Plant Production Science

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Diurnal Changes in Photosynthesis in Sugarcane

Leaves: I. Carbon dioxide exchange rate,
photosynthetic enzyme activities and metabolite
levels relating to the C4 pathway and the Calvin
cycle

Yu-Chun Du, Akihiro Nose, Ayumu Kondo & Kikuo Wasano

To cite this article: Yu-Chun Du, Akihiro Nose, Ayumu Kondo & Kikuo Wasano (2000)
Diurnal Changes in Photosynthesis in Sugarcane Leaves: I. Carbon dioxide exchange rate,
photosynthetic enzyme activities and metabolite levels relating to the C4 pathway and the
Calvin cycle, Plant Production Science, 3:1, 3-8, DOI: 10.1626/pps.3.3

To link to this article: https://doi.org/10.1626/pps.3.3

© 2000 Crop Science Society of Japan

Published online: 07 Dec 2015.

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 Plant Prod. Sci. 3 ( 1) : 3-8 (2000)

Diurnal Changes in Photosynthesis in Sugarcane Leaves

I. Carbon dioxide exchange rate, photosynthetic enzyme activities and
metabolite levels relating to the C 4 pathway and the Calvin cycle

Yu-Chun Du*, Akihiro Nose, Ayumu Kondo and Kikuo Wasano

(Faculty of Agriculture, Saga University, Honjo-machi, Saga 840-8502, Japan)

Abstract : Diurnal changes in carbon dioxide exchange rate (CER), stomatal conductance (g5 ) , photosynthetic
enzyme activities and metabolite levels relating to the C4 pathway and the Calvin cycle in sugarcane (Saccharum sp.
cv. NiF4) leaves were characterized during a natural 24 h day-night cycle. The activities of phosphoenolpyruvate
carboxylase (PEPcase), NADP-malic enzyme (NADP-ME), pyruvate, Pi dikinase (PPDK), ribulose-1,5-
bisphosphate carboxylase/ oxygenase (Rubisco) and chloroplast fructose- I ,6-bisphosphatase (FBPase) all exhibited
distinct diurnal changes. The levels of C4 metabolites, malate, phosphoenolpyruvate (PEP) and pyruvate, showed
large diurnal fluctuations, but the oxaloacetate levels were extremely low and exhibited no significant changes in the
day-night cycle. Diurnal changes in CER and g5 paralleled the changes in radiation of sunlight. The results in this
study suggest that at midday when CER is in steady-state and saturated by full sunlight, CER is limited by PPDK
activity; under lower radiation of sunlight, i.e., before CER is saturated in the morning and during the period when
CER is decreasing in the afternoon, CER is possibly limited by PEPcase activity or g8 •

Key words : C4 metabolites, C 4 photosynthesis, Diurnal changes, Photosynthetic enzymes, Sugarcane.

During the natural day-night cycle, light is the most high photosynthetic activity and high productivity
important environmental factor controlling the diurnal (Murata, 1981). It has been established that the high
changes of photosynthesis in C 4 plants (Moss et al., photosynthetic activity in c4 plants is mainly attributed
1961) . Among the photosynthetic enzymes, PPDK and to the c4 pathway function of concentrating atmospheric
NADP malate dehydrogenase in the C4 pathway and C0 2 for the fixation by Rubisco in the Calvin cycle.
chloroplast FBPase in the Calvin cycle are reported to be However, despite the importance of the C 4 pathway in
light-activated (Usuda et al., 1984a). Light-modulation c4 photosynthesis, the diurnal changes in enzyme activ~
of PEPcase is largely dependent on the pH at which it is ities and metabolite levels associated with the C 4 path-
assayed and other factors (Karabourniotis et al., 1985 ; way have not been well investigated.
Huber and Sugiyama, 1986 ; Doncaster and Leegood, In this study, in order to investigate the diurnal char-
1987). The response of Rubisco to light is different acteristics of photosynthesis, and hence to elucidate the
between C 3 and C 4 plants : Rubisco activity in C 3 plants regulation mechanisms of photosynthetic carbon assimi~
is largely modulated by light, but not as clearly in c4 lation in sugarcane leaves, we measured the diurnal
plants (Usuda, 1985b; Servaites et al., 1986). changes in gas exchange, enzyme activities and
In addition to light, photosynthetic enzymes in C 4 metabolite levels associated with the c4 pathway and the
plants are regulated by a number of factors, most of Calvin cycle during a 24h day-night cycle under natural
which are metabolites. For example, at a low PEP con- climatic conditions.
centration, PEPcase activity is strongly activated by
Materials and Methods
glucose-6-phosphate, fructose-6-phosphate and triose-
phosphates, but inhibited by malate (Doncaster and 1. Plant culture
Leegood, 1987). Therefore, the photosynthetic enzyme Sugarcane (Saccharum sp. cv. NiF4) seed cane sections
activity in vivo in plant leaves at any given point during were germinated in vermiculite at the end of April 1995
photosynthesis reflects the combined effects of light, in the glasshouse for two weeks, and then the seedlings
metabolites and other factors at that time. were transplanted to the field (Faculty of Agriculture,
Sugarcane is a typical NADP-ME subgroup C 4 plant. Saga University, Saga, Japan) at intervals of 0.4 meters
Along with other NADP-ME subtype C 4 plants, such as in the row, 1.2 meters apart. In the middle of June and
,Zea mays and Sorghum vulgare, sugarcane has been consid- August, a commercial fertilizer was applied to the soil at
ered a representative of this subgroup, and shows both the concentration of 4 kg nitrogen, 6.4 kg m- 2 each of
Received 4 August 1997. Accepted 16 July 1999. Corresponding author: A. Nose ([email protected], fax +81-952-28-8737).
*Present address: Department of Biochemistry, University of Nebraska, Lincoln, NE 68588·0664.
Abbreviations: CER, carbon dioxide exchange rate; Chl, chlorophyll; FBPase, fructose-1,6-bisphosphatase; gs, stomatal conductance;
NADP-ME, NADP-malic enzyme; PAR, photosynthetically active radiation; PEP, phosphoenolpyruvate; PEPcase, phosphoenol-
pyruvate carboxylase; PPDK, pyruvate, Pi dikinase; Rubisco, ribu1ose-1,5-bisphosphate carboxylase/oxygenase.

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 4 Plant Production Science Vol. 3, 2000

phosphorus and potassium per hectare. On dry days, tap 50

water was supplied to the plant roots. During the growth ....,__ 40
A 2000

period, the plants were not infected by any serious plant

diseases or insect pests.
~
1"4
'el 30
1500
-
....
'fll
1"4
'a '5
5 1000 'a
~ 20
2. Gas exchange E!
u~
~
All measurements were conducted on a clear day in
~~
10 500
early September 1995, about four and a half months
0 0
after planting. Gas exchange was measured with a
Portable Photosynthesis System (LI-6200, Li-COR, Inc., 0.30 8
USA) using the uppermost fully expanded leaves.
Measurements were conducted in the fields at a relative
-
't
0.20
~
humidity of 60%, a C0 2 concentration of 350 ,uL L - 1, 5
'a
and under natural sunlight. Gas exchange was measured 5
~ 0.10
at intervals of 1 h from predawn to evening darkness. be
0.00
3. Determinations of enzyme activities and 2 4 6 8 10 12 14 16 18 20 22 24 2
metabolite contents Time of day (hours)
All samples for the assays of enzyme activities were
taken at intervals of 2 h during a natural day-night cycle Fig. 1. Diurnal patterns of PAR and CER (A), and gs (B) in
sugarcane leaves during a natural day-night cycle. The results
from the uppermost fully expanded leaves, on which gas
in CER and g8 are means ± SE of four to six separate plants.
exchange was measured. Samples were taken just after
The solid bar and open bar at the bottom of the figure
gas exchange measurements during the day. Leaf sec- indicate darkness and light, respectively.
tions from three to four separate plants were cut off and
immediately immersed in liquid nitrogen. Before analysis,
leaf samples were stored at 80°C. Special care was
Results
taken to ensure that all samples were transferred directly
from liquid nitrogen to a freezer at 80°C without 1. Diurnal changes in CER and g5
exposing any sample to room temperature. Figure IA shows the diurnal patterns of PAR of
Leaf pieces (4 cm 2) were ground in 4 mL of extraction sunlight and CER in sugarcane leaves. The maximum
medium with 0.2 g sea sand and 40 mg of CER was observed at midday, and the diurnal CER
polyvinylpolypyrrolidone. The extraction medium changes paralleled the changes in PAR, indicating that
contained 50 mM Tris-HCl of pH 7.8, 8 mM MgC12 , 1 photosynthesis in sugarcane leaves was almost fully
mM EDTA-NaOH of pH 7.0, 5 mM dithiothreitol, 2.5 controlled by sunlight. This feature is different from C 3
mM pyruvate, 0.2% (w/v) bovine serum albumin and plants such as soybean, whose photosynthesis is saturat-
0.02% (w/v) Triton X-100. The homogenate was filter- ed during the early morning (Rufty et al., 1983). Figure
ed through one layer of Mira-cloth (Calbiochem- I B shows the diurnal changes in g5 • Like CER, g5 also
Novabiochem, La Jolla, CA, USA) and the filtrate was showed its maximum level at midday, and the diurnal
centrifuged at 12000 rpm for 30 s (Diskboy Kurabo changes in g5 were closely aligned with the changes in
Fb4000) at room temperature. The supernatant was CER and PAR. Under the field conditions, because leaf
used immediately for assays of PEPcase, NADP-ME, surfaces were wet with dew before 0800 h in the morning,
PPDK, Rubisco and chloroplast FBPase. it was difficult to measure the true g5 at 0600 hand 0700
PEPcase, NADP-ME, Rubisco and PPDK were as- h. For this reason, the data on g5 at 0600 h and 0700 h
sayed as described previously (Du et al., 1996). Chloro- were not used here.
plast FBPase activity was assayed in a mixture (I mL)
containing 50 mM Tris-HCl of pH 8.0, 10 mM MgC1 2 , 1 2. Diurnal changes in enzyme activities
mM EDTA-NaOH of pH 7.0, 0.5 mM NADP, 0.1 mM Figure 2 shows the diurnal changes in activities of
fructose-1,6-bisphosphate, 10 units each of G6P isomer- some important enzymes associated with the C 4 path-
ase and G6P dehydrogenase, and 20 Ji.L extract way and the Calvin cycle. PEPcase activity increased
(Holaday et al.,l992). rapidly in the morning and decreased slowly in the
For the determinations of metabolite contents, samples evening and at night. At 0400 h, PEPcase activity was
were taken and stored using the same protocol as was 134 ,umol m- 2 s- 1 • However, this activity increased by
used for the enzymes. The extraction and determinations 170% to 360 ,umol m- 2 s- 1 at 1400 h (Fig. 2A). Compar-
of the metabolites were carried out as described previous- ed with the changes in PEPcase, NADP-ME activity
ly (Du et al., 1998). showed fewer changes (Fig. 2A). The maximum activity
of NADP-ME at 1400 h was only 40o/0 higher than the
activity in the dark.

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 Du et al. --Diurnal Changes in C4 Photosynthetic Enzymes and Metabolites in Sugarcane 5
,-. 3000~------------------------------~
'7{ll
q'400 e PEPcase A iu 2500
N
{ll
0 NADP·ME
260
1:'! A

e 300
'! e
...'bll
220
-5 E2ooo
e
-5
200
~ 1
!,1500
~
Cll
180
! ~
z
i
'i1000
ft 100 140 ~

-
'7{ll
40 0 Chi FBPase
e PPDK B 30
....,-.
~
l:ll
N
e
e 30 '!
....
'bll 600
'! 20 e e
e 20 5 c
-5
~
i: 10
10
! ! 400

~ =
r,;r.
:a
~
ell
il>

-
'7{ll
0
60
0 Initial Rubisco
c
0
u
t
E
200

0
":'e so iu 70
c .... 60
e 40 'bll
-5 e 50
8
.~
.c 30 1
e
40
::1

~
=rli 30
w-..-.-~===============-_._..__._..
2 4 6 8 10 12 14 16 18 20 22 24 2 0 20
"=
Time of day (hours) i 10
~
0
Fig. 2. Diurnal changes in enzyme activities in sugarcane leaves ft 2 4 6 8 10 12 14 16 18 20 22 24 2
during a natural day-night cycle. A, PEPcase and NADP-
Time of day (hours)
ME; B, PPDK and chloroplast FBPase (Chl FBPase) ; C,
Rubisco. Data are means SE of three to four separate Fig. 3. Diurnal changes in metabolite levels in sugarcane leaves
plants. The solid bar and open bar at the bottom of the figure during a natural day-night cycle. A, malate ; B, pyruvate ; C,
indicate darkness and light, respectively. PEP and oxaloacetate (OAA). Leaf Chl content was 5.92 mg
dm- 2 • Data are means ± SE of three to four separate plants.
The solid bar and open bar at the bottom of the figure
Figure 2B shows the diurnal changes in the activities of
indicate darkness and light, respectively.
PPDK and chloroplast FBPase, both of which are known
to be light-activated (Usuda et al., 1984a). In the dark, 3. Diurnal changes in metabolite levels
PPDK was completely inactivated. However, the activity Figure 3 shows the diurnal changes in metabolite levels
increased rapidly with increasing PAR in the morning associated with the C 4 pathway. In the dark, the malate
and showed a maximum activity between 1000 h and pool size was approximately 850 nmol mg- 1 Chl, which
1400 h. After 1400 h, PPDK activity decreased sharply, was the largest pool size among the metabolites mea-
which also paralleled the changes in PAR. In contrast to sured (Fig. 3A). The malate level increased gradually
PPDK, chloroplast FBPase was not completely inactivat- through the morning until 1400 h, at which time malate
ed by darkness, but showed an activity of 10 ,umol m- 2 showed its peak of 2200 nmol mg- 1 Chl, or 160% more
s- 1 (Fig. 2B). Chloroplast FBPase activity increased with than the level in darkness. Between 1400 h and 1800 h,
increasing PAR in the morning, but the change was not the malate level decreased, and then was maintained at
as pronounced as that of PPDK, and the activity was not a relatively constant level in darkness. The pyruvate pool
saturated until 1200 h. After reaching a peak between size was small in the dark period, only 45 nmol mg- 1 Chl
1200 and 1400 h, chloroplast FBPase activity decreased (Fig. 3B). However, this level increased rapidly with
slowly and remained at a relatively constant activity of 10 increasing PAR in the morning, reaching its peak level of
,umol m- 2 s- 1 in darkness. 670 nmol mg- 1 Chi at 1200 h, a 15-fold increase. The
Figure 2C exhibits the diurnal changes in Rubisco pyruvate level then fell with decreasing PAR until 1800
activities. There were no significant differences between h, and was maintained at a low level of 50 nmol mg- 1 Chi
Rubisco initial and total activities at any time of the day, in darkness. After 0800 h the PEP level increased gradu-
in agreement with the results for Zea mays (Usuda, ally with increasing PAR until 1400 h, reaching its
1985b). In darkness, both initial and total Rubisco maximum level of 55 nmol mg- 1 Chi at 1400 h, and then
activities were about 30 ,umol m- 2 s- 1• Maximum activ- decreased slowly in the afternoon and during the night.
ities of about 50 ,umol m- 2 s- 1 were observed at between The oxaloacetate levels in sugarcane leaves were
1000 h and 1600 h, representing an increase of 65% extremely low and no significant diurnal changes were
relative to the activity in darkness. observed.

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 6 Plant Production Science Vol. 3, 2000

Table 1. Correlation coefficients between photosynthetic parameters in sugarcane leaves during a natural day-night cycle.

PAR g. PEPcase NADP-ME PPDK IRubisco TRubisco Chi FBPase Malate Pyruvate PEP OAA

CER 0.993** 0.936** 0.628 0.428 0.969** 0.640 0.683 0.954** 0.819* 0.950** 0.348 0.393
PAR 0.917** 0.650* 0.593 0.986** 0.669* 0.670* 0.938** 0.815** 0.958** 0.438 0.217
PEPcase 0.881 ** 0.574 0.976** 0.967** 0.745** 0.815** 0.712* 0.850** 0.696*
NADP-ME 0.531 0.854** 0.837** 0.675* 0.829** 0.667* 0.937** 0.609
PPDK 0.602* 0.615* 0.952** 0.773** 0.923** 0.336 0.167
IRubisco 0.991 ** 0.748** 0.751 * 0.669* 0.839** 0.691 *
TRubisco 0.788** 0.742* 0.711* 0.814** 0.641 *
Chi FBPase 0.847** 0.922** 0.521 0.396
Malate 0.869** 0.748* 0.600
Pyruvate 0.569 0.315
PEP 0.729*

1) Abbreviations used: IRubisco, initial Rubisco; TRubisco, total Rubisco; Chl FBPase, chloroplast FBPase; OAA, oxaloacetate.
2) *
and ** :
Significant at the 5% and 1% levels, respectively.

4. The relationships among the photosynthetic mays photosynthesis under atmospheric conditions
parameters (Usuda, 1987). Although the slower response of g8 to
Most of the photosynthetic parameters measured in changing PAR observed in this study, implies that g8
this experiment exhibited distinct diurnal changes. Diur- may limit CER in the morning and afternoon, i.e., under
nal changes in g8 , several enzyme activities and a low PAR, this response needs to be studied futher
metabolite levels were closely related to that of CER. because it is the inconsistent with the results from ,Zea
Table 1 shows the correlation coefficeints between the mays (Sharkey and Raschke 1981; Usuda, 1987).
photosynthetic parameters measured in this experiment. Usuda (1987) reported that PPDK activity was able
to limit the assimilation rate in ,Zea mays leaves under a
Discussion
low PAR, but Leegood and von Caemmerer (1989)
Before CER reached its peak rate at midday, both g8 suggested that PEPcase activity was the limiting factor
and CER increased with increasing PAR, but g8 in- on the assimilation rate in ,Zea mays and Amaranthus edulis
creased more slowly than CER (Fig. 1). For example, at leaves under a low PAR. In this study, PPDK activity
0800 h CER reached 64% of its maximum rate, while g8 increased faster than CER with increasing PAR in the
only reached 55% of its maximum conductance. morning and decreased faster than CER with decreasing
Compared with gs, PPDK and chloroplast FBPase activ- PAR in the afternoon. For example, at 0800 h in the
ities changed more rapidly in response to the change in morning PPDK activity reached 85% of its maximum
PAR. For instance, at 0800 h, PPDK and chloroplast activity, while CER only reached 64%; at 1600 h in the
FBPase activities reached more than 80% of their maxi- afternoon PPDK activity decreased by 42% relative to its
mum activities, respectively (Fig. 2). Similar patterns maximum activity, while CER had only decreased by
were also seen at other times in the morning and 32% relative to its maximum rate (Fig. 1A and 2B). If
afternoon (Fig. IB and 2B). These results suggest that gs PPDK was regulating the assimilation rate under those
may limit CER under a low PAR, i.e., before CER was conditions, CER and PPDK would be expected to
saturated in the morning and when CER was decreasing change at a similar pace. Therefore, the results in this
in the afternoon. However, Usuda and Edwards (1984) study suggest that PPDK does not limit sugarcane
reported that intercellular C0 2 pressure in ,Zea mays photosynthesis under a low PAR, i.e., before CER was
leaves was as high as 420 J.d L - 1 in darkness, far above saturated in the morning and when CER was decreasing
the intercellular C0 2 pressure during steady-state photo- in the afternoon.
synthesis under atmospheric C0 2 conditions approxi- In this study, the maximum PEP level in sugarcane
mately 150 ,u L L - 1 and that intercellular C0 2 pressure leaves was 55 nmol mg- 1 Chl, similar to that detected in
decreased during the period of light induction of photo- ,Zea mays (50 to 150 nmol mg- 1 Chi) leaves under high
synthesis. Sharkey and Raschke ( 1981) found that inter- light and atmospheric C0 2 conditions (Usuda, 1985a ;
cellular C0 2 pressure in ,Zea mays leaves was approxi- Usuda, 1987; Leegood and von Caemmerer, 1989). This
mately the same under low PAR (50 W m- 2 ) and under PEP concentration in plant leaves is considered to be
high PAR (620 W m- 2). Mainly based on the above kinetically insufficient for PEPcase (Leegood and von
results, it has been suggested that g8 does not limit ,Zea Caemmerer, 1989). In this study, the PEP level in sugar-

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 Du et al. --Diurnal Changes in C4 Photosynthetic Enzymes and Metabolites in Sugarcane 7

cane leaves was low in darkness and increased with greater differences in PEPcase activity during dark to
increasing PAR in the morning (Fig. 3C). However, light transition in Zea mays when assayed in simulated
after PAR reached more than one-half full sunlight, i.e., conditions of dark and light or in the presence of ef-
after 0800 h, the PEP level in leaves was still low, only fectors (Huber and Sugiyama, 1986; Doncaster and
37% of the maximum level (Fig. 3C). In C 4 photosynthe- Leegood, 1987).
sis, PEP mainly comes from two sources. First, PPDK Under full sunlight at midday from roughly 1100 h to
catalyzes the conversion of pyruvate to PEP. Second, 1400 h, CER in sugarcane leaves was saturated (Fig.
phosphoglycerate mutase and enolase create an equilib- lA). During this period, all light-modulated enzymes
rium of 3-phosphoglycerate and PEP (Leegood and von measured, i.e., PEPcase, PPDK, Chloroplast FBPase and
Caemmerer, 1988, 1989). In this study, the diurnal Rubisco, were almost fully activated (Fig. 2). Although
change of PEP in sugarcane leaves was markedly CER and photosynthetic enzyme activities were constant
different from that of PPDK. For example, PPDK activ- at their highest levels (except N ADP-ME) during this
ity increased rapidly after sunrise, but the PEP level period, metabolite levels fluctuated (Fig. 3), indicating
increased at a much slower pace; in the afternoon at that carbon flux was not in a steady-state. As discussed
around 1600 h, PPDK activity decreased sharply, while above, during photosynthesis, PEP mainly comes from
the PEP level decreased slowly (Fig. 2B and 3C) . These two sources, pyruvate or 3-phosphoglycerate. Although
results suggest that the PEP level in sugarcane leaves is we do not know how much of the total PEP in sugarcane
not closely related to PPDK activity. In other words, the leaves during saturation period of photosynthesis was
catalysis of phosphoglycerate mutase and enolase might derived from the catalysis of PPDK, the fact that 3-
be the main factor for maintaining the PEP level in phosphoglycerate content in leaves of C 4 plants becomes
sugarcane leaves under a low PAR. This result is consis- relatively constant under higher PAR (Usuda, 1987;
tent with Leegood and von Caemmerer's (1989) results Leegood and von Caemmerer, 1988,1989) suggests that
with Zea mays and Amaranthus edulis, which showed that a larger proportion of the total leaf PEP would depend
PEP and 3-phosphoglycerate levels in those plant leaves on the catalysis of PPDK under a high PAR. In other
were closely correlated. words, after the 3-phosphoglycerate content reaches the
PEPcase is a highly regulated enzyme with high affin- stable stage, a further increase of the PEP content in the
ity for bicarbonate but low affinity for PEP (O'Leary, leaves during photosynthesis may have to depend on the
1982). The S0. 5 (PEP) for PEPcase was estimated to be catalysis of PPDK. That is to say, PPDK may play a
in the range of 1-10 mM when the enzyme was measured regulatory role in sugarcane photosynthesis under a high
in a simulated cytosolic environment (Doncaster and PAR. The relationships between photosynthetic enzyme
Leegood, 1987). However, the actual PEP concentration activities and CER in C 4 plants under a high PAR have
in sugarcane leaves (Fig. 3) was much lower than the been well documented. PPDK has been viewed as a
PEP concentration needed for PEPcase activity, espe- limiting enzyme for C 4 photosynthesis under a high PAR
cially under a low PAR. For example, after PAR reached and atmospheric C0 2 partial pressures (Usuda, 1984 ;
more than one-half full sunlight, i.e., after 0800 h, the Usuda et al., 1984b). The results in this study are
PEP level was only 37% of the maximum level (Fig. 3C). consistent with those findings, though the approach to
Assuming that 50% of the PEP is in the cytosol (Usuda, the problem in this study was different from the
1988), the intracellular PEP concentration in sugarcane previous ones (Usuda, 1984; Usuda et al., 1984b).
leaves at 0800 h was estimated to be about 0.6 mM (ref.
Acknowledgments
Leegood and von Caemmerer, 1989). It is logical to
reason that at such a low PEP concentration the in vivo We thank Professor Y. Uchida (Faculty of Agricul-
PEPcase activity must be low. The present results sup- ture, Saga University, Japan) and Professor S. Mur-
port the previous hypothesis that PEPcase is an enzyme ayama (College of Agriculture, University of the Ryu-
that may regulate the assimilation rate in the leaves of C 4 kyus, Japan) for supporting this study, Mr. K. Oshima
plants under a low PAR (Leegood and von Caemmerer, for assistance in the field work, and Mr. K. Kinjo
1989). However, one may argue that since the PEPcase (Okinawa Agricultural Experiment Station) for supply-
activities detected in this study were much higher than ing the seed cane. We also thank Mr. John Szot
the corresponding CERs measured during the natural 24 (University of Nebraska-Lincoln, USA) for reading the
h day-night cycle (Fig. 1 and 2), how was PEPcase able manuscript and helpful discussions.
to limit CER? One possible explanation is that PEPcase
is a highly-regulated enzyme (O'Leary, 1982). Although
References
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between the maximum PEPcase activity in the daytime phoenolpyruvate carboxylase activity in maize leaves. Plant
and the minimum PEPcase activity in darkness (Fig. Physiol. 84 : 82-87.
2A), there may be more differences between the dark Du, Y.-C., Kawamitsu, Y., Nose, A., Hiyane, S., Murayama, S.,
and the light when the enzyme is measured in a more Wasano, K. and Uchida, Y. 1996. Effects of water stress on
precise way. It has been reported that there were much carbon exchange rate and activities of photosynthetic enzymes

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 8 Plant Production Science Vol. 3, 2000

in leaves of sugarcane (Saccharum sp.). Aust. J. Plant Physiol. diurnal changes in activities of enzymes involved in sucrose
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plants. An effect of pH. Plant Physiol. 77: 300-302. bisphosphate carboxylase in maize leaves in dark and light.
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