全 文 ：Comparative Profile of Rubisco-interacting
Proteins From Arabidopsis: Photosynthesis
Under Cold Conditions*
AN Bai-Yi1), LIU Xiao-Yu2), TAN Hua3), LIN Wei-Hong4)**, SUN Li-Wen1, 2)**
(1) Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130012, China;
2) Shijingshan District Center for Disease Control and Prevention, Beijing 100043, China;
3) Biotechnology Research Centre, Jilin Academy of Agricultural Sciences (JAAS), Changchun 130033, China;
4) The First Hospital of Jilin University, Changchun 130021, China)
Abbreviations: Rubisco, Ribulose 1, 5-bisphosphate carboxylase/oxygenase; Ru-L, Rubisco large subunit; PSAD-1,
Subunit D-1 of photosysmtem Ⅰ ; 2-DE, Two-dimensional electrophoresis; SDS-PAGE, Sodium dodecyl sulfate
polyacrylamide gel electrophoresis; IP, Immunoprecipitation; MALDI-TOF/TOF, Matrix-assisted laser desorption/
ionization tandem time-of-flight; PMF/MS, Peptide mass fingerprint/mass spectrum.
Abstract Rubisco (Ribulose 1, 5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39) is crucial in biological circumstance fluctuation.
Although disassembly of Rubisco after chill treatment has been reported in previous studies, there is only little known data on Rubisco
interactive proteins involved in the disassembly process of Rubisco. Both repression of net photosynthesis rate and disassembly
of Rubisco large subunits (Ru-L) have been investigated in the wild type, Arabidopsis thaliana (Col-0), treated at 4℃ for 4 h and
24 h together with their 24 h recoveries at 20℃ . Co-immunoprecipitation coupled with sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) analysis and MALDI-TOF MS identification was used to explore Rubisco-interacting proteins. Five
protein candidates were profiled. The identified AAA-type ATPase family and glycosyltransferase were determined crucial for Rubisco
activity. It is also strongly correlated to cold acclimation. Results suggest that the disassembly of Rubisco might have been the main
cause of photosynthesis rate reduction under chill conditions, rather than photosystem or biogenesis involvement.
Key words Arabidopsis, cold stress, Rubisco-interacting proteins, photosynthesis
DOI: 10.3724/SP.J.1206.2011.000009
生物化学与生物物理进展
Progress in Biochemistry and Biophysics
2011, 38(5): 455~463
www.pibb.ac.cn
研究报告Research Papers
Rubisco, the major stroma protein in higher
plants, is oligomeric and consists of eight large and
eight small subunits. During photosynthesis, the
catalytic site located in large subunit combines CO2
with ribulose-1, 5-bisphosphate to form glycerate-3-
phosphate; this is reduced to triose-phosphate with
NADPH and ATP generated by photosynthetic
electron transport. In order to catalyze photosynthetic
CO2 fixation at high rates, large amounts of Rubisco
are needed to compensate for the slow catalytic rate
(3～10 s -1) of the enzyme. Rubisco accounts for a
quarter of leaf nitrogen and up to half of the soluble
protein in leaves of C3 plants. Rubisco holoenzyme
assembly in chloroplast stroma is an ATP-dependent
process requiring the presence of other proteins with
chaperone function-Rubisco binding protein(RBP)/cpn
60 [1], as well as Rubisco activase (RA) possessing
ATPase activity [2]. In past years, investigations on
Rubisco and its changes under different stress
conditions have been reconsidered with special
emphasis on the importance of RA and RBP[3-6]. These
proteins might associate with each other by
protein-protein interactions facilitating direct Rubisco
* This work was supported by a grant from The National Natural Science
Foundation of China (30470159/C01020304).
**Corresponding author.
SUN Li-Wen. Tel/Fax: 86-10-88605115
E-mail: sunliwen1972@gmail.com
LINWei-Hong. Tel: 86-431-88782735, Fax: 86-431-85654528,
E-mail: linweihong321@126.com
Received: January 6, 2011 Accepted: March 18, 2011
生物化学与生物物理进展 Prog. Biochem. Biophys. 2011; 38 (5)
assembly and activation [7]. In consideration of its
complex structure and biological function, some
interacting proteins might have not been detected to
date. Rubisco is an enzyme with very complex and
poorly elucidated regulation in terms of activity and
quantity[8]. Supercomplexes containing variable amounts
of Rubisco and other stromal proteins that might allow
metabolic channeling and/or regulation have been a
recurring topic in Rubisco research. Rubisco itself is a
very thermostable enzyme, as revealed by studies
with isolated protein [2]. The major components of
photosynthesis typically affected by short-term light or
dark chills in thermophilic species are primarily
compromised by interference with carbohydrate
metabolism, inhibition of Rubisco activity, and
stomatal closure; these are with concurrent increase
in energy dissipation (i.e., heat) in the thylakoid
antennae [9-10]. Although these factors can also be
observed during concurrent chilling with incident
light, the potential for photodamage to PSⅡ is more
apparent, similar to the disruption of redox control of
stromal bisphosphatases SBPase and FBPase, and
possibly RA[11-12]. The effects of growth under chilling
conditions on PSⅠ have already been investigated[13-14].
Evidences showing PSⅠ activity declining at a greater
rate compared with PSⅡ[15-16] is insufficient to identify
PSⅠ as a primary target of chilling. One reason is that
the evidence does not exclude the possibility of
downstream chill-susceptible processes (carbon
metabolism and stomatal conductance; to be described
below) as the primary target; the observed changes in
PSⅠ and/or PSⅡ activities act as secondary response.
Recent studies with Arabidopsis have shown that a
sequence of events could reverse the inhibition of
photosynthesis as plants acclimate to low
temperatures. Interestingly, some changes in
photosynthetic metabolism occurring during cold
acclimation are reminiscent of responses to low Pi [17].
The recovery involves two important functions:
increasing sucrose production and protection against
photoinhibition by allowing the increased turnover of
the photosynthetic electron chain[18].
Carbohydrate metabolism has been reported to
have greater instantaneous low temperature sensitivity
compared with other components of photosynthesis [19].
However, when the oxygen sensitivity of CO2
assimilation was examined after return to permissive
temperatures, the persistent inhibition of photosynthesis
following dark chill seemed to be not directly related
to end-product inhibition.
Declines in photosynthesis after a chill under both
dark and light conditions have been attributed to loss
of Rubisco activity by various studies[20]. As previously
suggested, chilling could damage Rubisco protein [21].
However, a general consensus on the significance,
composition and function of Rubisco-containing
complexes still awaits future research. Reports have
also suggested that the activity repression of Rubisco,
along with disassembly, might be the dominant reason
of photosynthesis decrease under cold conditions [21].
Nevertheless, solid evidence supporting this hypothesis
still needs to be provided.
In this study, we used Arabidopsis in an attempt
to elucidate novel Rubisco-interacting protein
candidates under cold acclimation conditions.
Co-immunoprecipitation, followed by MALDI-TOF
MS, was used. Results suggest that the disassembly of
Rubisco might be the main cause of photosynthesis
rate reduction, rather than photosystem or biogenesis
involvement.
1 Materials and methods
1.1 Materials
Seeds of Arabidopsis thaliana Columbia ecotype
were obtained from the Arabidopsis Biological
Resource Center (Ohio State University, USA). Seeds
were geminated in mixture soil. The seedlings
routinely grew in a climate-simulated chamber at 75%
humidity with 16 h light(80 μE·s-1 m-2) at 22℃ and 8 h
dark at 19℃ . Plants were harvested after 4 weeks of
growth and washed with Milli-Q water in order to
remove the attached soil. Immediately thereafter, the
intact plants were frozen in liquid nitrogen and
stored at -80℃ prior protein preparation. For the cold
treatment, 3-week-old plants were placed in 4℃ for a
particular time span.
1.2 Photosynthesis measurement
Leaf net photosynthetic rates were measured by a
portable gas analysis system, cI-301PS (CID, USA).
Eight leaves for each sample were measured.
1.3 Anti-Rubisco immunoprecipitation
All steps were conducted at 4℃ . Anti-Rubisco
was subjected to IP from 20 g of Arabidopsis cells
sampled from the control at 4℃ for 4 h; 4℃ for 24 h;
and 24 h after re-shifting to normal condition. Crude
cell extracts were prepared in IP buffer (2 mmol/L
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Tris-HCl, pH 7.5, 150 mmol/L NaCl, 10% (v/v)
glycerol, 0.07% polyethyleneimine, 10 mmol/L
ethylenediaminetetra acetic acid/EDTA, 1% (v/v)
NP-40, 5 mmol/L dithiothreitol/ DTT, and 1 mmol/L
phenylmethylsulfoxyfluoride/PMSF). Clarified extracts
were obtained by homogenizing cells and centrifuged
for 15 min at 15 000 g. Supernatants were incubated
for 90 min with 100 μl of immune serum or pre-
immune serum. Antigen-antibody complexes were
incubated for 30 min with 500 μl protein G-Sepharose
beads (Watson, USA); previously, these were
equilibrated in IP buffer and centrifuged for 1 min at
15 000 g. Pellets were washed 5 times with 2 ml IP
buffer. Immunoprecipitates were eluted by 1 mol/L
NaCl, processed by organic extraction, and boiled
for 2.5 min in SDS sample buffer without DTT. The
immunoprecipitates were then immediately analyzed
by SDS-PAGE.
1.4 SDS-PAGE and two-dimensional gel
electrophoresis
The protocol was conducted following those by
Xi, et al. (2006)[22].
1.5 Western blotting
Following electrophoresis, either gels were
stained with silver (Bio-Rad protocol)/Brilliant Blue
or proteins were transferred to NC membranes
(Hybond-C Extra, Amersham Biosciences) using a
Trans-Blot Semi-Dry Electrophoretic Transfer Cell
(Bio-Rad) at 0.45 A for 30 min. Proteins separated
from membranes were blocked in freshly prepared
TBS containing 5% nonfat dry milk (Bio-Rad) for 1 h
at room temperature with constant agitation, and then
incubated in anti-Rubisco antibodies diluted in freshly
prepared TBS containing 5% nonfat dry milk for 2 h
with agitation at room temperature. Membranes were
subsequently washed twice with TBS, incubated in
goat anti-rat horseradish peroxidase-conjugated IgG
(upstate) diluted in freshly prepared TBS containing
5% nonfat dry milk for 1 h with agitation at room
temperature, and washed three times with TBS.
Membrane were developed using an ECL system
(SuperSignal West Pico Chemiluminescent Substrate,
Pierce).
1.6 Image analysis
Image analysis was accomplished using PDQuest
7.3 software (Bio-Rad). After automated detection and
matching, manual editing was conducted. Three
well-separated gels of each sample were used to
create replicate groups . Statistic, quantitative, and
qualitative analysis sets were created between
control group and each treated group. In the statistic
sets, students t-test at a significance level of 95% was
chosen. Only spots displaying reproducible change
patterns were considered as differentially expressed
proteins.
1.7 Protein identification based on PMF/MS
spectra
Protein identification based on PMF/MS spectra
was conducted following those by Sun (2009)[23].
1.8 Chilling injury indexes assessment of cold
responsive gene mutants
For cold responsive gene candidates, chilling
injury indexes between Arabidopsis col-0 (control) and
cold responsive gene T-DNA insertion mutants were
investigated as described by Semeniuk (1986)[24]. Seeds
of T-DNA insertion mutants were ordered from ABRC
stock of Tair. These were checked by PCR using
primers designed according to manual.
1.9 Protein analysis
Web-based TAIR Patmatch(http://www. arabidopsis.
org/cgi-bin/patmatch/nph-patmatch.pl) was used to
analyze the obtained proteins. Analysis of hypothetical
proteins was conducted using software at servers
accessible on the Internet (Blast, Pfam, Prosite,
Blocks, Prints, Prodom, and Proclass). Predictions
of functions were carried out using BLAST
(www.ch.embnet.org/software/BottomBLAST.html)
and InterProScan (www.ebi.ac.uk/interpro/scan.html)
tools. Predictions for chloroplast localization and
chloroplast were conducted using software programs,
PSORT (http://psort.nibb.ac.jp:8800/), ChloroP (http://
www.cbs.dtu.dk/services/ChloroP/), and SignalP(http://
www.cbs.dtu.dk/services/SignalP/).
The structure of PSAD-1 (e.g., Amunts et al.,
2007) and large subunit of Rubisco (e.g., Duff et al.,
2000) from X-ray diffraction were obtained from
RCSB Protein Data Bank (PDB) (http://www.rcsb.
org/pdb/home/home.do). Structures were then subjected
to the online Z-dock server (http://zdock.bu.edu/) for
Molecular modeling.
2 Results and discussion
2.1 Large subunits of Rubisco disassemble when
net photosynthetic rate decreases under cold
conditions
Photosynthesis in tropical warm climate plants
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生物化学与生物物理进展 Prog. Biochem. Biophys. 2011; 38 (5)
2.2 Effects of chill acclimation on Rubisco
interactive proteins
Given that Rubisco disassembles and its activity
declines, the effects of chill acclimation on Rubisco
complex were examined. Co-immunoprecipitation
techniques were used to study protein-protein
interactions of plant proteins with Rubisco. Compared
with conventional yeast two-hybrid system that use
domains as bait for screening proteins, this study uses
the full-length Rubisco as bait for screening for
proteins interacting directly with Rubisco, as well as
with second messenger proteins of Rubisco acting as a
protein complex. It has been previously reported that
Rubisco undergoes disassembly under sugar gradient
ultracentrifugation, and as such, plastid isolation of
Arabidopsis for IP probably lose some of
Rubisco-interacting proteins. The specificity of the
antibody employed for immunoprecipitation was
examined using dot blotting and Western blotting (data
not shown). Rubisco was recognized strongly by the
antibody.
Immunoprecipitated proteins were explored using
SDS-PAGE. Subsequent gel scanning and image
analysis identified five protein spots that appeared to
be expressed differentially compared with the controls,
while antibody and Rubisco acting as controls did not
change in the immunoprecipitation profile (Figure 2a).
Reproducible maps were obtained from more
than three independent experiments. In mock
immunoprecipitations using preserum, neither Rubisco
nor Rubisco-interacting proteins was adsorbed to the
antibody (Figure 2a).
The quantity of the particular band was evaluated
as a tense ratio to its lane (Figure 2b, c). The five
proteins could be classified into two groups: First,
bands 1 and 3 decreased along with chill time course
and recovered to normal quantities after 24 h recovery
(Figure 2b); their correlations to time course under
chill and Pn were observed as well. Second, bands 2, 4
and 5 seemed to increase at 4 h chill and increased
largely at 24 h recovery (Figure 2c).
is affected by chilling stress substantially. This
process is called low-temperature photoinhibition [25].
To evaluate the adverse effects of chilling stress
quantitatively, net photosynthetic rate (Pn) was
determined. Net photosynthetic rate decreased from
12.1 to 9.5 and 1.96 μmol·m-2·s-1 after 4 h and 24 h
of chill treatment, respectively, and returned to almost
basal level (11.7 μmol·m -2·s -1) after 24 h recovery
(Figure 1a). Previously, it was suggested that stomatal
conductance (Gs) was not the limiting factor for Pn in
this condition. The decreased Pn probably resulted
from other factors, such as Rubisco activity and ATP
availability. The disassembly and even degradation of
Rubisco were observed as well [26].
Then, the assembly of Rubisco was investigated
by 2-DE (Figure 1b). Ru-Ls were found smeared under
conditions of 24 h chill. This suggests that disassembly
has occurred with a change in proteins-Rubisco
interaction.
Fig. 1 Disassembly of Ru-L when Arabidopsis net photosynthesis rate (Pn) repression at 24 h chill
(a) Net photosynthesis rates are repressed at 4 h chill for 24 h and recovered after 24 h shift to normal incubate condition. (b) Disassembly of Ru-L at
24 h chill. Total protein extraction from control and 24 h chill were subjected to two-dimensional gel electrophoresis; the smear of Ru-L increased,
indicating disassembly of Ru-L.
2
0
Pn
/( μ
m
ol·
m
-2 ·
s-1
)
Control Chill
4 h
Chill
24 h
24 h
recovery
4
6
8
10
(a) (b) pH
4 6
Control Treatment
pH
4 6
11.6
6.6
4.5
3.5
2.5
1.8
1.4
ku
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2.3 The identification of Rubisco-interacting
proteins using PMF/MS
To identify the five co-immunoprecipitated
proteins, trypsin-digested peptides of protein were
analyzed with matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry. The
summary is presented in Table 1. AAA-type ATPase
family protein AT4G24860, α1,4-glycosyltransferase
family protein/glycosyltransferase sugar-binding DXD
motif-containing protein AT5G01250 were strongly
correlated to chill time course and Pn. The details are
shown in Figure 2b. By using database searches and
multiple sequence alignments, Neuwald [27] reported
that RA is related to an AAA family of proteins, a
class of chaperone-like ATPases associated with a
variety of cellular activities such as assembly,
operation, and disassembly of protein complexes. As
for α1, 4-glycosyltransferase family protein, at optimal
temperatures, it has an essential function in releasing
inhibitory sugar phosphates from the active site of
Rubisco, thereby allowing continuous CO2 fixation [3].
A previous report has shown that heat treatment
of thylakoid membranes would induce rapid
dephosphorylation of the PSⅡ reaction center protein
D1 [28], and that this is followed by rapid degradation of
damaged D1 copies. However, as a direct NADPH
donor of Rubisco CO2 fixation, PSⅠ was poorly
investigated and thus with limited information [29].
The present work found that PSAD-1 (photosystem Ⅰ
subunit D-1) interacting with Rubisco elucidate further
α helix of PSAD-1, directly binding to 60～80 and
100～120 AAs of Ru-L using Z-dock (Figure 3). The
α helix of PSAD-1 was originally bound to the PSⅠ
activity center; initially, it seemed that it was reversely
interacting with Ru-L, resulting in the feedback
inhibition of PSⅠ at 4 h chill. Along with degradation
of Ru-L, the photoinhibition by Ru-L might have
shifted to other photoprotective mechanisms at 24 h
chill. With prolonged heat stress and upon stress
repetition, the actual de novo synthesized heat shock
proteins were obtained to rescue thylakoid functions;
less RA was found associated with thylakoid protein
synthesis machinery [30]. At 24 h recovery, Ru-L was
synthesized and it up-tensed the interaction, avoiding
any XO access. To guarantee constant repair of
photosynthesis complexes, thereby avoiding damage, it
is crucial to maintain active protein synthesis in
chloroplasts. Hypothetical protein gi|7523687,
homologue to protein that is related to translation
machinery, was more active at 4 h chill and 24 h
recovery. Hence, specific association of Rubisco
with thylakoid-bound ribosome nascent chain
complexes could have exposed a chaperone-like
function of Rubisco; this was crucial in maintaining
Fig. 2 Profiles of Rubisco-interacting proteins from Arabidopsis under chill conditions
(a) Immunoprecipitates from anti-Rubisco of Arabidopsis total protein extracts under particular conditions subjected to SDS-PAGE. Bands 1～5 were
sliced and investigated using PMF/MS. Ru-L (56 ku), Ru-S (15 ku), Rubsico activase (43 ku), IgG large subunits (50 ku) and small subunits (26 ku) are
arrowed. (b) Changes in bands 1 and 3 ratios to lane quantities are negatively correlated to time course under chill conditions and positively correlated to
Pn. (c) In contrast to control, bands 2, 4, and 5 ratios to lane quantities increased at 4 h chill, recovered to normal quantities at 24 h chill, and increased
markedly at 24 h recovery after chill.
IP
pre-serum
(a) (b) (c)
1
2
3
4
5
11.6
6.6
4.5
3.5
2.5
1.8
1.4
kuControl
Chill
4 h
Chill
24 h
24 h
recovery
1.0
0Ra
tio
to
la
ne
qu
an
tit
y/
%
2.0
3.0
Control Chill
4 h
Chill
24 h
24 h
recovery
Band 1
1.0
0Ra
tio
to
la
ne
qu
an
tit
y/
% 2.0
Control Chill
4 h
Chill
24 h
24 h
recovery
Band 2
2
0Ra
tio
to
la
ne
qu
an
tit
y/
%
4
6
Control Chill
4 h
Chill
24 h
24 h
recovery
Band 3
1.0
0Ra
tio
to
la
ne
qu
an
tit
y/
% 1.5
Control Chill
4 h
Chill
24 h
24 h
recovery
Band 4
5
0Ra
tio
to
la
ne
qu
an
tit
y/
% 15
Control Chill
4 h
Chill
24 h
24 h
recovery
Band 5
Corrdlation
Time course
under chill
Pn
Band 1 -0.93 0.78
Band 3 -0.92 0.86
0.5
10
459· ·
生物化学与生物物理进展 Prog. Biochem. Biophys. 2011; 38 (5)
translation activity at transiently low temperatures.
Meanwhile, SEC14 cytosolic factor family protein/
phosphoglyceride transfer family protein AT1G55690
involved in lipid synthesis cooperated in this process.
Taken together, our results suggest a temperature-
dependent dual function for Rubisco. A switch of
protein from one function to another according to the
needs of cell under fluctuating environmental
conditions represents a great design, not only for cell
protection, but also for saving metabolic energy,
particularly if such environmental fluctuations are only
at short term. The first function involved feedback
Fig. 3 Molecular modeling between PSAD-1 (blue)
and Ru-L (green)
The interface covered the α helix of PSAD-1, 60～80, and 100～120
AAs of Ru-L.
Spot
No.
Identity
Accession
number
Mascot
score
Queries
matched
Sequence
coverage
Mass(ku)/
theoretical
ChloroP v1.1
prediction
Function prediction
1 AAA-type
ATPase family
protein
AT4G24860 69 10 10% 10/124871 0.512 Expected to ATP
binding, nucleoside-
triphosphatase tase
activity, nucleotide
binding and to
localize in chloroplast.
cadmium stress Aina
et al. (2007)
2 SEC14 cytosolic
factor family
protein /
phosphoglyceride transfer
family
protein
AT1G55690 65 10 14% 7.0/72041 0.443 Involved in
coordinating actin
remodeling guanine,
nucleotide exchange,
transport of secretory
proteins.
3 Alpha
1,4-glycosyltrans-
ferase family
protein /
glycosyltransferase
sugar-binding
DXD motif-
containing protein
AT5G01250 67 9 24% 3.5/46594 0.512 Expected to
transferase activity,
transferring glycosyl
groups,
galactosyltransferase activity.
4 Hypothetical
protein
gi|7523687 62 6 15% 2.5/31768 0.436 Reverse transcriptases
occur in a variety of
mobile elements,
including
retrotransposons, retroviruses,
groupⅡ
introns, caulimoviruses.
F-Box and BRCT
domain containing
plant proteins
associated with
nuclear functions.
5 PSAD-1
(PhotosystemⅠ
subunit D-1)
AT4G02770 92 8 33% 2.0/22641 0.570 Thylakoid electron
transfer chain
component
Table 1 Identification of Rubisco-interacting proteins under chill conditions
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After incubation under normal conditions for 4
weeks, the chilling injury indexes of Arabidopsis col-0
and cold responsive gene T-DNA insertion mutants
were measured after transferring the plants to 4℃ for
15 days. The experiment was repeated five times. SD
(±) is indicated.
2.5 Localization prediction
Majority of the chloroplast proteins are encoded
by the nuclear genome. The presequences of these
nuclear-encoded chloroplast proteins share common
features; these can be used to predict localization with
moderate confidence [33].
All proteins identified on the gels were used for
test predictions on chloroplast localization and transit
peptides. The software program, ChloroP(see in Table 1)
was utilized to evaluate chloroplast localization. The
ChloroP server predicts the presence of chloroplast
transit peptides (cTP) in the 5 protein sequences and
suggest them located in the chloroplast.
3 Conclusion
Co-immunoprecipitation coupled with SDS-PAGE
analysis and MALDI-TOF MS identification was used
in this study to explore Rubisco-interacting proteins
under Ru-L disassembly and Pn repression under chill
conditions. Cold acclimation had a marked effect on
respiration in A. thaliana. This suggests that the effects
of cold acclimation on Rubisco activity have not been
initiated by tight control (i.e., upstream or downstream
regulation); rather, regulation of Rubisco by
photoinhibition to PSⅠ through competitive binding
to PSAD-1; the second function, deduced from
observed association of Rubisco with thylakoid-bound
polysomes upon chill stress, probably occurred to
maintain translation of essential thylakoid proteins
during sudden exposure and lagging recovery of plants
to chill stress. The fast spatial segregation of Rubisco
to thylakoid membrane upon chill stress strongly
suggests the role of Rubisco as chaperone in
maintaining and protecting the thylakoid-associated
translation apparatus. An analogous situation has been
shown recently for HSP DegP, whose function
switches from chaperone at low temperature to
protease with increase in temperature [31]. The present
results indicate that Rubisco may form complexes with
thylakiod proteins in plant cells. In E. coli, the
capability of major cytosolic chaperones to cope with
protein misfolding and aggregation during heat shock
stress in vivo and in cell extracts has been
demonstrated [32].
2.4 Gene confirmation using T-DNA insertion
mutant
For chilling injury index assessment, only
up-regulated gene mutants are accessible for gene
chilling function. The PSAD-1 mutant has been
searched and ordered from ABRC stock, then the
T-DNA insertion mutants of PSAD-1 (SALK_090959C)
were selected for further phenotype observation under
4℃ cold conditions. The severity of symptoms of wild
type (control) and T-DNA insertion mutants was
assessed visually according to the five-stage scale. The
average extent of chilling-injury damage was
investigated. The chilling injury indexes are
summarized in Figure 4. The chilling endurances of
gene mutants sharply decreased, supporting the idea
that the gene has imposed a positive influence on the
chilling resistance of Arabidopsis.
Fig. 4 Chilling injury index of Arabidopsis col-0 and chill responsive gene T-DNA insertion mutants under 4℃ chilling stress
(a) The chilling injury indexes of the germplasm lines of Col-0(control) and SALK_090959C. (b) The pictures of the the germplasm lines of Col-0
(control) and SALK_090959C after 15 days chilling treatment.
SALK_090959C
Col-0
(b)(a)
Germplasm line Chilling injury index
1d 3d 5d 7d 9d 11d 13d 15d
Col-0
SALK_090959C 0.0 ± 0.0 4.1 ± 0.7 4.6 ± 0.4 5.0 ± 0.0 5.0 ± 0.0 5.0 ± 0.0 5.0 ± 0.0 5.0 ± 0.0
0.0 ± 0.1 2.3 ± 0.4 3.0 ± 0.6 3.3 ± 1.3 3.5 ± 1.1 3.7 ± 0.6 3.6 ± 0.6 3.6 ± 0.6
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生物化学与生物物理进展 Prog. Biochem. Biophys. 2011; 38 (5)
disassembly in cold-treated plants may be more
crucial, as shown by our results. It is Rubisco
disassembly, rather than Rubisco genesis or
degradation and feedback of photosystem, that resulted
in Rubisco activity repression, thus net photosynthesis
decreased under chill condition. The further
experiments using these chilling responsive gene
mutants should be explored for solidating the potential
relationship between Rubisco disassembly and net
photosynthesis reduction.
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安佰义, 等：冷胁迫条件下拟南芥 Rubisco相互作用蛋白质的比较分析2011; 38 (5)
冷胁迫条件下拟南芥 Rubisco相互作用
蛋白质的比较分析 *
安佰义 1) 刘小雨 2) 谭 化 3) 林卫红 4)** 孙立文 1, 2)**
(1)吉林大学生命科学学院，分子酶学工程教育部重点实验室，长春 130012；2)石景山区疾病预防控制中心，北京 100043；
3)吉林省农业科学院生物技术中心，长春 130033；4)吉林大学第一医院，长春 130021)
摘要 1, 5-二磷酸核酮糖羧化酶 /加氧酶(Rubisco，EC 4.1.1.39)在生物适应环境变化的过程中起到重要的作用．位于叶绿体
中与冷胁迫密切相关的非常重要的复合酶——Rubisco，其相互作用的蛋白质至今没有系统的研究．对拟南芥进行 4种处理：
a．持续在 20℃生长(对照)；b．4℃ 4 h冷胁迫；c．4℃ 24 h冷胁迫；d．4℃ 24 h冷胁迫后放入 20℃恢复 24 h．然后利用免
疫共沉淀、十二烷基硫酸钠聚丙烯酰胺凝胶电泳及基质辅助激光解析电离飞行时间质谱技术，在冷胁迫条件下研究了拟南芥
光合抑制与 Rubisco相互作用蛋白质解聚之间的关系．在鉴定出的 5个与冷胁迫相关的 Rubisco相互作用蛋白质中，AAA-
型 ATP酶家族蛋白和糖基转移酶对 Rubisco活性及植物适应冷胁迫起着重要的作用．研究结果表明，Rubisco复合酶体系的
解聚可能是低温胁迫下拟南芥光合速率降低的主要原因．
关键词 拟南芥，冷胁迫，Rubisco相互作用蛋白质，光合作用
学科分类号 Q71 DOI: 10.3724/SP.J.1206.2011.00009
*国家自然科学基金资助项目(30470159/C01020304).
**通讯联系人.
孙立文. Tel/Fax: 010-88605115, E-mail: sunliwen1972@gmail.com
林卫红. Tel: 0431-88782735, Fax: 0431-85654528, E-mail: linweihong321@126.com
收稿日期：2011-01-06，接受日期：2011-03-18
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