Copyright © Physiologia Plantarum 2010, ISSN 0031-9317
Physiologia Plantarum 139: 55–67. 2010
Two Rubisco activase isoforms may play different roles
in photosynthetic heat acclimation in the rice plant
Dun Wang, Xiao-Fei Li, Zheng-Jian Zhou, Xu-Ping Feng, Wan-Jun Yang and De-An Jiang∗
State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China
Correspondence
*Corresponding author,
e-mail: [email protected]
Received 3 August 2009;
revised 17 December 2009
doi:10.1111/j.1399-3054.2009.01344.x
Studies on some plant species have shown that increasing the growth
temperature gradually or pretreating with high temperature can lead to
obvious photosynthetic acclimation to high temperature. To test whether this
acclimation arises from heat adaptation of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) activation mediated by Rubisco
activase (RCA), gene expression of RCA large isoform (RCAL ) and RCA small
isoform (RCAS ) in rice was determined using a 4-day heat stress treatment
[40/30◦ C (day/night)] followed by a 3-day recovery under control conditions
[30/22◦ C (day/night)]. The heat stress significantly induced the expression of
RCAL as determined by both mRNA and protein levels. Correlative analysis indicated that RCAS protein content was extremely significantly related
to Rubisco initial activity and net photosynthetic rate (Pn) under both heat
stress and normal conditions. Immunoblot analysis of the Rubisco–RCA
complex revealed that the ratio of RCAL to Rubisco increased markedly
in heat-acclimated rice leaves. Furthermore, transgenic rice plants expressing enhanced amounts of RCAL exhibited higher thermotolerance in Pn
and Rubisco initial activity and grew better at high temperature than wildtype (WT) plants and transgenic rice plants expressing enhanced amounts
of RCAS . Under normal conditions, the transgenic rice plants expressing
enhanced amounts of RCAS showed higher Pn and produced more biomass
than transgenic rice plants expressing enhanced amounts of RCAL and wildtype plants. Together, these suggest that the heat-induced RCAL may play an
important role in photosynthetic acclimation to moderate heat stress in vivo,
while RCAS plays a major role in maintaining Rubisco initial activity under
normal conditions.
Introduction
Photosynthesis is one among the physiological processes
that are the most sensitive to high temperature stress
(Berry and Björkman 1980). High temperature damages
the permeability of thylakoid membranes resulting in
proton leakage and reduced electron flow (Bukhov et al.
1999, Schrader et al. 2004), followed by a reduction in
ribulose 1,5-bisphosphate (RuBP) regeneration (Kubien
and Sage 2008). Additionally, high temperature reduces
the activation state of ribulose 1,5-bisphosphate
carboxylase/oxygenase (Rubisco, EC 4.1.1.39), which
Abbreviations – BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; PBST, phosphate-buffered
saline with 0.05% Tween; Pn, net photosynthetic rate; PPFD, photosynthetic photon flux density; PS II, photosystem II;
PVDF, polyvinylidene fluoride; RCA, Rubisco activase; RCAL , Rubisco activase large isoform; RCAS , Rubisco activase small
isoform; RLS, Rubisco large subunit; RSS, Rubisco small subunit; Rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase;
RT-PCR, reverse transcriptase polymerase chain reaction; RuBP, ribulose 1,5-bisphosphate; SDS–PAGE, sodium dodecyl
sulfate–polyacrylamide gel electrophoresis; WT, wild-type.
Physiol. Plant. 139, 2010
55
is regulated by heat-labile Rubisco activase (RCA)
(Crafts-Brandner and Salvucci 2000, Feller et al. 1998,
Robinson and Portis 1989, Salvucci and Crafts-Brandner
2004, Salvucci et al. 2001). Although the primary reason for the rapid inhibition of the photosynthetic rate by
moderate high temperature is debated, many studies on
different species have shown that plants have the capacity to acclimate to moderate high temperature (reviewed
by Berry and Björkman 1980).
Photosynthetic acclimation to high temperature may
involve different mechanisms in different species. For
example, in winter wheat (Yamasaki et al. 2002) and
pea (Haldimann and Feller 2004), it was reported that
the major factor contributing to thermal acclimation of
photosynthesis was the improved thermal stability of
the thylakoid membranes and the plastic response of
photosynthetic electron transport to environmental temperature. On the other hand, in wheat, cotton (Law and
Crafts-Brandner 1999) and spinach (Yamori et al. 2006),
the activation state of Rubisco could acclimate to moderate heat stress, and the degree of acclimation was directly
related to photosynthetic heat acclimation. Hikosaka
et al. (2006) demonstrated that the activation energy of
the maximum rate of RuBP carboxylation was the most
important among several factors responsible for photosynthetic heat acclimation. Studies on wheat and cotton
(Law and Crafts-Brandner 1999), spinach (Yamori et al.
2006), black spruce (Way and Sage 2008) and creeping bentgrass (Liu and Huang 2008) provided evidence
supporting the view that enhanced thermotolerance of
the Rubisco activation state contributes to the heat acclimation of photosynthesis, suggesting that photosynthetic
heat acclimation is associated with the acclimation of
heat-labile RCA to high temperature. Recent studies on
Arabidopsis also showed that introducing more thermostable RCA increased photosynthesis and growth rate
under moderate heat stress (Kumar et al. 2009, Kurek
et al. 2007), which demonstrates that RCA is a major
limiting factor in plant photosynthesis under moderate heat stress and genetic manipulation of RCA could
feasibly provide the means to improve photosynthetic
thermotolerance.
RCA is a nuclear-encoded chloroplast enzyme usually
present as two isoforms in most species studied: a large
isoform of 45–48 kDa (RCAL ) and a small isoform
of 41–43 kDa (RCAS ). Studies on maize (Sánchez
de Jiménez et al. 1995), cotton (Law et al. 2001)
and wheat (Law and Crafts-Brandner 2001) showed
that high temperature induced a new isoform of
RCA that disappeared after recovery at the control
temperature, suggesting that RCA gene expression in
response to heat stress may constitute a mechanism of
photosynthetic acclimation to heat stress. A recent study
56
on red maple also indicated that the marked difference
in photosynthetic thermotolerance between different
genotypes was not because of obvious differences in rca
gene sequences but rather to the differential response of
rca gene expression to heat stress (Weston et al. 2007).
In vitro experiments have shown that both RCA large
isoform (RCAL ) and RCA small isoform (RCAS ) from
spinach are capable of promoting Rubisco activation
but they differ markedly in enzyme activity (Shen et al.
1991). Crafts-Brandner et al. (1997) found that RCAL
was more thermostable than RCAS and its optimum
temperature for ATP hydrolysis was much higher.
Subsequent studies demonstrated that light regulation
of RCA activity was achieved by the redox regulation
of RCAL via thioredoxin-f (Zhang and Portis 1999,
Zhang et al. 2001, 2002). Thus, these findings prompted
us to ask whether RCAL also plays a regulatory role
in response to high temperature. A recent study by
Salvucci et al. (2006) showed that transgenic Arabidopsis
plants only expressing either RCAL or RCAS exhibited
similar sensitivity to inhibition by high temperature,
suggesting that the amount rather than the form of
RCA might be the important factor for determining
photosynthetic heat sensitivity. However, this study
could not determine the interaction between the two
RCA isoforms, which was likely involved in the plant’s
response to heat stress. Hence, studies using plants
with different RCA isoform ratios will provide new
insights into the relationship between RCA isoforms and
photosynthetic heat sensitivity.
In this study, the gene expression of two RCA polypeptides that arise from one nuclear gene via alternative
splicing (To et al. 1999) and the interaction between
Rubisco and RCA were investigated during short-term
photosynthetic heat acclimation in rice plants. Furthermore, we also investigated the effect of increased RCAL
level on photosynthetic heat sensitivity using transgenic
rice plants expressing enhanced amounts of RCAL . Based
on the results of these experiments, we provide details
about the relationship between RCA isoforms and photosynthetic thermotolerance in rice plants.
Materials and methods
Plant material and growth conditions
Rice (Oryza sativa L. ‘Zhenong 952’) was used for
investigating the time course of photosynthetic acclimation to high temperature. Germinated rice seeds were
grown in International Rice Research Institute (IRRI) rice
nutrient solution (pH 5.0–5.5) consisting of 1.428 mM
NH4 NO3 , 0.323 mM NaH2 PO4 , 0.513 mM K2 SO4 ,
0.998 mM CaCl2 , 1.643 mM MgSO4 , 10.423 μM
MnCl2 , 0.0748 μM (NH4 )6 Mo7 O24 , 18.883 μM H3 BO3 ,
Physiol. Plant. 139, 2010
0.153 μM ZnSO4 , 0.155 μM CuSO4 , 35.581 μM FeCl3 ,
70.786 μM citric acid monohydrate and 1.6 mM
Na2 SiO3 . Rice seedlings were grown in a greenhouse
under a photosynthetic photon flux density (PPFD) of
500 μmol photons m−2 s−1 controlled at a day/night temperature regimen of 30/22◦ C. The solution was adjusted
to pH of 5.0–5.5 every 2 days and was renewed once in a
week. Heat stress was imposed on six-leaf-old seedlings
by increasing the temperature of the growth chamber
to 12 h light/12 h dark (40/30◦ C) for 1, 2, 3 or 4 days,
followed by a 3-day recovery at the control temperature
(30/22◦ C). The relative humidity in the growth chamber
was maintained at 80%.
To investigate if different RCA isoforms play different
roles in photosynthetic response to high temperature,
rca-transgenic homozygous T2 seeds with different
RCA isoform contents and wild-type (O. sativa L.
’Zhonghua11’) were used in this experiment. Rice plants
expressing increased levels of RCAL or RCAS were
produced by Agrobacterium-mediated transformation.
Full-length cDNA for the coding region of large isoform
(rca-L) or small isoform (rca-S) of RCA from rice was
cloned by reverse transcriptase polymerase chain reaction (RT-PCR) amplification. The resulting cDNA clones
were inserted into the binary vector pC1390 between a
maize ubiquitin promoter and nopaline synthase (NOS)
terminator. Each constructed plasmid (pC1390-rca-L or
pC1390-rca-S) was transferred, respectively, into rice
cultivar Zhonghua11 by Agrobacterium-mediated transformation. Positive transformants were identified by
selecting hygromycin-resistant seedlings and were verified by RT-PCR or western blotting. Six-leaf-old seedlings
were also used in the heat treatment. The growth and
heat stress conditions of transgenic rice plants were controlled, as described in the above experiments of heat
acclimation, except for that the recovery growth was
removed. After 8 days of growth at high temperature,
dry weight of each seedling was determined after drying
at 85◦ C for 2 days.
All the measurements on physiological and biochemical parameters were carried out on the fully expanded
fifth leaf. Immediately following each treatment, plant
material was frozen in liquid N2 and stored at −80◦ C
until extraction.
Gas exchange and chlorophyll fluorescence
measurements
To avoid the photosynthetic rate being disturbed by
respiration or other metabolic processes dependent on
temperature, measurements of net photosynthetic rate
(Pn) were made at 30◦ C in the morning of each treatment
day (08:00–09:00 h) using a portable photosynthesis
Physiol. Plant. 139, 2010
system (LICOR-6400; LICOR, Lincoln, NE). The other
conditions were controlled as follows: CO2 concentration of 380 ± 5 μl l−1 and PPFD of 1200 μmol
photons m−2 s−1 . Fv /Fm , the ratio of variable to maximum fluorescence was determined by measuring the
initial chlorophyll a fluorescence level Fo and maximum
fluorescence level Fm with the leaves that had been
kept in the dark for 30 min. This experiment lasted till
day 16. In the non-steady-state Pn measurements, different transgenic plant lines were adapted to a low PPFD
of 100 μmol photons m−2 s−1 for 30 min at 30◦ C; the
leaves were then exposed to the leaf chamber of the photosynthesis system controlled at 30 or 40◦ C with a PPFD
of 1200 μmol photons m−2 s−1 . Pn was determined
continuously during a 30-min time course.
Rubisco activity
Leaf samples for the Rubisco activity assay were
harvested and frozen in liquid N2 immediately after
Pn measurements. Initial and total Rubisco activities of
samples were measured by the photometric method
(Sharkey et al. 1991). The results presented are the
means ± SD of three to five individual plants.
Quantitative RT-PCR
To investigate the effect of heat stress on the
accumulation of RCA mRNA in rice leaves, total RNA
from each sample was extracted using the TaKaRa
RNAiso reagent (Otsu, Shiga, Japan) according to the
manufacturer instructions. The concentration of total
RNA was determined via Optical Density measurement.
First strand cDNA was synthesized from 3 μg of total
RNA using a PrimeScript 1st Strand cDNA Synthesis
Kit (TaKaRa) according to the supplier instructions.
Two microliters of cDNA was used as the template
for a 50-μl PCR amplification with rice actin primers
and rca gene-specific primers. Actin was used as an
internal standard (F 5 -TCCATCTTGGCATCTCTCAG-3 ;
R 5 -GTACCCGCATCAGGCATCTG-3 ). Because there
is no specific sequence for PCR amplification of
RCAL mRNA (To et al. 1999), the rca gene-specific
primers (F 5 -CGTGACGGGCGTATGGAGA-3 ; R 5 TTCCGGCACAGGCGGTTA-3 ) were designed for a
region that includes the gene-specific region of RCAS
mRNA. This resulted in an amplification product of
468 bp (RCAL mRNA) and another amplification product
of 550 bp (RCAS mRNA). The PCR amplification was set
up as follows: 94◦ C for 4 min; 25 cycles of 94◦ C for
30 s, 62◦ C for 30 s, 72◦ C for 1 min, followed by a
final extension at 72◦ C for 10 min. Taq polymerase was
purchased from TaKaRa, and the PCR mix was prepared
57
according to the manufacturer instructions. The PCR
products were assessed on ethidium bromide-stained
2% (w/v) agarose gel in 1× Tris–acetate–EDTA.
Rubisco and RCA protein quantification by ELISA
Samples were prepared as described above for the
Rubisco activity assay. The presence of the Rubisco
subunit and two RCA isoforms in the supernatant was
determined by direct enzyme-linked immunosorbent
assay (ELISA) using antibodies made by our laboratory
against Rubisco large subunit (RLS) or Rubisco small
subunit (RSS) and against different isoforms of RCA
(Appendix S1 C and D in Supporting information).
Extracted supernatant was diluted 640-fold for the
Rubisco content assay and 60-fold for the RCA content
assay using cover buffer. The cover buffer contained
50 mM carbonate/bicarbonate buffer (pH 9.6), 1 mM
phenylmethylsulfonyl fluoride, 2 mM benzamidine and
0.01 mM leupetine. The ELISA measurement was
developed based on the methods of DemirevskaKepova et al. (1990). In brief, flat-bottom immunoassay
polystyrene microplates (Costar, Cambridge, MA) were
used. The wells in each microtiter plate were coated with
100 μl of diluted sample. The plates were incubated at
37◦ C for 2 h and washed three times with phosphatebuffered saline with 0.05% Tween (PBST; 136.7 mM
NaCl, 2.68 mM KCl, 1.47 mM KH2 PO4 , 8.38 mM
Na2 HPO4 , 0.05% Tween 20, pH 7.4). The plates were
then incubated in 3% (w/v) bovine serum albumin (BSA)
and phosphate buffered saline (200 μl per well) for 0.5 h
at 37◦ C and washed three times with PBST. Horseradish
peroxidase-conjugated monoclonal antibodies (diluted
in PBST containing 3% BSA; 100 μl per well ) against
the RLS, RSS, RCA or RCAL from rice were added and
incubated for 1 h at 37◦ C. After washing the plates
five times with PBST, 100 μl of substrate solution
(0.1 M citric acid, 0.2 M Na2 HPO4 , 0.15% H2 O2 ,
3.699 mM o-phenylenediamine) was added to each
well. The reaction was allowed to proceed at 37◦ C
for 15 min and was stopped by the addition of 50 μl
of 2 M H2 SO4 to each well. The extent of enzyme
activity was monitored by measuring absorbance at
490 nm with a microplate reader (Model 680; Bio-Rad,
Hercules, CA). The background value resulting from
non-specific binding was obtained in control wells,
in which 3% (w/v) BSA was used instead of sample
solution. Quantities of Rubisco subunits and the two
RCA isoforms were calculated according to standard
curves plotted by parallel assays using serial dilutions
of standard Rubisco or RCA protein samples. The
purification of standard RLS and RSS was performed
by the method of Makino et al. (1983). An expressed
58
common segment of two rice RCA isoforms and a
specific segment of the C-end of the RCAL polypeptide
were used as RCA standard samples (Appendix S1 A and
B in Supporting information) for quantitative analysis of
total RCA content and RCAL content.
Co-immunoprecipitation and western blot analyses
In order to investigate the effect of heat stress on the
interaction between Rubisco and RCA, the Rubisco–RCA
complex was isolated from leaf extracts with ProFound™
Co-Immunoprecipitation Kit (Pierce, Rockford, IL)
according to the manufacturer instructions. Antibodies
to Rubisco were immobilized on a coupling gel to pull
down the Rubisco complex. To exclude non-specific
binding between the protein and the coupling gel or
between the protein and the antibodies, a non-relevant
antibody against 6× His taq was coupled to the antibody coupling gel in the parallel assay. Total soluble
protein from rice leaf samples was rapidly isolated with
extraction buffer as described in the Rubisco activity
assay, except that 5 mM ATP was included in the
extraction buffer. The antibody-coupled gels were incubated in the protein extraction supernatant and washed
five times with washing buffer (80 mM NaCl, 8 mM
sodium phosphate, 2 mM potassium phosphate and 10
mM KCl, pH 7.4), and then the Rubisco–RCA complexes were eluted from the coupling gel. The resulting
complexes were separated on 12.5% sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)
gel and transferred to polyvinylidene fluoride (PVDF)
membranes. Blots were probed with monoclonal antibodies to rice RCA. SDS–PAGE and western blot analysis
were performed as described by Sambrook and Russell
(2001). The Rubisco–RCA complexes were also isolated
using gels coupled with antibody to RCA, and the resulting captured complexes were then analyzed by western
blot using an antibody to RLS. To calculate how much
of the RCA protein was associated with the complexes,
the RCA fractions in the total soluble protein extract and
in the complexes were compared by immunoblotting.
Results
Effect of moderate heat stress on photosynthetic
rates, quantum yield of photosystem II (PS II)
photochemistry and Rubisco activation in rice
plants
Response of Pn to a short-term moderate heat stress
of 40/30◦ C (day/night) was investigated with six-leafold rice seedlings. At control temperature (30/20◦ C,
day/night), Pn, averaging 22.6 μmol CO2 m−2 s−1 ,
did not change significantly during the 4-day period
Physiol. Plant. 139, 2010
Fig. 1. Response of net photosynthesis (Pn), Fv /Fm and Rubisco activity to moderate heat stress in rice plants. (A) Pn, (B) Rubisco initial activity (C)
and Rubisco total activity were measured in rice plants in the control conditions at 30/22◦ C (open circles) and at moderate heat stress of 40/30◦ C
(closed triangles) for 1–4 days plus 3 days of recovery (day 7) at the control temperature. Pn was measured in the air at a PPFD of 1200 μmol
m−2 s−1 . Samples for measurement of Rubisco activity were harvested immediately after Pn measurement. (D) Fv /Fm was determined in dark-adapted
rice leaves during a 16-day time course. Symbols marked with the same letters were not significantly different (P < 0.05) for time course comparisons
for different samples. The results presented are the means ± SD of three to five individual plants.
(Fig. 1A). When rice plants were exposed to moderate
heat stress, Pn decreased sharply to 60% of the control
level on the first day. During prolonged heat stress,
an obvious acclimation of Pn to the high temperature
occurred gradually and Pn returned to 93% of the control on the fourth day. When the heat-stressed plants
were returned to the control temperature for 3 days, Pn
in the heat-stressed leaves fully recovered to the level of
control plants.
Like the change of Pn, Rubisco initial activity in
the heat-treated rice leaves was significantly inhibited
after 1 day of heat stress and then gradually increased
to the control level during the remaining 3 days of
treatment (Fig. 1B). In contrast, moderate heat stress had
no remarkable effect on Rubisco total activity (Fig. 1C).
These results indicated that the initial inhibition and
the subsequent acclimation of Pn under moderate heat
stress were closely related to the change of Rubisco initial
activity in rice leaves. The effect of 40/30◦ C heat stress
on maximum quantum yield of the PS II photochemistry
(Fv /Fm ) was also estimated by measuring chlorophyll
fluorescence in dark-adapted leaves (Fig. 1D). The
results showed that Fv /Fm was not affected by 40/30◦ C
heat stress until 16 days of treatment, suggesting that the
inhibition of Pn by short-term moderate heat was not
caused by the impairment of PS II activity.
Physiol. Plant. 139, 2010
Effect of moderate heat stress on rice rca gene
expression
To explore the cause of the change in Rubisco initial
activity, the quantitative changes of two Rubisco subunits
and two RCA isoforms in rice leaves during photosynthetic heat acclimation were determined by ELISA based
on specific monoclonal antibodies to RLS, RSS, RCAL
or both RCA isoforms. The results of ELISA showed that
4 days of heat stress (40/30◦ C) had little effect on RLS and
RSS levels (Fig. 2A, B). However, the two RCA polypeptides exhibited a different response to high temperature.
RCAL content was not influenced on day 1 but it was
significantly upregulated from day 2 to day 4 of heat
stress and increased by 40% compared with the control
on day 4 (Fig. 2C). RCAS content decreased by 18% at
day 1 of heat stress and then gradually recovered to
the control level over the remaining treatment (Fig. 2D).
After 3 days of recovery growth at the control temperature, both RCAL and RCAS contents returned to control
levels. Correlative analysis further showed that Pn was
significantly correlative to the Rubisco initial activity
(Fig. 3A, r = 0.958, P < 0.001) that was closely related
to the RCAS content at different temperatures (Fig. 3B,
r = 0.919, P < 0.001), suggesting that photosynthetic
heat inhibition and acclimation were associated with
59
Fig. 2. Responses of Rubisco subunit content and different RCA isoform contents to high temperature during heat acclimation and recovery of rice
plants. (A) Soluble RLS, (B) RSS, (C) RCAL and (D) RCAS content in rice leaves were measured in control conditions of 30/22◦ C (open squares) or
under moderate heat stress of 40/30◦ C for 1–4 days plus 3 days of recovery (day 7) in control conditions (closed circles). Symbols marked with the
same letters were not significantly different (P < 0.05) for time course comparisons for different samples. The results presented are the means ± SD
of four to six individual plants.
Fig. 3. (A) Pn as a function of Rubisco initial activity. (B) Rubisco initial
activity as a function of RCAS content at different temperatures. The
data of Pn, Rubisco initial activity and RCAS content in samples treated
at control temperature of 30/22◦ C (open circles) or moderate heat stress
of 40/30◦ C (closed circles) are taken from Fig. 1 and 2. The lines indicate
the linear fit for data (A, r = 0.958, P < 0.001; B, r = 0.919, P < 0.001).
60
the changes in Rubisco activation state and that the
initial inhibition of Rubisco initial activity by heat was
associated with the loss of RCAS content.
The mRNA sequence of the two rice RCA polypeptides
is 99% identical, the exception being a 82-bp addition
near the 3 end of small isoform, which contains an early
stop codon (To et al. 1999). Two PCR primers resulting
in a PCR product of 468 bp (denoting large RCA isoform mRNA, rca-L) and another PCR product of 550 bp
(denoting small RCA isoform mRNA, rca-S) were used to
investigate the effect of heat stress on gene expression of
the two RCA isoforms. At the control temperature, mRNA
accumulation of RCAS was much more than that of
RCAL (Fig. 4, left), which was consistent with the protein
accumulation (Fig. 2C). Unequal mRNA accumulation
indicated that the alternative splicing of RCA pre-mRNA
favored the product of RCAS mRNA at the control
temperature. When rice plants were exposed to high
temperature, the accumulation of RCAL mRNA increased
significantly after 1 day of heat treatment (Fig. 4, right).
This upregulation of RCAL mRNA persisted throughout
the 4-day heat treatment and disappeared after a 3day recovery, while the RCAS mRNA accumulation was
almost unaffected. These results suggested that high temperature induced more RCAL protein via transcriptional
regulation, but the initial loss and the following recovery
of RCAS content in response to high temperature were
not attributed to transcriptional regulation.
Physiol. Plant. 139, 2010
Fig. 4. RCA mRNA accumulation in response to moderate heat stress.
Rice plants were exposed to control (C, 30/22◦ C) and heat stress (HS,
40/30◦ C) conditions for 1–4 days followed by 3 days of recovery (re3)
at the control temperature. Total RNA from each sample was extracted
as described in section Materials and methods. RT-PCR analysis was
performed with actin as a control. The amplification products of both
the RCAL and the RCAS mRNA were separated on a 2% agarose gel. The
arrow indicates the position of the RCAL mRNA amplification product.
Effect of heat stress on the interaction between
Rubisco and RCA
RCA-mediated Rubisco activation necessarily requires
direct protein–protein interactions between the two
enzymes. To investigate the effect of heat stress on this
interaction, the Rubisco–RCA complex in leaf extract
was isolated by the method of co-immunoprecipitation.
Firstly, the complex was immunopurified using RCA
antibody, and the captured Rubisco–RCA complex
was then separated by SDS–PAGE and probed with
antibodies to RLS. The result showed that the amount of
Rubisco in the Rubisco–RCA complex declined slightly
in heat-acclimated leaves and heat-treated plus recovery
leaves (Fig. 5, top panel). Secondly, the complex was
also immunopurified using Rubisco antibody, and the
captured complex was then detected with antibodies
to RCA by western blot (Fig. 5, bottom panel). The
negative control (NC) using a non-relevant antibody
against 6× His taq instead of Rubisco or RCA antibodies
resulted in no immunoreaction, suggesting that nonspecific binding did not occur. In control leaves,
immunoblots of the eluted complex revealed that RCAS
was the major form present in the Rubisco–RCA
complex. In heat-acclimated leaves, the amount of RCAL
in the Rubisco–RCA complex increased significantly
and decreased to the control level after a 3-day
recovery, while the amount of RCAS in the Rubisco–RCA
complex rose only slightly. The obviously increased RCA
and slightly decreased Rubisco in the Rubisco–RCA
complex demonstrated that the ratio of RCA vs
Rubisco in Rubisco–RCA complex increased during heat
acclimation of rice leaves, and more of the RCAL isoform
was included in the Rubisco–RCA complex from heatacclimated leaves. These data suggest that the changes
in the make-up of the Rubisco–RCA complex may
be involved in the mechanism of photosynthetic heat
acclimation in rice plants. It should be noted that, based
Physiol. Plant. 139, 2010
Fig. 5. Effect of heat acclimation to Rubisco (top panel) and RCA
(bottom panel) protein in Rubisco–RCA complexes. Rice leaves from
control plants (C), 4-day heat-treated plants (HS) and heat-treated plus
3-day recovery plants (HS + Re) were used for Rubisco–RCA complex
analysis. NC samples were isolated with 6× His taq antibody instead of
Rubisco or RCA antibodies. Top panel, Rubisco–RCA complex in each
sample was isolated by co-immunoprecipitation using a monoclonal
antibody to rice RCA; and the eluted complex was separated by
SDS–PAGE, transferred to a PVDF membrane, then probed with
monoclonal antibody to RLS and visualized using alkaline phosphatase
conjugated to a secondary antibody. Bottom panel, the Rubisco–RCA
complex was isolated by co-immunoprecipitation using a monoclonal
antibody to Rubisco and detected by western blot using antibody to
rice RCA. The arrows indicate the position of RCAL (45 kDa) and RCAS
(41 kDa).
on quantitative analysis of the Rubisco–RCA complex,
the RCA in isolated complex from rice leaves is about
one fourth of the total soluble RCA (Appendix S2 in
Supporting information). Whether the RCA that does not
interact with Rubisco has additional functions requires
further study.
Transgenic rice plant with different RCAL levels
and comparison of photosynthetic heat sensitivity
of different plant lines
The upregulation of RCAL protein and the greater
inclusion of RCAL isoform in the Rubisco–RCA complex
at high temperature implied that RCAL might play an
important role in photosynthetic heat acclimation of rice
plants. To further clarify the relationship between RCAL
and photosynthetic heat sensitivity, transgenic rice plants
expressing different RCAL levels were used to investigate
the effect of different RCAL levels on photosynthetic
heat sensitivity. RCAL and RCAS sense gene constructs
were prepared by inserting a rice RCAL or RCAS cDNA
fragment in the binary vector pC1390 between a maize
ubiquitin promoter and a NOS terminator (Fig. 6A).
RT-PCR and western blot analysis of three selected
transgenic lines showed that the expression of target
genes was successfully enhanced in RCAL transgenic
rice plants (L1–L3) and in RCAS transgenic rice plants
(S1–S3) based on both mRNA (Fig. 6B) and protein
levels (Fig. 6C). To exclude the effect of increased
total RCA on photosynthetic thermotolerance, L1 plants
exhibiting the highest RCAL to RCAS ratio and S1 plants
expressing the similar amount of total RCA protein
61
Fig. 6. (A) rca-L or rca-S sense gene construct: rice rca-L or rca-S cDNA fragment was subcloned between the maize ubiquitin promoter and the NOS
terminator in the binary vector pC1390. (B) RT-PCR analysis of RCAL or RCAS mRNA from WT and three transgenic lines expressing the rca-L or rca-S
sense gene using actin as a control. A pair of primers resulting in one specific PCR product was used for amplification of RCAS mRNA (WT and S1–S3
plants), while a pair of primers resulting in amplification of both RCAL and RCAS mRNA was used for RCAL mRNA analysis (WT and L1–L3 plants).
The arrow indicates the PCR amplifying product of RCAL mRNA. (C) Immunoblot of leaf extracts from WT and transgenic (L1–L3, S1–S3) rice plants.
An equal amount of leaf tissues from each plant line was extracted in buffer, and the polypeptides were separated by SDS–PAGE and transferred
to a PVDF membrane. Blots were probed with antibody to rice RCA and visualized using alkaline phosphatase conjugated to a secondary antibody.
(D) Quantitative analysis of total RCA content and two isoform ratios in each plant line by ELISA. Symbols marked with the same letters were not
significantly different (P < 0.05). The results presented are the means ± SD of three individual plants.
with L1 plants were chosen for the photosynthetic heat
sensitivity assay.
A sudden increase in light flux upon dark- or
low light-adapted leaf usually leads to a subsequent
increase in Pn. This increase of Pn often comprises
two kinetically distinct phases before reaching the
steady-state Pn: a fast phase representing rapid RuBP
production (Woodrow and Mott 1992) and a slower
phase representing the activation of Rubisco from
inactive forms to active from mediated in part by RCA
(Hammond et al. 1998). This kinetic analysis of the
second, slower phase was used here to investigate the
effect of different RCAL levels on photosynthetic heat
sensitivity. Light-mediated kinetics of Pn following an
increase in light flux (from 110 to 1200 μmol m−2 s−1 )
was compared in L1, S1 and WT rice plants at different
temperatures. At control temperature (30◦ C), Pn of the
three plant lines increased rapidly and reached a steadystate level, respectively, after 15–30 min illumination
62
at a PPFD of 1200 μmol m−2 s−1 (Fig. 7A). At high
temperature (40◦ C), an obvious slow decline of Pn
occurred after the initial fast increase in all rice plants
(Fig. 7B). This decline of Pn partly reflected the limitation
of light-mediated Rubisco activation because of the fast
inhibition of RCA by heat. At the control temperature,
both L1 and S1 plants exhibited higher steady-state Pn
than WT plants, increasing by 12% in S1 plants and
3.8% in L1 plants; while at high temperature (40◦ C),
L1 plants exhibited the highest Pn in the whole time
course. When Pn reached a steady state after 30-min
illumination, it was inhibited by 32% in WT, by 30%
in S1 plants and by 12% in L1 plants (Table 1). These
data demonstrated that L1 plants with more amount of
RCAL exhibited better photosynthetic thermotolerance
than WT and S1 plants, while the S1 plants with more
amount of RCAS protein exhibited the highest Pn at the
control temperature.
Physiol. Plant. 139, 2010
Fig. 8. A comparison of RCA isoform in the Rubisco–RCA complex from
different plant lines. Rubisco–RCA complexes in leaf extracts of WT, L1
or S1 plants were isolated by co-immunoprecipitation using monoclonal
antibody to Rubisco. The eluted complex was separated by SDS–PAGE,
transferred to a PVDF membrane, then probed with monoclonal antibody
to rice RCA and visualized using alkaline phosphatase conjugated to a
secondary antibody.
Fig. 7. A comparison of the increase in Pn upon the illumination
of different rice plants at different temperatures. Wild-type (open
circle), L1 transgenic line (solid triangle) and S1 transgenic line (open
square) rice leaves were illuminated at a PPFD of approximately
110 μmol m−2 s−1 for 30 min at 30◦ C before the light flux was increased
to 1200 μmol m−2 s−1 at either (A) 30◦ C or (B) 40◦ C. Pn values were
plotted over time at a PPFD of 1200 μmol m−2 s−1 .
Table 1. Effect of moderate heat stress on steady-state Pn and Rubisco
initial activity in wild-type and transgenic rice plants. Pn was determined
when plants were exposed to the indicated temperatures at the
irradiance of 1200 μmol m−2 s−1 in air for 30 min, while the Rubisco
initial activity was determined after 1 h of treatment at a different
temperature with the same irradiance. The numbers in parenthesis
indicate the percent inhibition at 40◦ C. *Significantly different from
other plant lines at P < 0.01.
Plant line
Pn (μmol m−2 s−1 )
30◦ C
40◦ C
To understand the biochemical basis for enhanced
thermotolerance of Pn and Rubisco initial activity in
L1 plants, Rubisco–RCA complex in different lines was
isolated via co-immunoprecipitation for RCA isoform
analysis. Immunoblots showed that almost only RCAS
was detected in the Rubisco–RCA complex from WT and
S1 plants, while a similar amount of RCAL with RCAS was
detected in the Rubisco–RCA complex from L1 plants
(Fig. 8). This observation indicated that the mechanism
of enhanced photosynthetic thermotolerance in L1
plants was associated with more inclusion of RCAL
in Rubisco–RCA complex, which was consistent
with the situation in heat-acclimated rice leaves
(Fig. 5).
Fig. 9 shows the effects of extended heat stress on
growth of seedlings of WT plants and two transgenic
lines. When grown at control temperature, S1 plant
seedlings produced the most biomass. Eight-day heat
treatment significantly decreased the dry weight in WT
plants (decreased by 26%) and S1 plants (decreased
by 25%), while L1 plants were almost unaffected.
These results suggested that S1 plants produced the
Rubisco initial activity
(μmol m−2 s−1 )
30◦ C
40◦ C
Wild-type 25.6 ± 1.0 17.5 ± 0.6 (32) 36.3 ± 1.3 21.8 ± 1.0 (40)
L1
26.9 ± 1.3 23.6 ± 1.2 (12)* 38.2 ± 0.3 30.3 ± 1.9 (21)*
S1
28.3 ± 1.0 19.9 ± 1.1 (30) 40.8 ± 0.7 25.0 ± 1.4 (39)
Because high temperature (40◦ C) mainly inhibited the
Rubisco initial activity (Fig. 1B), we also determined the
effect of increased expression of RCAL protein on heat
sensitivity of Rubisco initial activity. After heat treatment
of 1 h in light, Rubisco initial activity was inhibited
by 40% in WT plants, 39% in S1 plants and 21% in
L1 plants compared with control plants, respectively
(Table 1), suggesting that the increased expression of the
RCAL protein indeed enhanced the thermotolerance of
Rubisco initial activity.
Physiol. Plant. 139, 2010
Fig. 9. Effects of moderate heat stress on WT and transgenic plant
seedling growth. Eighteen-day-old seedlings were transferred to heat
stress (40/30◦ C) or control (30/20◦ C) temperatures for 8 days. For
measurements of dry weight, the seedlings were dried at 85◦ C for
2 days. Columns marked with asterisk indicate a significant difference
(P < 0.05) between the control and heat stress seedling. The results
presented are the means ± SD of six individual plants.
63
most biomass at the control temperature but L1 plants
produced more biomass than WT and S1 plants under
moderate heat stress.
Discussion
Gene expression of RCA in response to high
temperature underlies a mechanism of
photosynthetic heat acclimation in rice plants
In this study, an obvious photosynthetic heat acclimation
in rice plants occurred during 4 days of moderate high
temperature of 40/30◦ C (Fig. 1A). Unaffected Fv /Fm
during a longer term of heat treatment indicated
that, in warm habitat rice plants, initial inhibition of
photosynthesis was not because of the impairment
of PS II (Fig. 1D). Our results showed that Rubisco
initial activity was significantly inhibited by 40◦ C high
temperature (Fig. 1B), while the activity of fully activated
Rubisco was hardly affected during the 4-day treatment
(Fig. 1C), which was consistent with the results obtained
in other studies (Craft-Brandner and Salvuuci 2000,
Haldimann and Feller 2004, Law and Craft-Brandner
1999). The markedly inhibited Rubisco initial activity
and unaffected Rubisco total activity suggested that
photosynthesis should be limited by the ability of RCA
to maintain Rubisco in a fully activated state, while heat
acclimation removed this limitation. Hence, our results
supported the view that RCA has the ability to acclimate
to moderate high temperature.
Previous studies on maize (Sánchez de Jiménez et al.
1995), wheat (Law et al. 2001) and cotton (Deridder
and Salvucci 2007) have shown that high temperature
induces a new form of RCA polypeptide, suggesting that
the response of RCA gene expression to high temperature
may contribute to acclimation of photosynthesis during
extended periods of heat stress. In rice plants, however,
our results showed that the accumulation of RCAL ,
which was much less than the accumulation of RCAS
at control temperature, was markedly upregulated by
heat (Fig. 2C). Analysis revealed that RCAL mRNA
accumulation increased significantly after 1 day of heat
stress (Fig. 4), implying that the alternative splicing of
RCA pre-mRNA tends to produce more large isoform at
high temperature. This upregulation of RCAL , shown at
both the mRNA and protein levels, persisted throughout
the whole heat treatment and disappeared after a 3-day
recovery growth at the control temperature (Fig. 2 and
4), which suggested that the enhanced accumulation of
RCAL was associated with the response of rice plants to
high temperature.
To our knowledge, no stable Rubisco–RCA complex
has been isolated. In our research, we used monoclonal
antibodies to Rubisco or RCA to detect the existence
64
of the Rubisco–RCA complex from rice leaves.
Quantitative analysis showed that the RCA in the
isolated complex was only about one fourth of the total
soluble RCA (Appendix S2 in Supporting information),
suggesting that a large portion of the RCA was not
in the Rubisco–RCA complex, or perhaps that this
complex was not stable. More interestingly, our results
showed that RCA content, especially the RCAL content,
in the Rubisco–RCA complex increased notably in heatacclimated rice leaves, while the Rubisco content in the
Rubisco–RCA complex decreased slightly (Fig. 5). This
finding provided direct evidence supporting the view
that the ratio of RCA to Rubisco in the Rubisco–RCA
complex increased at high temperature, which implied
that a larger RCA oligomer including more RCAL might
interact with Rubisco in heat-acclimated rice leaves.
Furthermore, L1 plants, which contained a similar
proportion of the two RCA isoforms in the Rubisco–RCA
complex (Fig. 8), exhibited enhanced thermotolerance
of the photosynthetic rate (Fig. 7) and Rubisco initial
activity (Table 1) and grew better than WT plants and
S1 plants at high temperature (Fig. 9). Together, these
results demonstrated that the proportion of RCAL in
the Rubisco–RCA complex played an important role
in regulating the photosynthetic heat sensitivity in rice
plants.
Previous studies have shown that the larger the RCA
oligomeric complex, the greater its ability to hydrolyze
ATP at high temperature ( Portis 2002, Salvucci 1992)
and that RCAL is more thermostable than RCAS (CraftsBrandner et al. 1997). Heat stress significantly induced
the expression of RCAL , which existed at a low level
at the non-stress temperature. Hence, it is likely that
interactions between different RCA isoforms lead to the
formation of a more stable RCA oligomeric structure in
heat-acclimated rice leaves and thus contribute to the
maintenance of Rubisco initial activity under moderate
heat stress. In addition, Salvucci (2008) has recently
found that chaperonin-60-β associates with RCA under
high temperature, suggesting that the chaperonin-60-β
may play a role in the assembly of oligomeric RCA.
Based on these observations, we conclude that the
upregulation of RCAL and its increased location in
Rubisco–RCA complex at high temperature underlie
a mechanism of photosynthetic heat acclimation in rice
plants.
Two RCA isoforms may have different roles in rice
plants
In most plant species studied, RCA contains two
isoforms arising from one gene via alternative splicing
or encoded by a separate gene (reviewed by Portis
Physiol. Plant. 139, 2010
2003). Unlike the cases of Arabidopsis and spinach,
which express equivalent amounts of the two RCA
isoforms (Salvucci et al. 1987), rice leaf contains a
much greater amount of small isoform than large
isoform at both the mRNA and protein levels (To
et al. 1999; Fig. 2 and 4), suggesting that the unequal
amounts of the two RCA isoform proteins in rice
leaves result from different alternative splicing efficiency.
Different protein abundance of two RCA isoforms may
reflect different physiological significance. At control
temperature, immunoblot analysis showed that RCAS
was the major form in isolated Rubisco–RCA complex
(Fig. 5). Furthermore, the Rubisco initial activity was
closely related to the soluble RCAS content at different
temperatures (Fig. 3). These results suggest that the
activation of Rubisco at the control temperature was
the main contributor to the action of RCAS in rice.
Our observation that, at the control temperature, the
transgenic rice plant line with increased RCAS content
(S1) exhibited higher steady-state Pn (Fig. 7A) and
produced more biomass (Fig. 9) than the transgenic rice
plant line with increased RCAL content (L1) also supports
this conclusion.
Interestingly, the accumulation of RCAL , which was
very low at the control temperature, was upregulated
significantly at the high temperature and resulted in the
increased participation of RCAL in the Rubisco–RCA
complex during heat acclimation. Furthermore, transgenic rice plants expressing more RCAL exhibited better
photosynthetic thermotolerance. These results indicate
that the RCAL may function as a heat-induced chaperone that contributes to the maintenance of Rubisco
initial activity and photosynthetic rate at moderate high
temperature.
Conclusions
In the present study, our findings in rice, including
photosynthetic heat acclimation and different RCA isoform sense genotypes, clearly indicate that the acquired
thermotolerance of RCA during heat acclimation is associated with heat-induced RCAL . Immunoblots of the
Rubisco–RCA complex reveal that Pn and Rubisco
initial activity at control temperature are primarily
maintained by RCAS , while in heat-acclimated rice
leaves RCAL plays an important role in regulating the
photosynthetic heat sensitivity by participating in the
Rubisco–RCA complex. Experiments using transgenic
rice plants demonstrated that increasing the proportion of
RCAL enhanced the thermotolerance of Pn and Rubisco
initial activity. Future studies on interactions between the
two RCA isoforms should provide new insights into the
Physiol. Plant. 139, 2010
mechanism of how RCAL works in response to moderate
high temperature.
Acknowledgements – This work was financially supported
by the National Natural Science Foundation (30971703
and 30471051) and National High Science and Technology
Program (20087AA10Z191).
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Appendix S1. Expressed protein fragment of RCA and
monoclonal antibodies to Rubisco subunits and RCA
isoforms.
Appendix S2. Stoichiometry of RCA protein in the
Rubisco–RCA complex.
Please note: Wiley-Blackwell are not responsible for
the content or functionality of any supporting materials
supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.
Edited by K.-J. Dietz
Physiol. Plant. 139, 2010
67