全 文 ：Received 16 Sept. 2003 Accepted 10 Nov. 2003
Supported by the National Natural Science Foundation of China (30370851).
* Author for correspondence.
http://www.chineseplantscience.com
.Rapid Communication.
Acta Botanica Sinica
植 物 学 报 2004, 46 (2): 137-141
Degradation of Rubulose-1, 5-Bisphosphate Carboxylase/Oxygenase in
Wheat Leaves During Dark-induced Senescence
RUI Qi, XU Lang-Lai*
(College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China)
Abstract: The degradation of Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39)
in wheat (Triticum aestivum L. cv. Yangmai 158) leaves during dark-induced senescence was studied. An in
vivo degradation product of Rubisco large subunit (LSU) with molecular weight of 50 kD was detected by
SDS-PAGE and immunobloted with antibody against tobacco Rubisco. This fragment could also be detected
in natural senescence. The result also suggested that the Rubisco holoenzyme had not dissociated when
LSU hydrolyzed from 53 kD to 50 kD. And LSU could be fragmented to 50 kD at 30-35 ℃ and at pH 7.5 in
crude enzyme extracts of wheat leaves dark-induced for 48 h, which suggested that maybe LSU was
degraded to 50 kD by an unknown protease in chloroplast.
Key words: Rubisco (EC 4.1.1.39); protease; proteolysis; leaf senescence; wheat
Ribulose-1,5-bisphosphate carboxylase/oxygenase
(Rubisco) (EC 4.1.1.39), accounting for more than 50% of
the soluble protein in the mature leaves of C3 plants, is the
most abundant protein in plant cells (Makino et al., 1983).
It is a bifunctional enzyme that catalyzes two competing
reactions, photosynthetic CO2 assimilation and
photorespiratory carbon oxidation in the stroma of the
chloroplasts. Thus the degradation of Rubisco is closely
related to both the rate of photosynthesis and nitrogen
economy in leaves. Although the synthesis, assembly,
structure and regulation of Rubisco have been studied
extensively, the mechanism of Rubisco degradation in
leaves are not clearly known yet (IIoutz and Portis, 2003).
Rubisco is a stromal protein that exists in chloroplasts.
The decrease of the amount of Rubisco was much faster
than the decrease of chloroplast number in many leaves
such as wheat and barley during senescence (Martinoia et
al., 1983; Mae et al., 1984). It has been suggested that the
initial step of Rubisco degradation in leaves must occur
within the chloroplasts and that the degradation may be
triggered by a Rubisco-specific protease or by a specific
modification of Rubisco protein, followed by general
proteolysis. Some researchers think that reactive oxygen
species (ROS) plays an important role in the Rubisco
degradation. Mehta (1992) observed that oxidative aggre-
gation between large subunits (LSU) and then transloca-
tion to the chloroplast membrane were crucial for its
degradation. But Peng and Peng (2000) pointed out that it
was not a necessary step for Rubisco breakdown. Now
many researchers used isolated chloroplasts or chloroplasts
lysates as model system, and treated them with some con-
ditions to study the degradation of Rubisco. Different deg-
radation products could be obtained under different condi-
tions (Mae et al., 1989; Desimone et al., 1996; Ishida et al.,
1997; Ishida et al., 1998; Kokubun et al., 2002). But so far,
there have been no reports in which in vivo degradation
products of Rubisco could be detected in leaves.
Proteolysis during leaf senescence is a genetically con-
trolled process. The results obtained using isolated chlo-
roplast as material cannot completely represent the condi-
tions in vivo. Using senescencing leaves as materials is
more important for studying the Rubisco degradation in
vivo. In this study, we investigated the degradation of
Rubisco in wheat leaves during dark-induced senescence.
An LSU fragment having an apparent molecular mass of 50
kD were detected in vivo.
1 Materials and Methods
Wheat (Triticum aestivum L. cv. Yangmai 158) seeds
were planted in pots with sandy clay and watered with
Hoagland’s complete nutrient solution. They grew in the
natural environment. Samples were harvested at approxi-
mately four weeks after planting and the fully expanded
leaves were used. Dark treatment was followed according
to Rui and Xu (2003). The leaves in vitro were stored in
dark at 25 ℃ to accelerate the senescence. To study the
Rubisco degradation in natural senescence, we selected
the leaves on different leaf positions as material (Zhang et
al., 2001). The wheat seedlings with five leaves were used.
The leaves from down to up were named as 1st, 2nd, 3rd,
Acta Botanica Sinica 植物学报 Vol.46 No.2 2004138
4th and 5th leaf and the 4th leaf was fully expanded. The 1st
leaf to the 4th leaf were selected.
The crude enzyme extract was prepared by grinding at a
ratio of 1 g leaf : 5 mL Tris-HCl buffer (50 mmol/L, pH 7.5)
containing 1 mmol/L EDTA, 3 mmol/L b-mercaptoethanol,
1% PVP and some sand quarts in ice bath. Homogenates
were centrifuged at 4 ℃ for 30 min at 10 000g. The superna-
tant was used as the crude enzyme extract or as sample for
SDS-PAGE after boiling for 5 min in equal volume of 2 ×
Laemmli sample buffer (Laemmli, 1970). The running gel
and stacking gel contained 12.5% and 3.5% acrylamide,
respectively. After electrophoresis, the gels were subjected
to staining with Coomassie Brilliant R-250 (CBB), or to
immunoblotting. If necessary, the gels stained with CBB
were scanned at 595 nm with a dual-wavelength TLC scan-
ner CS-930 (Shimadzu). For immunoblotting, the separated
polypeptides on the gel were electrophoretically transferred
to a nitrocellulose membrane with an electroblotting appa-
ratus (Biometra B33). The membrane was reacted with anti-
serum raised against the tobacco Rubisco (gifted from Dr.
CHEN G-Y), and further reacted with the second antibody
with horseradish peroxidase-conjugated goat anti-rabbit
IgG.
Rubisco was purified from wheat leaves dark-induced
for 0 h and 48 h as follows. The crude extracts were sub-
jected to natural PAGE (5%) (Davies, 1964). The region that
included the Rubisco was cut out from the gel plate and the
protein was electrophoretically recovered from the gel cut.
After elution, the protein was concentrated with concen-
trator (Amicon Microcon Devices) and then resolved by
SDS-PAGE.
The temperature effect on the formation of the degrada-
tion product was examined by incubation the crude extract
at 4, 25, 30, 35, 40, 45, 50 and 55 ℃ at pH 7.5 for 20 min
before SDS -PAGE.
Soluble protein content was determined as described
by Bradford (1976) using bovine serum albumin as a
standard.
Every experiment was carried out at least three times.
2 Results and Discussion
The contents of both total soluble protein and Rubisco
in wheat leaves decreased greatly during dark-induced se-
nescence (Figs.1, 2). The degradation of intrinsic Rubisco
large subunit (LSU) was analyzed by SDS-PAGE and
immunoblotting. An in vivo degradation product of LSU,
with an apparent molecular mass of 50 kD, appeared when
dark incubating for 24 h and its amount clearly increased
with dark treatment time from 24 h to 48 h (Fig.3A-C). Since
LSU and its degradation product have very near molecular
weight, they cannot be resolved clearly when the content
of LSU is high, especially when dark-induced for 24 h (Fig.
3A). While the applied sample was reduced to 5 mL, the 50
kD product in 24 h-dark-induced leaves was faintly visible
(Fig.3B). The antibody used in this experiment is against
tobacco Rubisco holoenzyme, which has high affinity to
LSU, but has no affinity to small subunit (SSU) (personal
communication with Dr. CHEN G-Y). So the Rubisco SSU
could not be detected by immunoblotting. The degrada-
tion of Rubisco during natural senescence was also
performed. The 50 kD fragment could be detected in the
1st, 2nd and 3rd leaves of wheat with five leaves (Fig.3D,
E), but not in the 4th leaves. That is to say, LSU was hydro-
lyzed to 50 kD in both natural and dark-induced senescence.
Many researchers had reported Rubisco degradation, but
none of them detected the in vivo degradation product. For
example, oxidative treatment could stimulate partial degra-
dation of LSU to 36 kD in isolated barley chloroplasts
(Desimone et al., 1996) and reactive oxygen species could
Fig.1. The changes of protein content during wheat leaf
senescence.
Fig.2. The changes of Rubisco in wheat leaves during dark-
induced senescence. Natural PAGE (5%) was used, in which about
30 mL sample was applied. The gel was stained with Coomassie
Brillient R-250 (CBB). Lanes 1-5 represent wheat leaves dark-
induced for 0, 24, 48, 72 and 96 h, respectively.
RUI Qi et al.: Degradation of Rubulose-1, 5-Bisphosphate Carboxylase/Oxygenase in Wheat Leaves During Dark-induced Senescence 139
directly fragment LSU to 37 kD and 16 kD (Ishida et al.,
1997). Additionally, EP1, a metalloprotease purified from
the pea chloroplast, could hydrolyze the LSU to 36 kD
(Bushnell, et al., 1993). Recently Kokubun et al. (2002) de-
tected a 44 kD fragment in wheat chloroplast when isolated
chloroplast was incubated in darkness for some time.
Obviously, our result is different from all the above and the
fragment which we detected is an in vivo product although
Kokubun et al. (2002) reported no in vivo degradation prod-
ucts of Rubisco could be detected in the extracts from dark-
induced or naturally senescing wheat leaves.
Rubisco is composed of eight LSUs and eight SSUs.
During leaf senescence, whether Rubisco was dissociated
or degraded firstly was not reported. In this study, Rubisco
holoenzyme was purified from natural gel and then sub-
jected to SDS-PAGE. From Fig.3 F and G, it could be seen
that only 53 kD was detected from Rubisco purified from
leaves dark-induced for 0 h; but for Rubisco purified from
leaves dark-induced for 48 h, both 53 kD and 50 kD frag-
ments could be detected. It implied that the fragment was
formed while the subunit was still integrated in the holoen-
zyme complex. Since these kinds of complexes have very
near molecular mass, they could not be obviously sepa-
rated by PAGE with low concentration (Fig.2).
Rubisco was rapidly degraded during leaf senescence.
But the degradation process is not very clear. Since the 50
kD fragment was not detected in young leaves, it might be
that LSU degradation from 53 kD to 50 kD should be a key
process for rapid Rubisco breakdown during leaf senes-
cence and the protease catalyzing this reaction should be
senescence-associated. To study the protease that func-
tioned during LSU degraded from 53 kD to 50 kD, effects of
temperature on the appearance of the 50 kD fragment were
tested. The optimal temperature for 50 kD fragment forma-
tion was 30-35 ℃ (Fig.4). When the crude extracts were
pretreated at 100 ℃ for 5 min and then incubated at 35 ℃
for 12 h, the amount of LSU fragment did not increase,
which suggesting the involvement of a protease in the ap-
pearance of the 50 kD fragment. The cleavage was trig-
gered by this unknown protease in the cell. And whether
Fig.3. Degradation of Rubisco during dark-induced senescence. Degradation product of Rubisco was detected by staining the gels (12.
5%) with Coomassie Brilliant R-250 (CBB) (A, B, D, F) or immunoblotting (C, E, G) after SDS-PAGE. For A, D and F, 20 mL sample
was applied to the lanes, but 5 mL of sample was applied to the lanes of B. For corresponding immunoblotting (C, E, G), 5 mL of sample
was applied. Lanes 1-5 in A, B and C represent wheat leaves dark-induced for 0, 24, 48, 72 and 96 h, respectively. Lanes 1-4 in D
represent the 4th leaf to 1st leaf, respectively. Lanes 1 and 2 in E represent the 4th leaf and the 3rd leaf. Lanes 1 and 2 in F and G represent
purified Rubisco from wheat leaves dark-induced for 0 and 48 h. LSU, larger subunit; M, mark; SSU, small subunit.
Acta Botanica Sinica 植物学报 Vol.46 No.2 2004140
the protease was expressed during senescence or existed
in the cell as a proenzyme was unknown. While the tem-
perature was above 45 ℃,the amount of both 53 kD and 50
kD decreased, and an emerging fragment with the molecu-
lar mass about 44 kD appeared (Fig.4). Whether it was the
fragment reported by Korimoto (2002) was not studied in
this study.
Since the decrease of the amount of Rubisco was much
faster than the decrease of chloroplast number during leaf
senescence, it was suggested that the initial step of Rubisco
degradation must occur within the chloroplasts (Martinoia
et al., 1983; Mae et al., 1984). To preliminarily study the
location of this unknown protease, which hydrolyzed LSU
to 50 kD fragment, we incubated the crude enzyme extract
at pH 5 and pH 7.5 for some time to observe the degrada-
tion of Rubisco. When 48 h dark-induced crude extracts
was incubated at pH 5 for 30 min, the 53 kD and 50 kD
fragments were degraded and could not be detected by
SDS-PAGE clearly (Fig.5). It had been tested that the en-
dopeptidase activity increased greatly and a number of en-
dopeptidase isoenzymes expressed during leaf senescence,
most of which have acidic pH values, about pH 5 (Rui and
Xu, 2002). So proteases activity at acidic pH was respon-
sible for the quick degradation of Rubisco when 48 h-dark-
induced crude enzyme extract was incubated at pH 5. While
at pH 7.5, near the physiological pH of the chloroplast
stroma, the LSU was hydrolyzed to 50 kD fragment in 30
min. And the amount of 50 kD fragment did not decrease
with further incubation, even for 5 h (Fig.5). It maybe that
the acidic proteases in the extract could not show their
activities greatly at pH 7.5, and an unknown protease with
optimal pH at 7.5 could specially hydrolyze LSU to 50 kD
fragment. This unknown protease with neutral pH value
that functioned during leaf senescence was probably lo-
cated in the chloroplast since chloroplast could supply such
environment. That is to say maybe LSU was initially de-
graded from 53 kD to 50 kD in the chloroplast，and then
Rubisco dissociated and were hydrolyzed by other pro-
teases in the cell. Further research to test this point is un-
der progress in our laboratory.
Rubisco is a major source of leaf nitrogen. During
senescence, it is degraded and the nitrogen is exported to
younger parts of the plant (Feller, 1986). But the mecha-
nism of Rubisco degradation is still in question. Studying
the enzymes that function in this important recycling event
is of great interest. In our study, future work will emphasize
on determination the site of the split in the Rubisco LSU
and identification of this unknown protease.
Acknowledgements: We are grateful to Dr. CHEN Gen-
Yun (Institute of Plant Physiology and Ecology, The Chi-
nese Academy of Sciences) for the generous gift of anti-
Rubisco antibody. We wish to thank Prof. PENG Xin-Xiang
from South China Agricultural University，Prof. WEI Jia-
Mian from Institute of Plant Physiology and Ecology for
constructive advice and help, Prof. YAN Qing and Ms.
CHEN Min from Nanjing Agricultural University for techni-
cal assistance during our experiments.
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