Plant Cell Physiol. 47(3): 380–390 (2006)
doi:10.1093/pcp/pcj004, available online at www.pcp.oupjournals.org
JSPP © 2006
Contribution of Fructose-1,6-bisphosphatase and Sedoheptulose-1,7bisphosphatase to the Photosynthetic Rate and Carbon Flow in the Calvin
Cycle in Transgenic Plants
Masahiro Tamoi, Miki Nagaoka, Yoshiko Miyagawa and Shigeru Shigeoka *
Department of Advanced Bioscience, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara, 631-8505 Japan
;
ase; PGA, 3-phosphoglycerate; PRK, phosphoribulokinase; PVP,
polyvinylpyrrolidone; Rubisco, ribulose-1,5-bisphosphate carboxylase/
oxygenase; RuBP, ribulose-1,5-bisphosphate; SBP, sedoheptulose 1,7bisphosphate; SBPase, sedoheptulose-1,7-bisphosphatase; SPS,
sucrose phosphate synthase; X5P, xylulose 5-phosphate.
To clarify the contributions of fructose-1,6-bisphosphatase (FBPase) and sedoheptulose-1,7-bisphosphatase (SBPase) separately to the carbon flux in the
Calvin cycle, we generated transgenic tobacco plants
expressing cyanobacterial FBPase-II in chloroplasts (TpF)
or Chlamydomonas SBPase in chloroplasts (TpS). In TpF11 plants with 2.3-fold higher FBPase activity and in TpS11 and TpS-10 plants with 1.6- and 4.3-fold higher SBPase
activity in chloroplasts compared with the wild-type plants,
the amount of final dry matter was approximately 1.3-, 1.5and 1.5-fold higher, respectively, than that of the wild-type
plants. At 1,500 µmol m–2 s–1, the photosynthetic activities
of TpF-11, TpS-11 and TpS-10 were 1.15-, 1.27- and 1.23fold higher, respectively, than that of the wild-type plants.
The in vivo activation state of ribulose-1,5-bisphosphate
carboxylase/oxygenase (Rubisco) and the level of ribulose1,5-bisphosphate (RuBP) in TpF-11, TpS-10 and TpS-11
were significantly higher than those in the wild-type plants.
However, the transgenic plant TpF-9 which had a 1.7-fold
higher level of FBPase activity showed the same phenotype
as the wild-type plant, except for the increase of starch content in the source leaves. TpS-11 and TpS-10 plants with
1.6- and 4.3-fold higher SBPase activity, respectively,
showed an increase in the photosynthetic CO2 fixation,
growth rate, RuBP contents and Rubisco activation state,
while TpS-2 plants with 1.3-fold higher SBPase showed the
same phenotype as the wild-type plants. These data indicated that the enhancement of either a >1.7-fold increase of
FBPase or a 1.3-fold increase of SBPase in the chloroplasts
had a marked positive effect on photosynthesis, that
SBPase is the most important factor for the RuBP regeneration in the Calvin cycle and that FBPase contributes to the
partitioning of the fixed carbon for RuBP regeneration or
starch synthesis.
Introduction
The Calvin cycle is the primary pathway of carbon fixation in chloroplasts of C3 plants (Sharkey 1985). It is considered to have three stages, the first of these being carboxylation
of the CO2 acceptor molecule ribulose-1,5-bisphosphate
(RuBP) by the enzyme ribulose-1,5-bisphosphate carboxylase/
oxygenase (Rubisco), resulting in the formation of 3-phosphoglycerate (PGA). The second stage is the reduction phase,
which produces triose phosphate by consuming ATP and
NADPH. The final stage of the cycle is the regenerative phase,
in which triose phosphates are used to produce RuBP. In the
cycle, the triose phosphates are key intermediates, and they are
also available for allocation to either the starch or sucrose
biosynthetic pathway (Woodrow and Berry 1988, Geiger and
Servaites 1994, Quick and Neuhaus 1997). It is extremely
important to maintain a balance between export and regeneration in order that the cycle does not become depleted
of intermediates. To achieve this balance, the catalytic activities of certain enzymes within the cycle are highly regulated
(Fridlyand et al. 1999, Raines et al. 1999). In particular, the
activities of some enzymes, including sedoheptulose-1,7bisphosphatase (SBPase) and fructose-1,6-bisphosphatase
(FBPase), are regulated by the redox potential via the ferredoxin/
thioredoxin system, which modulates the enzyme activities in
response to light/dark conditions (Scheibe 1990, Buchanan
1991). In addition, FBPase functions at the branch point
between the regenerative phase of the Calvin cycle and starch
biosynthesis, and SBPase and FBPase catalyze irreversible
reactions (Koßmann et al. 1994).
In order to determine the limiting steps of photosynthesis
and factors that influence the carbon allocation, a considerable
number of studies have been carried out on the regulation of
carbohydrate metabolism in photosynthetic CO2 fixation in
plant leaves (Stitt and Sonnewald 1995, Fridlyand et al. 1999).
We have demonstrated that transgenic tobacco (TpFS) plants
Keywords: Calvin cycle — Fructose-1,6-bisphosphatase —
Sedoheptulose-1,7-bisphosphatase — Transgenic plant.
Abbreviations: AGPase, ADP-glucose pyrophosphorylase; BSA,
bovine serum albumin; DHAP, dihydroxyacetone phosphate; DTT,
dithiothreitol; E4P, erythlose 4-phosphate; F6P, fructose 6-phosphate;
FBP, fructose 1,6-bisphosphate; FBPase, fructose-1,6-bisphosphatase;
FBP/SBPase, fructose-1,6-/sedoheptulose-1,7-bisphosphatase; G6P, glucose 6-phosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogen*
Corresponding author: E-mail, [email protected]; Fax, +81-742-43-8976.
380
Role of FBPase and SBPase in the Calvin cycle
expressing cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) in chloroplasts exhibit increased
photosynthetic CO2 fixation capacity and final dry matter under
atmospheric conditions (360 ppm CO2) (Miyagawa et al.
2001). In TpFS-6 and TpFS-3 plants with 1.7- or 2.3-fold
higher FBPase and SBPase activities in the chloroplasts, the
final dry matter was 1.4- and 1.5-fold more, respectively, than
that of the wild-type plants. The light-saturated photosynthetic
CO2 fixation rate on a leaf area basis of TpFS-6 and TpFS-3
plants was shown to be 1.20- and 1.24-fold higher, respectively, than that of the wild-type plants. Interestingly, the initial
activity of Rubisco in TpFS plants, reflecting the degree of
activation in vivo, was 1.2-fold that of the wild-type plants.
The content of RuBP in TpFS-6 and TpFS-3 plants was 1.5and 1.8-fold that of wild-type plants, respectively. It is therefore conceivable that the increase in RuBP results in the high
activity of Rubisco activase, leading to the increase in the initial activity of Rubisco in the transgenic plants. These phenotypes were correlated with the increase in FBPase and SBPase
activities. Recently, Lefebvre et al. (2005) have reported that
increased SBPase activity stimulated photosynthesis and the
growth rate of transgenic tobacco plants overexpressing an
Arabidopsis cDNA. In the young expanding leaves of the transgenic plants, photosynthetic rates were increased, sucrose and
starch were accumulated in the photoperiod, and leaf area and
biomass were increased up to 30% compared with the wildtype plants. These data suggest that SBPase is one of the limiting factors of the Calvin cycle. However, no significant
increase in CO2 assimilation rate and dry weight was observed
in the fully expanded leaves of the plants where SBPase was
introduced, unlike the TpFS plants. These facts suggest that
both FBPase and SBPase are important factors participating in
the regulation of the carbon flow through the Calvin cycle in
mature leaves. It is debatable how SBPase and FBPase contribute to photosynthesis and carbon metabolism and how different
increased levels of FBPase and/or SBPase cause different effects
on photosynthetic activity and carbon flow in the Calvin cycle.
We have reported previously that cyanobacterium
(Synechococcus PCC7942) cells contain two FBPase isozymes, designated FBP/SBPase and FBPase-II. The deduced
amino acid sequence of FBPase-II has been found to be quite
similar to those of the cytosolic forms from eukaryotic cells
(Tamoi et al. 1996). However, the enzymatic properties of
FBPase-II were more similar to those of chloroplastic FBPase
than to the cytosolic FBPase in higher plants. FBP/SBPase
could hydrolyze both 1,6-P2 and Sed 1,7-P2, while FBPase-II
could hydrolyze only Fru 1,6-P2. Furthermore, we have isolated
and characterized SBPase from halotolerant Chlamydomonas
W80 (CW80), which were resistant to H2O2 up to 1 mM
(Tamoi et al. 2001). The activities of FBP/SBPase, FBPase-II
and CW80 SBPase were not regulated by redox conditions via
the ferredoxin/thioredoxin system. In this study, we generated
transgenic tobacco plants expressing cyanobacterial FBPase-II
or Chlamydomonas SBPase in chloroplasts to clarify the contri-
381
Fig. 1 Protein levels and activities of FBPase-II (A) and SBPase (B)
in the transgenic and wild-type tobacco plants. The white bar represents the total activity of FBPase or SBPase. The gray bar represents
the initial activity of FBPase or SBPase derived from endogenous and
exogenous enzymes in the chloroplasts. The hatched bar represents the
in vivo inactivated state of plastidic FBPase or SBPase. The dark gray
bar shows the endogenous cytosolic FBPase. Values without a common
letter are significantly different according to the t-test (P < 0.05). (C)
Changes of FBPase-II and SBPase protein levels in the transgenic (T2)
and wild-type tobacco plants. The total soluble proteins (30 µg) were
prepared from the wild-type and transgenic plants at the times indicated.
bution of FBPase and/or SBPase to CO2 fixation, RuBP regeneration and biosynthesis of photosynthates.
Results
Analysis of transgenic tobacco plants expressing FBPase-II
The fbp-II gene from Synechococcus PCC7942 was introduced into tobacco (Nicotiana tabacum cv. Xanthi) nuclear
DNA using a fusion construct with the tomato rbcS promoter
and transit peptide. By Western blot analysis of the protein
extracted from isolated chloroplasts using an antibody raised
against Synechococcus PCC7942 FBPase-II, we confirmed that
the FBPase-II protein was localized in the chloroplasts of six
382
Role of FBPase and SBPase in the Calvin cycle
transformants carrying the fbp-II transgene (TpF-1, -4, -8, -9,
-10 and -11; T2 progeny) (Fig. 1A). The tomato rbcS promoter
regulates light-dependent transcription mainly in leaves
(Sugita et al. 1987). Protein levels of FBPase-II in the transgenic plants were decreased to 40% under dark conditions
compared with those under light conditions (Fig. 1C). Activities of FBPase in the fourth leaf from the top of six transformants and the wild-type plants were determined (Fig. 1A);
total FBPase activities derived from endogenous plastidic
FBPase, cytosolic FBPase and cyanobacterial FBPase-II in all
transgenic plants were 1.2- to 1.8-fold higher than those in wildtype plants. In TpF-8, TpF-9, TpF-11 and wild-type plants, the
total FBPase activities were 6.71 ± 0.31, 7.55 ± 0.36, 9.30 ±
0.51 and 5.39 ± 0.43 µmol m–2 s–1, respectively. On the basis of
the characteristics of plastidic FBPase in higher plants, the
activities of cytosolic FBPase, plastidic FBPase and FBPase-II
in chloroplasts were assayed separately by the method
described previously (Miyagawa et al. 2001). The activity of in
vitro-activated plastidic FBPase was found to be almost
identical to that of the cytosolic FBPase in wild-type plants
(Fig. 1A). Accordingly, the FBPase activity in chloroplasts of
TpF-8, TpF-9 and TpF-11 was approximately 1.4-, 1.7- and
2.3- fold, respectively, of that in the wild-type plants. The ratio
of the initial FBPase activity, reflecting the degree of enzyme
activation in vivo, to the total activity was ∼64% in the wildtype plants. The FBPase activities in the chloroplasts of TpF-9
and TpF-11 were ∼2.1- and ∼3.1-fold higher, respectively, than
that in the wild-type plants. There were no significant differences in SBPase activity between the TpF and wild-type plants.
Analysis of transgenic tobacco plants expressing Chlamydomonas W80 SBPase
Western blot analysis of the protein extracted from isolated chloroplasts using an antibody raised against CW80
SBPase showed that the SBPase protein was localized in the
chloroplasts of 12 transformants carrying the sbp transgene
(TpS-1 to -12; T2 progeny) (Fig. 1B). Protein levels of the
CW80 SBPase in the transgenic plants were decreased to 39%
under dark conditions compared with those under light conditions (Fig. 1C). The activities of SBPase in the leaves of seven
transformants and the wild-type plants were determined (Fig.
1B); the total SBPase activities derived from endogenous plastidic SBPase and CW80 SBPase in all transgenic plants were
1.3- to 4.3-fold higher than those in the wild-type plants. In
TpS-2, TpS-11, TpS-10 and wild-type plants, the total SBPase
activities were 3.38 ± 0.41, 4.32 ± 1.00, 11.83 ± 0.73 and 2.73
± 0.44 µmol m–2 s–1, respectively. The ratio of the initial
SBPase activity to the total activity was ∼47% in the wild-type
plants. The SBPase activities in the chloroplasts of TpS-2,
TpS-11 and TpS-10 were ∼1.5-, ∼2.2- and ∼8.1-fold higher,
respectively, than that in the wild-type plants. There were no
significant differences in FBPase activity between the TpS and
wild-type plants.
Fig. 2 Development of wild-type and transgenic plants. (A) The
wild-type and transgenic plants were grown hydroponically under a
12 h light (25°C) and 12 h dark (20°C) cycle with a light intensity of
400 µmol m–2 s–1, 360 ppm CO2 and 60% relative humidity. Each data
point represents the mean ± SD of measurements from eight wild-type
plants and 12 transformants. (B) The total dry weight of the wild-type
and transformed tobacco plants after 18 weeks of culture. The white
bar represents the total dry weight of shoots and the gray bar represents the total dry weight of roots. Asterisks indicate that the difference between transgenic plants and wild-type plants was significant
according to the t-test (P < 0.05).
Plant growth
TpF-11, TpS-11 and TpS-10 were phenotypically distinguishable from the wild-type plants in terms of the growth rate,
height, size and dry weight when grown hydroponically under
normal conditions (360 ppm CO2, light intensity: 400 µmol
m–2 s–1) (Fig. 2, 3, and Table 1). The transgenic plants had
grown significantly larger than the wild-type plants 12 weeks
after planting, and thus the heights of TpF-11, TpS-11 and
TpS-10 were approximately 1.2-, 1.4- and 1.3-fold, respectively, those of the wild-type plants at 18 weeks after planting
(Fig. 2A, 3D). The total dry weight of TpF-11, TpS-11 and
TpS-10 was increased approximately 1.3-, 1.5- and 1.5-fold,
Role of FBPase and SBPase in the Calvin cycle
383
Fig. 3 Phenotype of the wild-type and
transgenic tobacco plants. Whole parts
(A), the fourth leaves from the top (B)
and roots (C) of the wild-type and transformed tobacco plants grown for 12
weeks. (D) Whole parts of the wild-type
and transformed tobacco plants grown
for 18 weeks.
Table 1
Effect of increased FBPase and SBPase activities on plant growth
–2 –1
Chloroplast FBPase (µmol m s )
Chloroplast SBPase (µmol m–2 s–1)
Plant height (cm)
Total dry weight (g)
Shoot/root ratio
Wild type
TpF-9
TpF-11
TpS-2
TpS-11
TpS-10
2.93
2.73
77.7 ± 1.53
13.5 ± 1.61
1.84
5.00
n.d.
78.9 ± 1.25
13.2 ± 1.21
1.91
6.75
n.d.
88.9 ± 3.75
17.4 ± 2.29
1.96
n.d.
3.38
81.3 ± 3.51
13.8 ± 1.90
2.57
n.d.
4.32
98.0 ± 3.46
20.7 ± 1.46
2.24
n.d.
11.8
100.7 ± 1.53
20.9 ± 2.49
2.23
n.d., not determined.
respectively, compared with that of the wild-type plants
(Fig. 2B). TpF-10, having FBPase activity similar to TpF-11,
showed the same growth rate and photosynthesis (data not
shown). TpS-3, -1 and -4 having 2.6-, 3.1- and 3.4-fold higher
SBPase activity, respectively, showed an increased growth rate
and photosynthesis, which were roughly in agreement with the
384
Role of FBPase and SBPase in the Calvin cycle
data for TpS-10 (data not shown). On the other hand, TpF-9
(1.7-fold higher FBPase activity) and TpS-2 (1.3-fold higher
SBPase activity) were phenotypically indistinguishable from
the wild-type plants (Fig. 3). Another three transgenic plants
(TpF-1, -4 and -8), exhibiting a similar or lower level of FBPase
activity compared with TpF-9, were phenotypically the same
(data not shown). Accordingly, we selected two lines of TpF
plants (TpF-9 and -11) and three lines of TpS plants (TpS-2,
-11 and -10) with different FBPase or SBPase activities and
subjected them to further analysis.
Photosynthesis
The photosynthetic activities in the fourth leaves of the
TpF-9, TpF-11, TpS-2, TpS-11, TpS-10 and wild-type plants
were measured under atmospheric conditions (360 ppm CO2)
and various light intensities (50–1,500 µmol m–2 s–1) at 25°C
(Fig. 4A, B). At irradiances below 200 µmol m–2 s–1, the photosynthetic activities were not significantly different among TpF
plants, TpS plants and the wild-type plants. However, the
photosynthetic activities of TpF-11, TpS-11 and TpS-10 were
increased significantly compared with that of the wild-type
plants when the light intensities were in excess of 200 µmol
m–2 s–1. At a saturating irradiance (1,500 µmol m–2 s–1), the
photosynthetic activities of TpF-11, TpS-11 and TpS-10 were
1.15-, 1.27- and 1.23-fold, respectively, that of the wild-type
plants. On the other hand, no differences were observed in the
photosynthetic activities between TpF-9, TpS-2 and wild-type
plants. The same data had also been obtained in transgenic
plants (TpF-1, -4, -8 and -10, and TpS-5 and -8) exhibiting a
similar or lower level of FBPase or SBPase activities (data
not shown).
A/Ci response analysis
The response of the saturation level of CO2 assimilation to
a range of intracellular CO2 concentrations (Ci) was assessed
by measuring the CO2 uptake in vivo at an irradiance of
400 µmol m–2 s–1 (Fig. 4C). Under low CO2 conditions, the initial slopes of A–Ci curves of TpF-11, TpS-11 and TpS-10 were
significantly larger than that of the wild-type plants. Under
CO2-saturated conditions, the photosynthetic activities of TpF11 and TpS-11 were approximately 1.20- and 1.30-fold higher,
respectively, than that of the wild-type plants.
Fig. 4 Photosynthetic parameters of the wild-type and transgenic
plants. The CO2 assimilation rate was measured in the fourth leaves
from the top of the wild-type (open circle), TpF-9 (filled triangle),
TpF-11 (inverted filled triangle), TpS-2 (filled circle), TpS-11 (filled
square) and TpS-10 (filled diamond) plants after 12 weeks of culture.
(A and B) Effect of increasing irradiance on CO2 assimilation at
360 ppm of CO2. (C) Response of CO2 assimilation rates to increasing
intercellular CO2 concentrations (Ci) at irradiance (400 µmol m–2 s–1).
Each data point represents the mean ± SD of measurements on the
fourth leaf from the top of 12 separate plants.
Activities of enzymes involved in the Calvin cycle and carbon
metabolism
Total extractable activities of phosphoribulokinase (PRK),
NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH), total Rubisco, aldolase, sucrose phosphate
synthase (SPS) and ADP-glucose pyrophosphorylase (AGPase)
were not altered in either of the transformants. Interestingly, a
10–20% increase relative to the initial activity of Rubisco was
observed in TpF-11, TpS-11 and TpS-10 compared with that in
the wild-type plants, indicating that the in vivo activation state
of Rubisco in transgenic plants was increased (Fig. 5A). On the
Role of FBPase and SBPase in the Calvin cycle
385
from the bottom (lower leaves) at 12 h in the light period and
12 h in the dark period (Fig. 6). There were no significant differences in the levels of hexose, sucrose and starch between the
transgenic and wild-type plants in the dark period. In the light
period, however, sugar contents in the TpF and TpS plants in
the upper leaves were significantly different from those in the
wild-type plants. In the upper leaves of the TpF-9 and TpF-11
plants, the sucrose contents were almost the same as those in
the wild-type plants, but the hexose and starch contents were
significantly increased compared with the wild-type plants. On
the other hand, in the upper leaves of TpS-11 and TpS-10
plants, the hexose, sucrose and starch contents were 1.5–2.0
times higher than those in the wild-type plants. The contents of
hexose and sucrose in the lower leaves of all transgenic plants
were almost the same as those in the wild-type plants. The
starch contents in TpF-11 were higher than those in the TpS-11
and TpS-10 plants, although the photosynthetic activity in TpF11 was lower than those in the TpS-11 and TpS-10 plants.
Relationships between FBPase or SBPase activity and leaf
characteristics
The relationships between the FBPase or SBPase activity
and the photosynthetic activity, RuBP content, Rubisco activation state, dry weight, hexose content, sucrose content and
starch content are summarized in Fig. 7. The increases in
almost all parameters, except for the sucrose content, in the
upper leaf of TpF plants were associated with the increase of
FBPase activity. However, there were no relationships between
these parameters and the enzyme activities in the TpS plants
with a >1.6-fold increase in the SBPase activity.
Discussion
Fig. 5 Rubisco activity and RuBP content. (A) Rubisco activity was
measured in the same extract prepared from the fourth leaflet from the
top on 12-week-old plants. The results are given as the mean ± SD of
12 extracts, each from a separate plant. Each shaded bar represents the
initial activity. The white bar represents the in vivo inactivated state.
(B) The level of RuBP was measured in the fourth leaf from the top at
6 h after illumination. Values without a common letter are significantly different according to the t-test (P < 0.05).
other hand, there were no differences in the initial activities of
Rubisco among the TpF-9, TpS-2 and wild-type plants.
Levels of RuBP and carbohydrates
The contents of RuBP were compared in the fourth leaves
of the wild-type and transgenic plants at 6 h in the light period.
In TpF-11, TpS-11 and TpS-10 plants, the contents of RuBP
were 1.4-, 1.7- and 1.7-fold those of the wild-type plants, respectively (Fig. 5B). On the other hand, there was no difference in
the content of RuBP between TpF-9, TpS-2 and wild-type plants.
The levels of hexose, sucrose and starch were measured in
the fourth leaf from the top (upper leaves) and the third leaf
In a previous report, we demonstrated that the overexpression of a cyanobacterial FBP/SBPase in chloroplasts
enhanced photosynthesis and the growth rate (Miyagawa et al.
2001). Thus, we suggested that FBPase and/or SBPase, which
are involved in the regeneration of RuBP, are the limiting factors that participate in the regulation of the carbon flow through
the Calvin cycle. To examine the substantial contribution of the
level of chloroplastic FBPase and/or SBPase to CO2 fixation,
RuBP regeneration and biosynthesis of photosynthates, and the
mechanisms of increased photosynthesis, here we generated
transgenic tobacco plants expressing cyanobacterial FBPase-II
or Chlamydomonas SBPase in chloroplasts.
The transformants with increased FBPase or SBPase
activity in chloroplasts (TpF-11, TpS-11 and TpS-10) showed
enhanced phothosynthetic activity under saturated light conditions (1.15-, 1.23- and 1.27-fold) and growth characteristics
under atmospheric conditions (360 ppm CO2) (Fig. 4). The
levels of RuBP in TpF-11, TpS-11 and TpS-10 were 1.4-, 1.7and 1.7-fold higher, respectively, than those in the wild-type
plants (Fig. 5B). In these lines, the Rubisco activation states
were 1.1- to 1.2-fold that of the wild-type plants. These find-
386
Role of FBPase and SBPase in the Calvin cycle
Fig. 6 Contents of hexose, sucrose and
starch. The levels of hexose, sucrose
and starch were measured in the fourth
leaf from the top (upper leaves) and
third leaf from the bottom (lower
leaves) at 12 h in the light period (open
bars) and 12 h in the dark period
(shaded bars). Values without a common letter are significantly different
according to the t-test (P < 0.05).
ings clearly indicated that the enhancement of either more than
a 1.7-fold increase of FBPase or a 1.3-fold increase of SBPase
in the chloroplasts had a marked positive effect on the process
of RuBP regeneration, resulting in an enhancement of the level
of RuBP, an increase in the initial activity of Rubisco and thus
an increase of the photosynthetic rate in the chloroplasts of the
transgenic plants.
TpS-11 showed the same dry weight as TpS-10 given that
it had less of an increase in SBPase activity. Furthermore, as
shown in Fig. 4–7, the hexose content (upper leaves), sucrose
content (upper and lower leaves) and starch content (lower
leaves) in TpS-10 were almost the same as those in TpS-11.
However, the photosynthetic activity under saturated light
conditions, RuBP content, Rubisco activation state and starch
content (upper leaves) in TpS-11 were significantly higher
than those in TpS-10, indicating that the increases in these
parameters were not correlated with the increase in the
SBPase activity. Accordingly, it seems likely that an increase of
carbon flow in the Calvin cycle with an increase in SBPase
activity is not necessarily associated with increases of sucrose
biosynthesis and biomass under the present experimental
conditions.
Role of FBPase and SBPase in the Calvin cycle
387
Fig. 7 Comparison of FBPase- and SBPase-dependent
changes of photosynthetic characteristics in transgenic
plants. Photosynthetic activities under saturated light
conditions (light intensity of 1,500 µmol m–2 s–1 in Fig.
4) and the contents of hexose, sucrose and starch measured at 12 h in the light period (Fig. 5) were plotted.
Fructose 6-phosphate (F6P), the product of the reaction
catalyzed by FBPase, is the branch point for metabolites leaving the Calvin cycle and moving into starch biosynthesis
through the conversion into glucose 6-phosphate (G6P), and
phosphorylated intermediates are converted to sucrose and
exported from the leaf or stored as starch and remobilized during the subsequent night, and used for plant growth (Koßmann
et al. 1994). In TpF-11 plants, the excess F6P generated with
cyanobacterial FBPase-II may be converted into erythlose 4phosphate (E4P) and xylulose 5-phosphate (X5P) in the Calvin
cycle or into G6P in the starch biosynthesis pathway. E4P may
be immediately converted into sedoheptulose 1,7-bisphosphate
(SBP) by aldolase, and SBP may accumulate in the chloroplasts. It has been reported previously that in aldolase antisense plants, the activities of FBPase and SBPase are
decreased, accompanied by a decrease in the plastid aldolase
activity, suggesting that the change of the levels of FBP and
SBP caused by the decrease in plastid aldolase activity has a
marked effect on the stability of FBPase and SBPase (Haake et
al. 1998). Thus, in the TpF-11 plants, the accumulated SBP
may contribute to the stability and in vivo activity of endog-
enous SBPase in the chloroplasts. As a result, the RuBP regeneration and photosynthesis were increased in the TpF-11 plants
with only the enhancement of FBPase.
On the other hand, we found that there were no differences in the photosynthetic activity or the growth between the
wild-type plants and the TpF-9 plants, although the chloroplastic FBPase activity of the transgenic plants was 1.7-fold
higher than that of the wild-type plants (Fig. 2, 4). The initial
activity of Rubisco in TpF-9 was the same as that in wild-type
plants (Fig. 5A). Moreover, TpF-9 plants did not show any
changes in the contents of RuBP compared with the wild-type
plants (Fig. 5B). In our previous paper, however, TpFS-6,
which has both 1.7-fold higher FBPase and SBPase activity
than wild-type plants, showed increased levels of both RuBP
and photosynthetic CO2 fixation capacity under atmospheric
conditions (Miyagawa et al. 2001). Accordingly, it is likely that
enhancement of the FBPase activity by <1.7-fold has no effect
on the level of RuBP and thus on the photosynthetic activity
without an increase in SBPase activity.
Interestingly, the levels of starch in the upper leaves of
TpF-9 plants were slightly increased compared with those in
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Role of FBPase and SBPase in the Calvin cycle
wild-type plant leaves (Fig. 6). A decrease in plastidic FBPase
activity or aldolase activity led to the strong inhibition of starch
synthesis (Koßmann et al. 1994, Haake et al. 1998). FBPase
and aldolase lie upstream of the site at which carbon was withdrawn for starch synthesis. These data indicated that the increase
in the FBPase activity in chloroplasts leads to the increase in the
amount of F6P and then the increase in the level of F6P changes
carbon flux into starch synthesis in chloroplasts in the absence
of enhancement of the photosynthetic activity.
In the case of TpS-2, a 1.3-fold only increase in the
SBPase activity did not accelerate the photosynthesis or
growth. In TpS-2 plants, slightly increased SBPase may function to catalyze the dephosphorylation of SBP into sedoheptulose 7-phosphate (S7P), and then S7P was used for the
regeneration of RuBP. This phosphorylated intermediate was
immediately changed into dihydroxyacetone phosphate
(DHAP), and then DHAP was transferred into the cytosol and
used for sucrose biosynthesis. As a result, the concentration of
SBP was decreased, leading to the instability of endogenous
SBPase in the chloroplasts of TpS-2 plants.
It is interesting to note that the 1.6-fold increase in the
SBPase activity causes increased photosynthesis, while the 1.7fold increase in the FBPase activity did not (Fig. 4). Antisense
potato plants that displayed 36% of the wild-type level of
FBPase activity showed photosynthetic activity similar to the
wild-type plants, whereas the photosynthesis and growth rate
were drastically inhibited when the FBPase activity was
decreased to <14% of the wild-type level (Koßmann et al.
1994). The antisense inhibition of SBPase activity had a
stronger effect on photosynthesis (Harrison et al. 1998, Harrison et al. 2001, Ölçer et al. 2001). In the SBPase antisense
plants that retained 71% of the wild-type level of SBPase activity, the light-saturated photosynthetic activity was reduced by
36%. These data indicate that the photosynthesis, carbon partitioning, carbon flow of the Calvin cycle and plant growth are
remarkably sensitive to small decreases of SBPase activity.
Modeling studies predicted that SBPase has the greater control
over the photosynthetic flux (Fridlyand et al. 1999, Poolman et
al. 2001).
The data reported here, together with the findings reported
so far, suggest that an increase in the chloroplastic FBPase or
SBPase level correlates with an increase of the RuBP level
through the regeneration of RuBP, and thus affects the photosynthetic capacity and the growth in transgenic plants, and that
the SBPase is the primary limiting factor for the RuBP regeneration in the Calvin cycle, and a >1.6-fold increase of SBPase
activity caused a shift to FBPase as the limiting step of the
photosynthetic carbon fixation (Fig. 7). A slight increase in the
FBPase activity seems to contribute to starch synthesis rather
than to RuBP regeneration in chloroplasts (Fig. 7), indicating
that the level of chloroplastic FBPase strictly controls the regeneration of RuBP in the Calvin cycle and the starch synthesis.
Raines (2003) and Lefebvre et al. (2005) have reported
that in Arabidopsis SBPase-overexpressing tobacco plants with
10–65% increased activity, the photosynthetic rate in the young
expanding leaves was 12% higher than that in the equivalent
leaves on the wild-type plants. Furthermore, shoot biomass in
the transgenic plants was increased by 40% compared with the
wild type. There was no significant difference in SBPase activity between the young and old leaves of the wild type or
Arabidopsis SBPase-overexpressing plants. However, no significant increases in photosynthesis and biomass were observed
in fully expanded leaves. They supposed that, in the mature
leaves of Arabidopsis SBPase-overexpressing plants, additional
limiting factors, such as the availability of water, control the
flux of carbon through the Calvin cycle. In contrast to their
data, however, photosynthetic activities and biomass in fully
expanded leaves in TpFS, TpF and TpS plants were higher than
those in the wild-type plants (Miyagawa et al. 2001, and this
study). Lefebvre et al. (2005) considered that these differences
could be due to the fact that a bifunctional cyanobacterial
enzyme is introduced, that transgenic plants are grown in
hydroponic culture under low light conditions and/or that the
tobacco cultivar used is Xanthi. Recently, we found that the
same phenomena are observed in transgenic plants grown in
soil culture under normal light conditions or in hydroponic
culture under high light conditions (data not shown). Furthermore, they have used the cauliflower mosaic virus promoter so
that Arabidopsis SBPase is constitutively expressed in the
transgenic plants (Lefebvre et al. 2005). It is well known that
the transcriptional levels of SBPase are regulated under
light/dark conditions in the chloroplasts of higher plants
(Willingham et al. 1994, Hahn et al. 1998) and the constitutive
expression of thiol-modulated enzymes, including SBPase, in
the dark has negative effects on the carbon flow in chloroplasts
of higher plants (Buchanan 1991, Scheibe 1990). In contrast,
we have used the rbcS promoter that regulates light-dependent
transcription (Miyagawa et al. 2001, and this study) and found
that protein levels of the exogenous FBPase and SBPase significantly decreased in the dark periods (Fig. 1C). These facts also
seem to account for the differences in photosynthetic activities
and biomass in fully expanded leaves between tobacco plants
overexpressing cyanobacterial enzymes (TpFS, TpF and TpS)
and Arabidopsis SBPase-overexpressing tobacco plants. Judging from the data reported here, together with the findings
reported so far, it is clear that increases in SBPase and/or
FBPase activities in the Calvin cycle impact on the photosynthesis and biomass of plants. Accordingly, there is fairly general agreement that the manipulation of one enzyme involved
in the Calvin cycle improves crop yield by manipulating photosynthetic carbon fixation capacity.
Materials and Methods
Construction and transformation of chimeric fbp-II and sbp genes
The fbp-II and sbp genes were fused, respectively, with the gene
encoding the promoter and transit peptide of a tomato rbcS3C gene
(Sugita et al. 1987) and cloned into pBI101 (data not shown). These
Role of FBPase and SBPase in the Calvin cycle
constructs were then introduced into leaf disks derived from tobacco
(N. tabacum cv. Xanthi) using Agrobacterium-mediated transformation. Transgenic plants were regenerated from kanamycin-resistant
calli and planted in the soil as described previously (Shikanai et al.
1998).
Growth conditions
Wild-type tobacco plants and transgenic plants overexpressing
the cyanobacterial fbp-II gene (TpF; T2) and sbp gene (TpS; T2) were
hydroponically grown in a growth chamber under a 12 h light (25°C)
and 12 h dark (20°C) cycle with a light intensity of 400 µmol m–2 s–1,
360 ppm CO2 and 60% relative humidity (Miyagawa et al. 2001).
Homozygous T2 generation plants were used for all experiments. Photosynthesis, enzyme activities and RuBP were measured in the same
fourth leaflet from the top on 12-week-old plants. Carbohydrates were
measured in the fourth leaf from the top (upper leaves) and third leaf
from the bottom (lower leaves) on 12-week-old plants. Samples were
taken for enzyme assays and the determination of metabolite and carbohydrate levels from the same leaves on the same plants used for gas
exchange and chlorophyll fluorescence measurements. Root tissue was
separated from the growth medium by washing the roots with a running stream of water and soaking the roots in water several times.
Roots and shoots were dried for 3 d at 130°C in a forced air-drier.
Measurement of photosynthetic activity
CO2 fixation was measured as previously described (Miyagawa
et al. 2001) using a portable photosynthesis system, the LI-6400
(Li-Cor, Lincoln, NE, USA). Net CO2 assimilation rates were measured using fully expanded leaves under the following conditions: 0–
1,500 µmol m–2 s–1, 0–1,000 µmol CO2 mol–1, 25°C and 60% relative
humidity.
Protein extraction and immunoblotting
Protein extraction and immunoblotting were carried out according to previously described methods (Miyagawa et al. 2001). Total soluble proteins (30 µg) of chloroplasts isolated from wild-type and
transgenic tobacco plants were used for Western blotting with the
mouse antibodies raised against Synechococcus PCC7942 FBPase-II
and Chlamydmonas SBPase, as previously described (Tamoi et al.
1996, Tamoi et al. 2005).
Preparation of crude extract and enzyme assays
Leaf tissues (1.1 cm2×10 disks) were harvested at 6 h in the light
regime and ground to a fine powder in liquid N2 using a mortar and
pestle. For all enzyme assays, except for the Rubisco assay, leaf tissues were homogenized with 1 ml of 100 mM Tris–HCl buffer (pH
8.0), 16 mM MgCl2, 1 mM EDTA, 20 mM dithiothreitol (DTT), 2%
(w/v) insoluble polyvinylpyrrolidone (PVP), 0.05% Triton X-100 and
2 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged for 10 min at 15,000×g and the supernatant was used for enzyme
assays. Preparation and assays of the initial and total activities of
Rubisco and FBPase were carried out according to the cited methods
(Sawada et al. 1990, Holaday et al. 1992). Rubisco activity was measured by coupling the activity of Rubisco to NADH oxidation using
phosphoglycerate kinase and NAD-GAPDH as the decrease in absorbance at 340 nm. Leaf tissues (1.1 cm2×5 disks) were ground to a fine
powder in liquid N2 and then homogenized with 3 ml of 100 mM Tris–
HCl buffer (pH 7.8), 5 mM MgCl2, 1 mM EDTA, 5 mM DTT, 0.02%
bovine serum albumin (BSA) and 2% PVP. The homogenate was centrifuged for 30 s at 15,000×g. Aliquots of the supernatant were assayed
to determine the initial activity of Rubisco. To measure the total
Rubisco activity, 1 ml of the initial extract was transferred to a new
tube, the MgCl2 concentration was brought to 20 mM, and then the
389
NaHCO3 concentration was brought to 10 mM. This mixture was kept
on ice for 10 min before assaying for total Rubisco activity. The reaction mixture contained 50 mM HEPES-KOH (pH 8.0), 15 mM MgCl2,
20 mM NaCl, 10 mM DTT, 0.5 mM ATP, 0.2 mM NADH, 5 mM
phosphoenol-pyruvate, 5 mM creatine phosphate, 10 mM NaHCO3,
20 U of pyruvate kinase, 2 U of phosphocreatine kinase, 18 U of
NAD-GAPDH, 18 U of phosphoglycerate kinase, 1 mM ribulose 1,5bisphosphate and the enzyme solution. The SBPase activity was determined by measuring the rate of NADH oxidation as the decrease in
absorbance at 340 nm (Tamoi et al. 1998). The reaction mixture contained 100 mM Tris–HCl buffer (pH 8.0), 10 mM MgCl2, 20 mM KCl,
20 mM DTT, 0.1 mM ATP, 0.15 mM NADH, 1 mM phosphoenolpyruvate, 2 U of pyruvate kinase, 2 U of lactate dehydrogenase,
0.5 U of 6-phosphofructokinase, 0.1 mM SBP and the enzyme solution. SBP was synthesized from DHAP and E4P with an enzyme reaction with recombinant cyanobacterial aldolase (Tamoi et al. 2005).
Assays of enzyme activities involved in carbon metabolism were carried out according to previously described methods (Miyagawa et al.
2001). Protein was determined using the method of Bradford (1976)
with BSA as a standard. Chlorophyll was measured according to the
method of Arnon (1949).
Determination of metabolite and carbohydrate levels
Plant tissues were harvested in the growth chamber after a 12 h
light period or 12 h dark period. They were plunged immediately into
liquid N2 and stored at –80°C until required under respective light or
dark conditions. The tissues (0.2 g FW) were ground in liquid N2 using
a mortar and pestle with 2 ml of 5% HClO4. The extract was allowed
to thaw and was then was centrifuged at 12,000×g for 10 min. Next,
the supernatant fraction was brought to a neutral pH value by adding
5 M KOH–1 M triethanolamine solution and then centrifuged at
12,000×g for 10 min. The supernatant was used for the determination
of soluble sugars (hexose and sucrose) and some metabolites. The levels of sugars (hexose and sucrose) as well as RuBP were measured
enzymatically, as described by Leegood (1993). The level of starch
was measured using the method of Lin et al. (1988). Plant leaves and
roots (0.2 g FW) were extracted three times with 80% ethanol–50 mM
HEPES (pH 7.5) at 70°C. The extract was suspended in 5 ml of 0.2 M
KOH, incubated at 100°C for 30 min and adjusted to pH 5.5 with 1 M
acetic acid. Starch was hydrolyzed with α-amylase and amyloglucosidase, and the released glucose was analyzed enzymatically, as
described by Jones et al. (1977).
Acknowledgments
This work was supported by the Japan Society for the Promotion
of Science Research for the Future Program grant (No. JSPS-RFTF
00L01604) and in part by an ‘Academic Frontier’ Project for Private
Universities: matching fund subsidy from MEXT 2004–2008, and by
CREST, JST.
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(Received October 5, 2005; Accepted January 3, 2006)