 6.8), and 2 mg protein was separated by SDS-
PAGE with Bio-Rad apparatus (Yu et al., 2001). Proteins
were then transferred to nitrocellulose filter at 100 V for 2 h.
The filter blots were incubated with 1:500 dilution of anti-
bodies against TPT and the bands were visualized with
goat anti-rabbit IgG-AP referred to the procedure of Brodzik
et al. (2000).
1.6 Labeling and characterization of wheat TPT
A volume of 5 mmol/L [3H]2-DIDS was added to an equal
volume of chloroplast suspension (1 mg chl/mL) and incu-
bated at 4 ℃ for 30 min to make the [3H]2-DIDS/TPT complex.
The isolation of the labeled chloroplast envelope membrane
by [3H]2-DIDS was as same as the method of 1.5 and ana-
lyzed by SDS-PAGE. Sample loading is 2 mg total proteins
in each lane. After electrophoresis, one lane is cut into gel
slices and dissolved in NCS (Amersham, UK). Then 3H
radioactivity of each slice was measured by liquid scintilla-
tion counter (Model 1414 WinSpectralTM, Wallac, Finland)
(Wu et al., 1996; Chen et al., 1997). Another lane was stained
with Coomassie Brilliant Blue R-250. The 35 kD polypep-
tide into which 3H radioactivity was incorporated was
Acta Botanica Sinica 植物学报 Vol.46 No.3 2004296
quantitated by densitometry.
1.7 Measurement of transport activity of chloroplast
TPT
A single silicone-oil-layer centrifugation system was
used to analyze the transport activity of Pi with substrate
[32P]-KH2PO4, and a double silicone-oil-layer centrifuga-
tion system was employed to measure the transport activ-
ity of DHAP, phosphoenolpyruvate (PEP) and glucose-6-
phosphate (G6P) by counter exchange with [32P]-KH2PO4
(Gross et al., 1990). Eppendorf reaction vessels (400 mL) of
the single silicone-oil-layer centrifugation system were filled
with three layers (20 mL of 10% perchloric acid; 70 mL of
silicone oil (DC 200:AR 200 =1:4, V/V, Fluka); and 200 mL of
chloroplast suspension, from the bottom to the top of
vessels, respectively). Eppendorf reaction vessels (400 mL)
of the double silicone-oil-layer centrifugation system were
filled with five layers (20 mL of 10% perchloric acid; 70 mL
of silicone oil (DC 200:AR 200 =1:4, V/V, Fluka); 75 mL 10 %
Percoll containing substrates; 30 mL of silicone oil (DC 200:
AR 200 = 4:1, V/V, Fluka); and 105 mL of chloroplast
preloaded with [32P]-KH2PO4, from the bottom to the top of
vessels, respectively). After centrifugation for 30 s in a
Beckman Microfuge E equipped with a horizontal rotor, the
sample-containing tubes were frozen in liquid N2, and the
suspension solutions and denaturing ones containing the
chloroplast pellet were removed with a tubing cutter to sub-
ject to scintillation counting. The sorbitol and water perme-
able spaces were measured with [14C]-sorbitol and 3H2O,
respectively, as described by Heldt (1980). The space inac-
cessible to [14C]-sorbitol but accessible to 3H2O represented
the combined internal volume of the intact chloroplasts.
The transport activity of TPT was measured with chloro-
plasts under light (500 mmol.m-2.s-1) with or without in-
hibitor DIDS (5 mmol/L).
1.8 Measurements of starch content and enzyme activity
in chloroplasts
In light (500 mmol.m-2.s-1), chloroplasts were treated
for 6 h with or without 5 mmol/L DIDS. The measurements
of starch content and enzyme activities related to Calvin
cycle and starch synthesis in chloroplasts were based on
the methods of Häulser (2000) and Batz et al. (1992).
2 Results
2.1 Chloroplasts preparation and photosynthetic activity
Based on the measurement of specific enzymes of the
isolated chloroplasts, it could be seen that the purity of the
chloroplasts was very high, with only a minor contamina-
tion of cytosol (Table 1). The intactness of the chloroplasts
isolated from wheat by us was typically between 91% and
95% calculated by the Hill reaction activity of the unbroken
and broken chloroplasts, which was higher than that of the
chloroplasts isolated from tobacco and spinach leaves by
Rangasamy and Ratledge (2000) (between 80% and 88%
intact). The prepared chloroplasts were also with a high
photosynthetic rate (27.05 mmol CO2.mg-1 chl.h-1). Pho-
tosynthetic rate of the chloroplasts was decreased only
1% after treated with DIDS, showing that the TPT inhibitor
DIDS has little effect on the photosynthetic rate and the
production of photosynthetic assimilates in chloroplasts.
Taken together, these results demonstrated that the pre-
pared photosynthetic chloroplasts from wheat seedling
leaves were suitable for the study of transport activity and
function of TPT, and the modified Percoll density gradient
centrifugation was better improved for the preparation of
wheat chloroplasts.
2.2 Characterization and subcellular location of TPT protein
Western blotting analysis (Fig.1) showed that immune
Table 1 Characterization of purity, intactness and photosynthetic rate of chloroplasts isolated from wheat leaves
Enzyme Hill reaction activity Intactness Photosynthetic rate
(mU/mg chl) (mmol K4Fe(CN)6.mg-1 chl.h-1) (%) (mmol CO2.mg-1 chl.h-1)
Chloroplast Cytosol Mitochondrial Vacuole Broken Unbroken -DIDS +DIDS
27.32± 1.84 0.06± 0.01 Undectable Undectable 17.93± 0.71 195.95± 2.58 91-95 27.05±2.36 26.74± 2.03
Marker enzyme: NADP-GAPDH for chloroplast, UDP-Glc-pyrophosphorylase for cytosol, citrate synthase for mitochondrial, and a-
mannosidase for vacuole. Values were the means of four independent experiments ± SE.
Fig.1. Western blotting analysis for the subcelluar localization
of triose phosphate translocator (TPT). 1, mitochondrial
membrane; 2, tonoplast; 3, chloroplast envelope membrane; M,
protein marker.
SUN Jin-Yue et al.: Characteristics of Triose Phosphate /Phosphate Translocator from Wheat and Its Role in the Distribution of
Assimilates 297
reaction was only detected in chloroplasts (lane 3), and the
molecular weight of the band was 35 kD. So it seemed that
TPT proteins exist only in the envelope membrane of
chloroplasts, but not in tonoplast and mitochondria.
SDS-PAGE analysis of chloroplast membrane proteins
labeled with [3H]2-DIDS also showed that only one band
with a molecular weight of 35 kD was detected to be radio-
active (Fig.2A). Figure 2B showed that TPT is the main
protein located in the envelope membrane of wheat
chloroplasts. The densitometry scanning of the gel sug-
gested that TPT protein is about 15.1% of the total enve-
lope membrane proteins.
2.3 Kinetic parameters of the TPT from wheat chloro-
plasts
The internal volume of the intact wheat chloroplasts
was determined to be 25.0 mL/mg chl (SE 0.79 mL, n = 6).
This was 40% of the total chloroplast volume (3H2O-per-
meable space). The ratio of stroma to intermembrane space
is thus in the same range as has been determined for spin-
ach chloroplasts (Heldt, 1980) and for etioplasts from bar-
ley leaves (Batz et al., 1992).
The kinetic constants of the TPT from wheat chloro-
plasts for DHAP, PEP, and G6P were measured by back-
exchange experiments using the double silicone-oil-layer
system, and for Pi by forward-reaction experiments using
the single silicone-oil-layer system. The substrate phases
contained 0.01- 0.20 mmol/L substrates. With increasing
DHAP concentrations, the uptake of DHAP into the inter-
nal volume of wheat chloroplasts became clearly saturated
with an apparent Km value of 0.017 mmol/L and a Vmax of
33.1 mmol.mg-1 chl.h-1 in light (Table 2). The Km value of
TPT for Pi uptake was 0.122 mmol/L and a Vmax of 48.4
mmol.mg-1 chl.h-1 in light.
Compared with other substances transported, DHAP
exhibited the lowest Km value (0.017 mmol/L). This indi-
cated that the substrates transported by TPT are mainly
DHAP, and Pi, which is counter exchanged with DHAP.
The transport activity of PEP and G6P was lower, therefore
the most preferred substrate of TPT is DHAP.
To further characterize the uptake of DHAP into iso-
lated chloroplasts, inhibitor known to act specifically upon
the chloroplast TPT, namely, DIDS (Rumpo et al., 1988),
was added. When chloroplasts were treated with DIDS, the
transport of DHAP, PEP, G6P and Pi was severely inhibited,
resulting in 94.5% and 90.5% inhibition of the uptake rate
of TPT for DHAP and Pi, respectively.
2.4 Effects of changes of TPT transport activity on the
distribution of assimilates
Inside chloroplasts, the assimilates, TPs, produced dur-
ing photosynthesis are utilized in the regeneration of RuBP
in the Calvin cycle, the export out of chloroplasts to cyto-
sol for the synthesis of sucrose and a variety of other meta-
bolic pathways, and the synthesis of transient starch in
chloroplasts. TPT, sited in the envelope membrane of
chloroplasts, is the key point of the regulation of the three
ways. The concentration of starch in wheat chloroplasts
treated with or without DIDS was measured (Table 3). Re-
sults showed that there was only a little starch (1.62 mmol
Glc/mg chl) accumulated in chloroplasts treated without
DIDS when the three ways were smooth. However when
the transport activity of TPT was inhibited (95%), namely
that TPs were almost not transported to cytosol from stroma,
a great deal of starch was accumulated in chloroplasts
(24.12 mmol Glc/mg chl, increased by 13.89 times). In the
situation this TPs have two ways to go, one is for the re-
generation of RuBP and another is for synthesizing starch.
These suggested that when chloroplasts were treated with-
out DIDS as in vivo TPs equaling the starch content of
23.68 mmol Glc/mg chl (Calculated by 95% inhibition of the
TPT transport activity) were exported out of chloroplasts.
Namely, there were 93.6% of TPs exported out of chloro-
plasts into cytosol for synthesizing sucrose and participat-
ing in other metabolic pathways, therefore the TPs utilized
for the accumulation of transient starch in chloroplasts were
only 6.4%, except for the TPs needed for the normal run-
Fig.2. Analysis by SDS-PAGE of envelope membrane proteins
from wheat chloroplasts labeled by [H3]2-DIDS. A. Densitom-
etry scanning of the radioactivity of the gel slices of SDS-PAGE
of envelope membrane proteins from wheat chloroplasts labeled
by incubation with 5 mmol/L [3H]2-DIDS as outlined in Materials
and methods. B. Envelope membrane proteins from wheat chlo-
roplasts were analyzed by SDS-PAGE stained with Comassie
Brilliant Blue R-250. Chl, envelope membrane protein of wheat
chloroplasts; M, protein marker.
Acta Botanica Sinica 植物学报 Vol.46 No.3 2004298
ning of Calvin cycle.
2.5 Effects of DIDS on the activity of enzymes related to
starch synthesis
One of the possible reasons for the accumulation of
transient starch in chloroplasts when they were treated with
DIDS was that inhibition of DIDS on the transport activity
of TPT resulted in the depression of export of TP; another
may be that DIDS increased the activity of enzymes in-
volved in starch synthesis pathway. To explore the truth
for the accumulation of transient starch in chloroplasts was
whether DIDS increased the activity of enzymes involved
in starch synthesis pathway or not, we determined the ac-
tivity of enzymes involved in starch synthesis. The func-
tion of fructose 1,6-bisphosphatase (FBPase) is to catalyze
fructose 1,6-bisphosphate (FBP) coming from TP to pro-
duce fructose-6-phosphate, to ensure the regeneration of
RuBP. Phosphoglucoisomerase (PGI) is the key enzyme in
the pathway of starch synthesis in chloroplasts, and its
function is to catalyze fructose-6-phosphate (F6P) to pro-
duce glucose-6-phosphate (G6P). Phosphoglucomutase
(PGM) catalyzes G6P to produce G1P, and then G1P, as raw
material, is synthesized into starch catalyzed by ADP-glu-
cose pyrophosphorylase and starch synthase. Results
showed that the activity of all enzymes was inhibited with
various extents, FBPase 5.46%, PGI 11.58%, PGM 35.25%
and starch synthase 20.68% (Fig.3). The activity of all the
four enzymes was not improved by the treatment with DIDS,
on the contrary they decreased with different extents (5%-
35%). These suggested that the accumulation transient
starch in chloroplasts was the results of the inhibition of
the transport activity of the TPT, but not the improvement
of the activity of enzymes related to starch synthesis
pathway. Except for the need of participating in the Calvin
cycle, the ratio of TP exported out of chloroplast to the one
used for synthesizing starch was, at least, 93.6:6.4.
3 Discussion
Our results (Figs.1, 2) suggested that the molecular
weight of TPT in wheat is 35 kD, which is in agreement with
the mature peptide following elimination of the signal pep-
tide sequence of putative precursor peptide according to
the cloned tpt cDNA (Wang et al., 2002). The deduced
mature TPT protein in wheat contains about 67.5% nonpo-
lar amino acids and possesses eight hydrophobic
segments, indicating the hydrophobic nature of the pro-
tein (Wang et al., 2002). This was further proved by us that
the TPT protein in wheat is a kind of membrane protein and
Table 2 Kinetic constants of the TPT from wheat chloroplasts and the effect of DIDS on them measured by silicone-oil-layer
centrifugation system
Vmax (mmol.mg-1 chl.h-1) Km (mmol/L)
DHAP PEP G6P Pi DHAP PEP G6P Pi
Light 33.1±3.2 17.2±1.4 15.6±1.1 48.4±2.5 0.017±0.002 0.224±0.014 0.280±0.019 0.122±0.008
Light+DIDS 1.7±0.4 1.5±0.3 0.7±0.1 4.6±0.7 - - - -
DHAP, dihydroxyacetone phosphate; G6P, glucose-6-phosphate; PEP, phosphoenolpyruvate; Pi, inorganic phosphate; Vmax, the maximal
transport velocity; Km, the apparent half-maximal saturating substrate concentration. Data were the means of four independent experiments
± SE. DIDS, 4, 4-diisothiocyano-2, 2-stilbenedisulfonate; TPT, triose phosphate translocator.
Table 3 Effects of changes of TPT transport activity on the distribution of assimilates

Treatments
Relative rate of CO2 Relative transport Starch content in chloroplasts
TP exported out of
fixation (%) activity of TPT (%) (mmol Glc/mg chl)
chloroplasts/TP for
synthesizing transitory
starch in chloroplasts
-DIDS 100 100 1.62±0.11a
93.6:6.4
+DIDS 99 5 24.12±1.36 a
The relative rate of CO2 fixation was 100% in light when chloroplasts were treated without DIDS. The relative transport activity of TPT was
100% in light when chloroplasts were treated without DIDS. a, values were the means of four independent experiments ± SE.
Fig.3. Effects of DIDS on the activity of enzymes related to
starch synthesis in chloroplasts. 1, fructose 1,6-bisphosphatase,
FBPase; 2, phosphoglucoisomerase, PGI; 3, phosphoglucomutase,
PGM; 4, starch synthase. The relative activity of every enzyme
in chloroplasts is100 in light，respectively. Data were the means
of four independent experiments ± SE.
SUN Jin-Yue et al.: Characteristics of Triose Phosphate /Phosphate Translocator from Wheat and Its Role in the Distribution of
Assimilates 299
located in chloroplasts of wheat.
The TPT of wheat chloroplasts exhibited an affinity to-
ward DHAP (Table 2) in a similar range as the TPT of millet
(Panicum miliaceum L.) mesophyll chlorolplasts, which is
half saturated at 0.019 mmol/L DHAP (Ohnishi et al., 1990).
The TPT from wheat chloroplasts in our results had been
characterized by an almost 8-fold higher affinity than the
TPT from spinach chloroplasts (Km 0.13 mmol/L), and a
nearly 5-fold higher affinity than the TPT from maize meso-
phyll chloroplasts (Km 0.084 mmol/L) (Wang et al., 2001).
TPT protein of wheat chloroplasts has a higher transport
activity than that of many other plants, including some of
C4 plants with high photosynthetic rate. The rate of G6P
and PEP uptake is significantly lower than for DHAP and Pi
(Table 2). These results are in contrast to the findings for
G6P and PEP transport across envelopes of other het-
erotrophic plastids such as amyloplasts purified from pea
roots, but in correspondence to the findings for DHAP and
Pi transport across envelopes of spinach chloroplasts and
millet mesophyll chloroplasts (Wang et al., 2001).
Obviously, TPT from wheat chloroplast envelope has a
high specificity and transport activity for triose phosphates,
and the transport of DHAP is coupled with the counter
exchange of DHAP against Pi. This is more beneficial to the
export of DHAP out of chloroplasts in wheat than in maize
and in spinach.
To probe into the effects of TPT activity alteration on
metabolism, we treated wheat chloroplasts with DIDS to
change the transport activity of TPT. The TPT from C3 and
C4 mesophyll chloroplasts had been specifically labeled by
[3H]2-DIDS, an inhibitor acting upon the chloroplast enve-
lope TPT protein (Rumpo et al., 1988). Table 2 shows that
the strong inhibition (95%) of DHAP uptakes into wheat
chloroplasts in the presence of 0.5 mmol/L DIDS. This also
proved that DIDS is a strong and special inhibitor of TPT in
wheat. The photosynthetic rate decreased only 1 % when
wheat chloroplasts were treated with DIDS (Table 1), there-
fore DIDS had little effects on the photosynthetic rate of
wheat chloroplasts. The activity of enzymes related to
starch synthesis was not improved by DIDS treatment; on
the contrary they decreased to some extents (Fig.3). As an
inhibitor of wheat TPT, DIDS could severely depress the
transport activity of wheat TPT, whereas it had little effects
on the photosynthesis activity and at the same time it did
not increase the activity of enzymes involved in starch syn-
thesis but decrease the activity of them. Therefore DIDS is
a useful tool for the study on the characteristics and func-
tion of TPT in wheat.
In the cytosol, TPs occupy a central position in
glycolysis and gluconeogenesis. TPs in glycolysis are used
for energy and reducting potential production and carbon
backbones for a variety of metabolites. In gluconeogenesis,
TPs lead to glucose formation that in turn leads to sucrose
synthesis, translocation. Photosynthesis, starch synthesis,
glycolysis and gluconeogenesis are connected through the
TPT residing in the chloroplast envelope that helps to bal-
ance two TPs pools, one in the chloroplasts and another in
the cytosol, with export of DHAP to the cytosol. TPT ac-
tivities are central in connecting the two subcellular pools.
Recently Schneider et al. (2002) proved that TPT played a
key role in the regulation on the partitioning of photosyn-
thate with an Arabidopsis thaliana knock-out mutant of
the TPT. In the day when TPs cannot be exported out of
chloroplasts in time into cytosol in the mutant, more starch
was accumulated than the wild types. Table 3 shows that
except for the need of participating in the Calvin cycle, the
ratio of TP exported out of chloroplast to the one used for
synthesizing starch was, at least, 93.6:6.4. Since the TPs
transported by TPT into cytosol is mainly used for the
synthesis of sucrose (Häusler et al., 1998), this further dem-
onstrated that wheat was a kind of sugar-leaf plant, in which
the synthesis of photosynthetic products and export take
places nearly at the same time to ensure the photosynthate
to be exported rapidly from chloroplast into cytosol. How-
ever the TPT protein from wheat had much high transport
efficiency in light (Table 2), so most of the TPs was trans-
ported into cytosol during the day and little starch was
accumulated in chloroplasts. The main function of wheat
TPT is to ensure the photosynthate to be exported rapidly
from chloroplast into cytosol, and where they are converted
into sucrose and further transported to other sink organelles
and tissues until finally they are converted into starch, the
main component of wheat seeds, but not accumulated in
the form of starch. TPT is a key point for partitioning of
photosynthate between the source and sink.
Many factors affect the accumulation of starch in chlo-
roplasts when the transport activity of TPT is inhibited. In
higher plants the production of starch is orchestrated by
chloroplast-localized biosynthetic enzymes, namely starch
synthases, ADPglucose pyrophosphorylase, and starch
branching and debranching enzymes. Diurnal regulation of
these biosynthetic enzymes, as well as starch degrading
enzymes, e.g. amylase, a-glucosidase, and starch
phosphorylase, influences both the levels and composi-
tion of starch. Regulation of the above enzymes involved
in starch synthesis is affected by allosteric effectors, sub-
strate levels, and product levels, as well as by phosphory-
lation (Sehnke et al., 2001). In our results, we speculated
Acta Botanica Sinica 植物学报 Vol.46 No.3 2004300
that the improvement of substrate levels possibly promoted
the accumulation of transient starch in chloroplasts. In the
plastids of nongreen tissues, the precursors for the syn-
thesis of starch are predominantly G1P and G6P (Rubay,
1998). However, our results demonstrated the preferred
substrate for TPT was DHAP, not G6P; thus, we speculate
that the synthesis of starch in wheat chloroplasts is not the
main metabolism pathway. Because starch can be synthe-
sized in wheat chloroplasts under certain experimental
conditions, this indicates that it is possible to realize the
relationship conversion between source and sink each other
after induced. Quick et al. (1995) fed spinach leaves with
glucose in both light and dark, and induced the synthesis
of starch. All these data demonstrate that the relationship
between the source and sink is not always kept
unchangeable. But additional studies are needed to make
sure if wheat chloroplasts can use G6P or G1P as raw mate-
rials for synthesis of starch. During the whole life cycle of
plants, all the organelles and tissues continuously change
the status between source and sink to satisfy the growth
and development of plants. Understanding the flux pro-
cess of metabolites between source and sink will be key to
search the factors limiting crop yield.
References:
Batz O, Scheibe R, Neuhaus H E. 1992. Transport processes and
corresponding changes in metabolite levels in relation to starch
synthesis in barley (Hordeum vulgare L.) etioplasts. Plant
Physiol, 100:184-190.
Brodzik R, Koprowski H, Yusibov V, Sirko A. 2000. Production
of urease from Helicobacter pylori in transgenic tobacco plants.
Cell Mol Biol Lett, 5:357-366.
Chen J, Wang X-C , Wang Q-M. 2001. The regulation of metabo-
lite translocators on photosynthesis. LOU Cheng-Hou. Physi-
ological Bases for the Formation of Crop Yield. Beijing: China
Agricultural Press. 7-26. (in Chinese)
Chen J, Wu Z-Y , Zhu M-J. 1997. Solubilization and characteris-
tics of ABA binding proteins from plasma membrane of maize
roots. Acta Bot Sin, 39:1015-1021. (in Chinese with English
abstract)
Chen S , Chen J , Wang X-C. 2002. Existence and characteristics
of tonoplast-bound protein kinase in the tip cell of maize
root. Acta Bot Sin , 44:661-666. (in English with Chinese
Abstract)
Gross A, Rucker G, Heldt H W, Flügge U I. 1990. Comparison of
the kinetic properties, inhibition and labeling of the phos-
phate translocators from maize and spinach mesophyll
chloroplasts. Planta, 180:262-271.
Häulser R E, Fisher K L, Flügge U I. 2000. Determination of low-
abundant metabolites in plant extracts by NAD(P)H fluores-
cence with a microtiter plater reader. Anal Biochem, 281:1-8.
Häusler R E, Schlieben N H, Nicolay P, Fischer K, Fischer K L,
Flügge U I. 2000. Control of carbon partitioning and photo-
synthesis by the triose phosphate/phosphate translocator in
transgenic tobacco plants (Nicotiana tabacum L.). Ⅰ. Com-
parative physiological analysis of tobacco plants with
antisense repression and overexpression of the triose phos-
phate/phosphate translocator. Planta, 210:371-382.
Häusler R E, Schlieben N H, Schulz B, Flügge U I. 1998. Com-
pensation of decreased triose phosphate/phosphate
translocator activity by accelerated starch turnover and glu-
cose transport in transgenic in tobacco. Planta, 204:366-376.
Heldt H W, Flügge U I. 1992. Metabolite transport in plant cells.
Tobin A K. Plant Organelles. Cambridge, UK: Cambridge Uni-
versity Press. 21-47.
Heldt H W. 1980. Measurement of metabolite movement across
the envelope and of the pH in the stroma and the thylakoid
space in intact chloroplasts. Meth Enzymol, 69:604-613.
Ohnishi J, Heldt H W, Flügge U I. 1990. Phosphate translocator
of mesophyll and bundle sheath chloroplasts of a C4 plant,
Panicum miliaceum L., identification and kinetic
characterization. Plant Physiol, 91:1507-1511.
Quick W P, Scheibe R, Neuhaus H E. 1995. Induction of
hexosephosphate translocator activity in spinach chloroplasts.
Plant Physiol, 109:113-121.
Rangasamy D, Ratledge C. 2000. Compartmentation of ATP:
citrate lyase in plants. Plant Physiol, 122:1225-1230.
Rumpo M E, Edwards G E, Yousif A E, Keegstra K. 1988. Spe-
cific labeling of the phosphate translocator in C3 and C4 meso-
phyll chloroplasts by tritiated dihydro-DIDS (1, 2-ditritio-1,
2-(2, 2-disulfo-4, 4-diisothiocyano) diphenylethane). Plant
Physiol, 86:1193-1198.
Schneider A, Häusler R E, Kolukisaoglu Ü, Kunze R, van der
Graaff E, Schwacke R, Catoni E, Desimone M, Flügge U I.
2002. An Arabidopsis thaliana knock-out mutant of the chlo-
roplast triose phosphate/phosphate translocator is severely
compromised only when starch synthesis, but not starch
mobilisation is abolished. Plant J, 32:685-699.
Sehnke P C, Chung H J, Wu K, Ferl R J. 2001. Regulation of
starch accumulation by granule-associated plant 14-3-3
proteins. Proc Natl Acad Sci USA, 98:765-770.
Sun Y, Chen J, Sun D-Y . 1998. Extra cellular calmodulin stimu-
lates the transplasma membrane redox reaction of root proto-
plasts in Zea mays. Acta Bot Sin , 40:437-441. (in Chinese
with English abstract)
Walker D A, Baldry C W, Cockburn W. 1968. Photosynthesis by
isolated chloroplasts, simultaneous measurement of carbon
assimilation and oxygen evolution. Plant Physiol, 43:1419-
SUN Jin-Yue et al.: Characteristics of Triose Phosphate /Phosphate Translocator from Wheat and Its Role in the Distribution of
Assimilates 301
1422.
Wang Q-M , Chen J, Wang X-C, Sha W , Sun J-Y. 2002. Cloning
and expression analysis of triose phosphate/phosphate
translocator gene from wheat. Acta Bot Sin, 44:67-71.
Wang Q-M, Yang S-D , Chen J. 2001. Triose phosphate
translocator in the inner membrane of chloroplast. Chin Bull
Bot , 18:11-15. (in Chinese with English abstract)
Wang Q M, Chen J, Wang X C, Sun J Y, Sha W. 2002. Molecular
cloning and expression analysis of the rice triose phosphate/
phosphate translocator gene. Plant Sci, 162:785-790.
Wiese A, Gröner F, Sonnewald U, Deppner H, Lerchl J, Hebbeker
U, Flügge U I, Weber A. 1999. Spinach hexokinase I is located
in the outer envelope membrane of plastids. FEBS Lett, 461:
13-18.
Wu Z-Y , Chen J , Zhu M-J. 1996. Identification of the
photoaffinity-labelled abscisic acid binding proteins from
Maize microsome. Acta Biochim Biophys Sin , 28:694-697.
(in Chinese with English abstract)
Yu H-F , Chen J , Wang X-C . 2001. Effects of salt stress on the
activity and the amount of tonoplast H+-ATPase from pea
roots. Acta Bot Sin , 43:586-591. (in English with Chinese
abstract)
Zhu M-J , Chen J. 2001. Subcellular localization and kinetic char-
acterization of ABA binding proteins in maize roots. Acta Bot
Sin, 37:942-949. (in Chinese with English abstract)
Zou J, Qi Q, Katavic V, Marillia E F, Taylor D C. 1999. Effects
of antisense repression of an Arabidopsis thaliana pyruvate
dehydrogenase kinase cDNA on plant development. Plant Mol
Biol, 41:837-849.
Zubay G L. 1998. Biochemistry. 4th ed. McGraw-Hill, Boston,
USA: William C Brown Publishers. 265-438.
(Managing editor: HE Ping)