Downregulation of Chloroplast RPS1 Negatively Modulates Nuclear Heat-Responsive Expression of HsfA2 and Its Target Genes in Arabidopsis
et al. (2012) Downregulation of Chloroplast RPS1 Negatively Modulates Nuclear Heat-Responsive
Expression of HsfA2 and Its Target Genes in Arabidopsis. PLoS Genet 8(5): e1002669. doi:10.1371/journal.pgen.1002669
Downregulation of Chloroplast RPS1 Negatively Modulates Nuclear Heat-Responsive Expression of HsfA2 and Its Target Genes in Arabidopsis
Hai-Dong Yu 0
Xiao-Fei Yang 0
Si-Ting Chen 0
Yu-Ting Wang 0
Ji-Kai Li 0
Qi Shen 0
Xun-Liang Liu 0
Fang- Qing Guo 0
Li-Jia Qu, Peking University, China
0 The National Key Laboratory of Plant Molecular Genetics and National Center for Plant Gene Research (Shanghai), Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai , China
Heat stress commonly leads to inhibition of photosynthesis in higher plants. The transcriptional induction of heat stressresponsive genes represents the first line of inducible defense against imbalances in cellular homeostasis. Although heat stress transcription factor HsfA2 and its downstream target genes are well studied, the regulatory mechanisms by which HsfA2 is activated in response to heat stress remain elusive. Here, we show that chloroplast ribosomal protein S1 (RPS1) is a heat-responsive protein and functions in protein biosynthesis in chloroplast. Knockdown of RPS1 expression in the rps1 mutant nearly eliminates the heat stress-activated expression of HsfA2 and its target genes, leading to a considerable loss of heat tolerance. We further confirm the relationship existed between the downregulation of RPS1 expression and the loss of heat tolerance by generating RNA interference-transgenic lines of RPS1. Consistent with the notion that the inhibited activation of HsfA2 in response to heat stress in the rps1 mutant causes heat-susceptibility, we further demonstrate that overexpression of HsfA2 with a viral promoter leads to constitutive expressions of its target genes in the rps1 mutant, which is sufficient to reestablish lost heat tolerance and recovers heat-susceptible thylakoid stability to wild-type levels. Our findings reveal a heat-responsive retrograde pathway in which chloroplast translation capacity is a critical factor in heatresponsive activation of HsfA2 and its target genes required for cellular homeostasis under heat stress. Thus, RPS1 is an essential yet previously unknown determinant involved in retrograde activation of heat stress responses in higher plants.
Funding: This research was supported in parts by the Ministry of Science and Technology of China (2012CB944802, 2007CB10880, and 2007AA10Z111), the
National Natural Science Foundation of China (90817015 and 30770198), and the Bairen Project of Chinese Academy of Sciences. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
It is generally accepted that a temperature upshift, usually 10
15uC above an optimum temperature for growth, is considered as
heat stress for leaf photosynthesis in higher plants [1,2].
Photosystem II (PSII) is the most heat sensitive apparatus within
the chloroplast thylakoid membrane protein complexes involved
in photosynthetic electron transfer and ATP synthesis .
Chlorophyll fluorescence, the ratio of variable fluorescence to
maximum fluorescence (Fv/Fm) and the base fluorescence (Fo) are
used as common indicators of heat stress-induced damages that
have been shown to correlate with alterations of photochemical
reactions in thylakoid lamellae of chloroplast [1,2,6,7]. Oxygen
evolving complex (OEC) in PSII is highly thermolabile and heat
stress may cause the dissociation of OEC, resulting in an
imbalance in the electron flow from OEC toward the acceptor
side of PSII in the direction of PSI reaction center [13,8,9].
Studies on spinach thylakoids subjected to heat stress have shown
that heat stress causes cleavage of the reaction center-binding
protein D1 of PSII and induces dissociation of a manganese
(Mn)stabilizing 33-kDa proteins from PSII reaction center complex
. Besides the disruption of OEC in PSII, heat stress also leads
to dysfunction in the system of carbon assimilation metabolism in
the stroma of chloroplast . The rate of
ribulose-1,5-bisphosphate (RuBP) regeneration is limited by the disruption of
electron transport and inactivation of the oxygen evolving
enzymes of PSII [7,11]. It is known that under heat stress, the
decline in ribulose-1,5-bisphosphate carboxylase/oxygenase
(Rubisco) activity is mainly due to inactivation of Rubisco activase
that is extremely sensitive to elevated temperatures because the
enzyme Rubisco of higher plants is heat stable [5,11]. In addition
to the early effects on photochemical reactions and carbon
assimilation, heat stress usually leads to alterations in the
microscopic ultrastructures of chloroplast and the integrity of
thylakoid membranes, including membrane destacking and
Because temperature elevations represent a fundamental
challenge to all sessile organisms, higher plants are capable of a
variety of heat shock responses characterized by a rapid expression
reprogramming of a set of proteins known as heat shock proteins
(HSPs) [16,17]. Analysis of Arabidopsis genome-wide expression
profiles to heat stress has shown that the transcripts of the
wellAs a consequence of global warming, increasing
temperature is a serious threat to crop production worldwide and
may influence the objectives of breeding programs. As a
universal cellular response to a shift up in temperature, the
heat stress response represents the first line of inducible
defense against imbalances in cellular homeostasis in the
prokaryotic and eukaryotic kingdoms. Given that
components of the photosynthetic apparatus housed in the
chloroplast are the primary susceptible targets of thermal
damage in plants, the chloroplasts were proposed as
sensors to a shift up in temperature. However, the
mechanism by which chloroplasts regulate the expression
of nuclear heat stressresponsive gene expression
according to the functional state of chloroplasts under heat stress
remains unknown. In this study, we have identified
chloroplast ribosomal protein S1 (RPS1) as a
heat-responsive protein through proteomic screening of
heat-responsive proteins. We have established a previously
unrecognized molecular connection between the downregulation
of RPS1 expression in chloroplast and the activation of
HsfA2-dependent heat-responsive genes in nucleus, which
is required for heat tolerance in higher plants. Our data
provide new insights into the mechanisms whereby plant
cells modulate nuclear gene expression to keep
accordance with the current status of chloroplasts in response
to heat stress.
characterized HSPs increased dramatically, including Hsp101,
Hsp70s and small HSPs, which are proposed to act as molecular
chaperones in protein quality control under heat stress . In
addition to classical heat stress responsive genes, these studies have
also revealed the involvement of factors in heat tolerance,
including members of the dehydration-responsive element-binding
transcription factor 2 (DREB2) family of transcription factors,
GALACTINOL SYNTHASE 1 (GolS1) in the raffinose
oligosaccharide (RFO) pathway, and ASCORBATE PERROXIDASE2 (APX2)
. The accumulation of HSPs is assumed to counteract the
detrimental effects of protein misfolding and aggregation that
result from heat stress [28,29]. Genetic analyses demonstrate that
Hsp101 is required for heat tolerance, functioning in cooperation
with the small HSPs to resolubilize protein aggregates after heat
stress in higher plants [30,31]. Small HSPs may function as
membrane stabilizers and possibly as site-specific antioxidants to
protect thylakoid membranes against heat stress [2,32]. Several
small HSPs have been reported to protect thylakoid stability from
heat or oxidative stresses in photosynthetic organisms such as
higher plants  and cyanobacteria [36,37].
Expression of HSP genes is orchestrated mainly at the
transcriptional level by heat shock transcription factors (HSFs)
that recognize cis-elements (heat shock elements; HSEs)
conserved in HSP gene promoters [16,17]. HSFs play a central role
in heat shock response in many species. In contrast to Drosophila,
Caenorhabditis elegans and yeast that have a single HSF, the
Arabidopsis genome contains 21 HSFs that are assigned to 3
classes, A, B and C, based on the structural features of their
oligomerization domains . At least 23 and 18 HSF genes were
identified in rice (Oryza sativa) and tomato (Lycopersicon esculentum),
respectively [39,40]. In comparison with class A HSFs, the
members of class B lack the structural motif (aromatic,
hydrophobic and acidic amino acids) present in the C-terminal
domain crucial for the activator activity of class A HSFs . In
agreement with the structural difference between class A and B
HSFs, the transient overexpression of several class B HSFs failed
to activate heat shock responsive promoters in tobacco
protoplasts [42,43], indicating that certain class B HSFs may function
as coactivators to enhance transcriptional levels of house-keeping
genes during heat stress. Recently, consistent with having
repressive activities , Arabidopsis HsfB1 and HsfB2b were
reported to act as repressors that negatively regulate the
expression of HSFs, including HsfA2, in response to heat stress,
indicating that these two B class members may interact with class
A HSFs in regulating the shut-off of the heat shock response
As one of the most intensely studied HSFs, HsfA2 is
considered as a key regulator of heat tolerance in tomato
[41,47,48] and Arabidopsis [21,49,50] owing to its high activator
potential for transcription of HSP genes and its continued
accumulation during repeated cycles of heat stress and recovery
. In tomato, HsfA1a, a constitutively expressed HSF,
regulates the transcriptional activation of HsfA2 and HsfB1 in
response to heat stress, indicating that these three HSFs seem to
form a regulatory network to regulate the expression of
downstream heat shock-responsive genes [40,51]. In contrast to
tomato, Arabidopsis HsfA2 as a transcriptional activator can
localize to the nucleus and is regulated by a complex master
switch containing HsfA1a-e . Interestingly, Arabidopsis
ROF1 (AtFKBP62, a peptidyl prolyl cis/trans isomerase) also
modulates thermotolerance by interacting with HSP90.1 and
affecting the accumulation of HsfA2-regulated sHSPs . The
major target genes regulated by HsfA2 in Arabidopsis have been
identified by analyzing HsfA2 knockout mutant and
overexpression transgenic plants [21,49]. These target genes encode
APX2, GolS1, several small Hsps and individual isoforms of
the Hsp70 and Hsp101 families. In addition to the induction by
heat stress, the expression levels of HsfA2 were also up-regulated
in response to high light and H2O2 . Interestingly, a recent
report has shown that sumoylation of HsfA2 by the small
ubiquitin-like modifier protein (SUMO) regulates its activity in
connection with heat stress response and heat tolerance in
Although the accumulating literatures on the functions of HSPs
and HSFs have substantially extended our understanding of heat
stress response in plants, its regulatory network is far from
completely understood. In this study, we have identified
chloroplast ribosomal protein S1 (RPS1) as a heat-responsive protein
through proteomic screening of heat-responsive proteins. In
Escherichia coli, RPS1, the largest ribosomal protein, is involved in
the process of mRNA recognition and binding by the 30S
ribosomal subunit to the translation initiation site . The
RPS1 in E. coli consists of six repetitions of a conserved structural
domain, called S1 domain, which is found in many other proteins
involved in RNA metabolism in all organisms [57,58]. In bacteria,
RPS1 is believed to facilitate the binding of the 30S small
ribosomal subunit near the initiation codon of the transcripts
[59,60]. A homologue of the bacterial S1 protein was found in
spinach chloroplast [61,62], cyanobacteria  and Chlamydomonas
With the identification of RPS1 as a heat-responsive protein, we
have further demonstrated that knockdown of RPS1 expression
leads to inhibition of transcriptional activation of HsfA2 and its
target genes in the rps1 mutant, which confers a heat-sensitive
phenotype. Furthermore, our findings support that the capacity of
plastid protein translation is critical for retrograde activation of
HsfA2-dependent heat tolerance pathway. Our findings shed new
light on the mechanisms whereby plant cells modulate nuclear
gene expression to keep accordance with the current status of
chloroplasts in response to heat stress.
RPS1 Is a Heat-Responsive Protein Required for Heat
The objective of this study was the identification of novel genes
and pathways that contributed to the regulatory networks
involved in the development of heat tolerance in Arabidopsis.
During our initial studies, we were particularly interested in the
responsive proteins that are predicted to be targeted to
chloroplast since photosynthesis housed in chloroplast is
extremely sensitive to heat stress [1,2]. To uncover how plant cells
modulate protein expression in response to heat stress, we
performed a proteomic screen for heat-responsive proteins. One
heat-responsive protein was identified as the chloroplast RPS1
(Figure 1A, 1B, Figures S1 and S2), equivalent to the plastid
ribosomal protein S1 orthologues CS1 in spinach [61,62] and
CreS1 in C. reinhardtii  (Figure S3), which have been reported
to specifically bind chloroplast mRNA during translation
initiation. Western blot analysis further confirmed that the
protein levels of RPS1 gradually increased to peak at 2 h after
heat treatment, indicating that RPS1 is a heat-inducible protein
(Figure 1C and Figure S4). Interestingly, RPS1 responded to heat
stress at protein level, but not at transcriptional level since the
expression levels of RPS1 slightly decreased during 2-h heat
treatment (Figure S4), suggesting that RPS1 may not be identified
as a heat-responsive protein through the analysis of
heatresponsive transcriptome because the correlation between mRNA
and protein levels is not sufficient to predict protein expression
levels from the quantitative mRNA data [66,67]. Given that
RPS1 was induced by heat (Figure 1A1C and Figure S4) and its
highly conserved orthologues are involved in the direct control of
chloroplast gene expression, we reasoned that RPS1 might act as
a retrograde communication coordinator to trigger nuclear gene
expression critical for heat tolerance. We first examined whether
RPS1 plays a role in heat tolerance by identifying a homozygous
knockdown mutant of rps1 with a T-DNA insertion at 6 bp from
the 59-untranslated region (59-UTR) of the RPS1 gene (Figure 2A
and Figure S5). The substantial reduction in RPS1 expression in
the rps1 mutant compared with wild type was verified using
RTPCR, western blots and quantitative PCR with reverse
transcription (qRT-PCR) (Figure 2B and 2C). To analyze RPS1
expression, we generated transgenic Arabidopsis plants carrying
pRPS1:GUS constructs and the GUS staining signals were highly
observed in cotyledons and true leaves (Figure S6). The rps1
Figure 1. RPS1 is a heat-responsive protein required for heat tolerance. (A) Zoom-in view of the RPS1 spot in the images of 2D-PAGE
electrophoresis separation of proteins in response to heat treatment (38uC, 2 h) in dark. The fully-expanded leaves were detached from 21-d-old wild
type (WT) plants for control or heat treatment. (B) The quantified silver-staining signal intensity of the RPS1 spot according to the images shown in
(A). (C) Western blot analysis showing RPS1 protein levels in wild type leaves in response to heat treatment (38uC) in dark for the indicated time with
an anti-RPS1 polyclonal antibody. Equal protein loading was confirmed with antiserum against a-Tubulin. (D) to (F) The rps1 mutant plants showing
heat-sensitive phenotypes compared with WT plants as examined with young seedling (D), whole plant (E) and detached leaf (F). Heat treatments
were performed as described in Materials and Methods. (G) Trypan blue staining of the heat-challenged detached leaves of WT and the rps1 plants as
described in (F).
Figure 2. RPS1RNA interference leads to defects in heat tolerance. (A) Phenotypes of WT, rps1, rps1 complemented with RPS1 genomic DNA,
and two RPS1-RNA interference lines RNAi 1 and 2 as 21-d-old plants. (B) to (C) RT-PCR, western blot and qRT-PCR analysis showing expression and
translation levels of RPS1 in leaves excised from different genotypes as described in (A). For qRT-PCR analysis, Actin2 was used as the internal
standard. Error bars indicate standard deviations of three technical replicates, and the results were consistent in three biological replicates. (D) to (E)
Heat-sensitive phenotypes of different genotypes as described in (A) as examined with detached leaf and whole plant assays performed as described
in Materials and Methods.
mutant plants appeared slightly pale green with a reduced plant
size (Figure 2A). When 2.5-d-old seedlings were exposed to
transient increases in temperature, almost none of the mutant
seedlings survived after a 7-d recovery, compared with a survival
rate of greater than 90% for wild type seedlings (Figure 1D). In
addition, both mature plants and detached leaves of the rps1
mutant exhibited heat-sensitive phenotypes compared with wild
type plants after heat treatment and recovery (Figure 1E and 1F).
Cell death was examined by Trypan blue staining in the detached
leaves challenged with heat stress shown in Figure 1F, and the cell
death phenotype of the rps1 mutant was considerably more severe
than that of wild type (Figure 1G). Given the diminished
expression of RPS1 in the rps1 mutant, these results indicate that
RPS1 is required for heat tolerance. To test whether RPS1 is
generally involved in abiotic stress responses, we monitored the
sensitivity of wild type and rps1 mutant seedlings to salt and
osmotic stresses and observed no significant difference under
either treatment between wild type and rps1 mutant plants
(Figures S7 and S8). These data support the assumption that the
alteration in RPS1 expression affects cellular heat stress response
by disrupting specific machinery rather than through general
To further confirm the relationship existed between the
downregulation of RPS1 expression and the loss of heat tolerance
observed in the rps1 mutant, we investigated whether RNA
interference (RNAi)-mediated gene silencing of RPS1 alters the
heat-responsive behavior of transgenic plants harboring RNAi
constructs. As expected, we found that the RNAi lines, in which
downregulation of RPS1 expression was validated using RT-PCR,
western blots and qRT-PCR (Figure 2B and 2C), appeared pale
green like those of the rps1 mutant (Figure 2A) and exhibited a heat
sensitive phenotype in detached leaves and whole plants in
comparison with wild type plants (Figure 2D and 2E).
Furthermore, transforming genomic fragments of RPS1 complemented the
defects of the rps1 mutant in heat tolerance (Figure 2D and 2E).
These results demonstrate that RPS1 is the gene responsible for the
deficiency in heat tolerance exhibited in the rps1 mutant. In
addition, three genomic complementation lines (Comp rps1-2, 1-3
and 1-4) were generated in which the expression level of RPS1
dramatically increased when compared with wild type (Figure 2B,
2C and Figure S9). To test if the increased expression levels of
RPS1 could enhance the heat tolerance, we performed the heat
tolerance assay using the seedlings of wild type and the
complementation lines. The result showed that when 2.5-d-old
seedlings were exposed to transient increases in temperature,
almost none of the wild type seedlings survived after a 7-d
recovery, compared with a survival rate of 4050% for the
complementation line seedlings (Figure S9). On the other hand, we
also analyzed the translation efficiency of the representative
thylakoid membrane proteins encoded by chloroplast DNA,
including D1, D2, CP43, CP47, PsaA, PsaB and b-subunits of
ATPase between wild type and the genomic complementation line
Comp rps1-3 in which the transcript level and protein level of RPS1
increased dramatically compared with wild type (Figure 2B and
2C). In agreement with the enhancement in heat-tolerance, the
translation efficiency of the representative thylakoid membrane
proteins in complementation line Com rps1-3 increased
substantially in comparison with the wild type seedlings under both
control and heat stress conditions (Figure S9C). These data further
support that RPS1 is required for heat tolerance.
Knockdown of RPS1 Expression Inhibits Activation of
HsfA2-Dependent Heat Stress Responses
Based on heat-responsive transcriptional analysis, HsfA2 is the
most inducible HSF gene and appears to play a key role not only in
the triggering of cellular responses to heat stress, but also in the
amplification of the signal in the responses [49,50]. HsfA2
knockout mutant displays a heat-sensitive phenotype ,
indicating that HsfA2 is a key heat tolerance regulator that cannot
be replaced by other HSF genes. We verified the expression
pattern of HsfA2 in the rps1 mutant in response to heat by
qRTPCR. At a heat stress temperature (38uC), the levels of HsfA2
mRNA rapidly increased and peaked 1 h after treatment in wild
type plants whereas the heat-activated expression of HsfA2 was
severely inhibited in the rps1 mutant (Figure 3A). In agreement
with the HsfA2 expression pattern, the heat-responsive expression
of a subset of representative HsfA2 target genes, including APX2,
GolS1 and several HSPs (Hsp17.7-CII, Hsp18.1-CI, Hsp25.3-P,
Hsp70 and Hsp101) , was nearly abolished in the rps1 mutant
and did not match the transcriptional activity of these genes in
wild type plants (Figure 3B and 3C). In addition, we examined the
heat-responsive expression levels of 15 members in class A HSF by
qRT-PCR analysis. In agreement with the previous reports
[19,49], we found that HsfA2 is the most inducible HSF gene
among 15 members in class A and the mutation of RPS1 leads to
the most pronounced inhibitory effect on heat-responsive
transcriptional activation of HsfA2 in comparison with the rest of
the class A members whose relative expression levels are less than
100 with no or marginal difference between wild type and the rps1
mutant after heat treatment (Figure S10). These results indicate
that downregulation of RPS1 expression considerably inhibits the
transcriptional activation of HsfA2 and its target genes in response
to heat stress, which are required for establishing cellular heat
Constitutive Expression of HsfA2 Is Sufficient to
Reestablish Lost Heat Tolerance in rps1 Mutant Plants
Many of HsfA2 target genes are involved in protective
environmental stress responses, including APX2 encoding a
enzyme scavenging stress-induced reactive oxygen species (ROS)
, GolS1 encoding a enzyme catalyzing the synthesis of
protective osmolyte such as RFO  and several HSPs encoding
chaperone proteins stabilizing damaged proteins . In this view,
the defects in heat tolerance observed in the rps1 mutant are likely
to be due to the repressed expression of HsfA2 and its target genes
in response to heat stress. To further define the role of HsfA2 as a
mediator in the activation of RPS1-dependent heat-responsive
processes, we generated transgenic rps1 plants with the HsfA2 gene
under the control of constitutive CaMV35S promoter (Figure 4A).
The constitutive expression of HsfA2 in the 35S:HsfA2 rps1 mutant
was verified using RT-PCR and qRT-PCR (Figure 4B and 4C).
Notably, constitutive expression of HsfA2 almost totally restored
the heat-sensitive phenotype of the rps1 mutant compared with
wild type in young seedlings, detached leaves and whole plants
(Figure 4D4F). Furthermore, we have performed qRT-PCR and
western blot analysis to examine the expression levels of HsfA2
target genes and the protein levels of thylakoid membrane proteins
in 35S:HsfA2 rps1 plants, respectively. The results showed that the
expression levels of a subset of representative HsfA2 target genes,
including APX2, GolS1 and several HSPs (Hsp17.7-CII, Hsp18.1-CI,
Hsp25.3-P, Hsp70 and Hsp101), constitutively increased in
35S:HsfA2 rps1 plants compared with that in wild type and rps1
plants (Figure 5A). These results further confirm that the reduced
expression levels of HsfA2 and its target genes under heat stress are
responsible for the heat-sensitive phenotype of the rps1 mutant.
Previous studies indicate that HSP21 is targeted to chloroplast and
functions in protecting chloroplasts from heat or oxidative stresses
in higher plants [34,35]. Importantly, by performing western blot
analysis, we further confirmed that the protein level of HSP21,
encoded by the HsfA2 target gene Hsp25.3-P, also increased
dramatically in 35S:HsfA2 rps1 plants in comparison with WT and
the rps1 mutant plants (Figure 5C). On the other hand, we found
that the protein levels of thylakoid membrane proteins represented
by D1, D2, CP43, CP47, PsaA, PsaB and b-subunits of ATPase in
35S:HsfA2 rps1 plants substantially increased in comparison with
the rps1 mutant, and were restored to near the wild type levels
(Figure 5B). These data reveal that HsfA2 acts downstream of
RPS1 and plays an essential role in mediating chloroplast
RPS1initiated transcriptional reprogramming of downstream target
genes critical for heat tolerance.
Disturbance of RPS1 Expression Destabilizes Thylakoid
It is well known that heat stress leads to a loss of thylakoid
membrane integrity, especially destacking of thylakoid membranes
[2,1214], suggesting that the maintenance of thylakoid stability in
response to heat stress is a key sign of heat tolerance in plants. To
confirm that RPS1 is the chloroplast orthologue of CS1 in spinach
[61,62] and CreS1 in C. reinhardtii , we determined its
localization in mesophyll cell protoplasts and guard cells prepared
from the transgenic plant leaves harboring 35S:RPS1-GFP
Figure 3. Downregulation of RPS1 expression inhibits transcriptional activation of HsfA2 and its target genes in response to heat
stress. qRT-PCR analysis of mRNA levels of HsfA2 (A), its representative target genes, including GolS1, APX2 (B) and HSP genes (C) in detached,
fullyextended WT and rps1 leaves challenged with heat treatment (38uC) for the indicated time in dark. Actin2 was used as the internal standard. Error bars
indicate standard deviations of three technical replicates, and the results were consistent in three biological replicates.
constructs and its role in synthesis of thylakoid membrane proteins
encoded by chloroplast genes. As predicted, RPS1 is targeted to
chloroplasts (Figure 6A and Figure S11) and the downregulation of
RPS1 in the rps1 mutant caused a substantial reduction (5060%)
in the protein levels of thylakoid membrane proteins, represented
by D1, D2, CP43, CP47, PsaA, PsaB and b-subunits of ATPase
(Figure 6B). To further determine the function of RPS1 in plastid
protein translation, we examined the differences in translation
efficiency of thylakoid membrane proteins encoded by chloroplast
DNA between wild type and the rps1 mutant by employing the
pulsed stable isotope labeling assay with amino acids . As
shown in Figure 6C, the translation efficiency of D1 and CP43
proteins in chloroplast was reduced by 56% and 45% respectively
in rps1 mutant leaves incorporated with medium heavy
isotopelabeled amino acids (M), compared with that in wild type leaves
incorporated with heavy isotope-labeled amino acids (H). The
ratio of peak intensities of M versus H peptides reflects difference
in translation of the corresponding proteins D1 and CP43 between
wild type and the rps1 mutant. These results demonstrate that
RPS1 plays a critical role in biosynthesis of thylakoid membrane
proteins encoded by chloroplast genes.
Having established that RPS1 functions in synthesis of thylakoid
membrane proteins, we next investigated whether the
downregulation of RPS1 altered the stability of thylakoid membranes. We
generated estrogen-inducible RPS1-RNAi and 35S:RPS1
cosuppressed transgenic lines (CS1 and CS2) and in both cases the
transgenic plants exhibited a variegated phenotype (Figure 7A and
7C). We attempted to examine the phenotypes of RPS1
overexpression transgenic lines and were not able to obtain such
overexpression lines mainly owing to co-suppression caused by
overexpressing RPS1 under the control of constitutive CaMV35S
promoter. Estrogen treatment-induced downregulation of RPS1
expression was validated in estrogen-inducible RPS1-RNAi lines
using RT-PCR and qRT-PCR (Figure 7B). Furthermore, western
blots with polyclonal antisera against RPS1 confirmed the highly
reduced levels of RPS1 in the white sectors but the relatively higher
levels in the green sectors of the representative variegated CS1
leaves (Figure 7D). Detailed examinations of a representative
variegated leaf detached from a mature CS1 plant using
transmission electron microscopy (TEM) revealed that chloroplast
structures in the green sector had normally developed granal
stacks, but granum-stroma thylakoid membranes were severely
disrupted in the transition sector with broken stromal membranes
and large, thick granal stacks; the configurations of thylakoid
systems nearly disappeared in the white sector, with the thylakoids
decomposed into vesicles (Figure 7E). These results further support
the conclusion that RPS1 plays a critical role in synthesis of
thylakoid membrane proteins that are required for maintaining
Figure 4. Overexpression of HsfA2 complements the heat-sensitive phenotype of rps1 mutant plants. (A) Phenotypes of WT, rps1 and
rps1 with a 35S:HsfA2 cDNA transgene as 21-d-old plants. (B) to (C) HsfA2 mRNA levels in leaves of the plants described in (A) were analyzed by RT-PCR
(B) and qRT-PCR (C). Actin2 was used as the internal standard. Error bars indicate standard deviations of three technical replicates, and the results
were consistent in three biological replicates. (D) to (F) Heat-challenged phenotypes of different genotypes as described in (A) as examined with
young seedling (D), detached leaf (E) and whole plant (F) assays performed as described in Materials and Methods.
the stability and integrity of thylakoid membranes in a RPS1
expression level-dependent manner.
Overexpression of HsfA2 in rps1 Mutant Restores
HeatSusceptible Thylakoid Stability to Wild-Type Levels
As the most sensitive component to the inhibiting action of heat
stress in chloroplasts, thylakoid membrane system is vulnerable to
be destabilized under heat stress conditions . We next addressed
whether the restoration of heat tolerance in the rps1 mutant by
HsfA2 overexpression would be correlated with the improvement in
thylakoid membrane stability under heat stress. To this end, we
conducted TEM examinations. The thylakoid membranes in the
chloroplasts of the rps1 mutant appeared distorted (38uC for 1 h)
and began to decompose 4 h after heat stress, whereas the wild type
thylakoid systems, including grana and stroma membranes, retained
their initial configurations (Figure 8A and 8B). More importantly,
overexpressing HsfA2 restored thylakoid stability in the rps1 mutant
to wild type levels (Figure 8A and 8B). To further corroborate TEM
ultrastructural findings, we studied the surface topography of
thylakoids in de-enveloped chloroplasts by atomic force microscopy
(AFM). AFM images also revealed that heat stress led to a
considerable loss of granum structures in de-enveloped chloroplasts
from the rps1 mutant, observed as a substantial reduction in the size
of de-enveloped chloroplasts, but little reduction was observed for
wild type and the rps1 mutant with overexpressed HsfA2 (Figure 8C).
These observations further confirmed that the rps1 mutant thylakoid
membranes are susceptible to heat stress, which is mainly caused by
the inhibition of the heat-responsive activation of HsfA2-dependent
heat tolerance pathway. Furthermore, the heat-induced dramatic
decrease in Fv/Fm values in the rps1 mutant was reverted by
overexpressing HsfA2, reflecting a substantial improvement in
thylakoid stability and indicating that HsfA2-dependent restoration
of thylakoid stability in the mutant is physiologically relevant
(Figure 8D). Therefore, these findings have established a previously
unrecognized genetic connection between the RPS1 expression in
chloroplast and the activation of HsfA2-dependent heat-responsive
gene expression in nucleus, which is required for heat tolerance in
In recent years, proteomics has provided a powerful approach
to discovering the genes and pathways that are crucial for heat
Figure 5. Overexpression of HsfA2 leads to constitutive expression of its target genes in rps1 mutant plants. (A) qRT-PCR analysis of
mRNA levels of the representative HsfA2 target genes in the fully-extended leaves of WT, rps1 and rps1 with a 35S:HsfA2 cDNA transgene plants.
Actin2 was used as the internal standard. Error bars indicate standard deviations of three technical replicates, and the results were consistent in three
biological replicates. (B) Western blot analysis of thylakoid membrane proteins extracted from the leaves of the genotypes indicated in (A). Equal
protein loading was determined by contents (2 mg) of chlorophyll in thylakoid membrane extracts according to (Peng et al., 2006). (C) Protein levels
of HSP21, encoded by HsfA2 target gene Hsp25.3, in leaves of the plants described in (A) were analyzed by western blots with a polyclonal antibody
stress responsiveness in a variety of plant species . In contrast
to many studies on analysis of Arabidopsis transcriptome in
response to heat stress , a few reports have focused on
proteome analysis of heat stress-responsive proteins in Arabidopsis
[72,73]. In this study, we have demonstrated that RPS1 is a
heatresponsive protein based on two lines of evidence from proteomic
screen and western blot analysis with a RPS1 polyclonal antibody.
Although numerous studies on the proteomic response to heat
shock were reported on a variety of plant species such as rice,
wheat, barley and Arabidopsis , the physiologically relevant
roles of the proteins identified in these studies have hardly been
examined by analyzing heat stress-related phenotypes of
corresponding mutant plants. This difficult situation is probably due to
the limited mutant resources and the functional redundancy for
most of the heat-responsive genes such as HSPs [16,17].
Fortunately, we identified a homozygous knockdown mutant of
rps1, which enabled us to do reverse genetic analysis to define the
entirely novel roles of RPS1 in cellular heat stress response. We
have answered the question of whether downregulation of RPS1
expression alters plant responses to heat stress by studying the
knockdown mutant of rps1 and RPS1-RNAi transgenic plants; both
the mutant and RNAi plants displayed sensitivity to heat stress.
These results provide strong genetic evidence to support that
RPS1 is required for heat tolerance.
It is generally accepted that the chloroplasts in modern plants
and algae are the descendants of the ancient photosynthetic
bacteria. The modern chloroplast maintains a circular genome
and transcription and translation machinery similar to that of its
evolutionary precursor [74,75]. In E. coli, ribosomal protein S1
contains six S1 domains that are essential for RNA binding and is
an essential protein required for the translation of most transcripts
[57,59,60]. Based on the molecular diversity analysis, RPS1s in
prokaryotes have been classified into four types depending on their
functional reliability of translation initiation . According to the
Figure 6. RPS1 functions in biosynthesis of chloroplast proteins. (A) RPS1-GFP signals in mesophyll cell protoplasts. (B) Western blot analysis
of thylakoid membrane proteins extracted from WT and rps1 leaves. Equal protein loading was determined by contents (2 mg) of chlorophyll in
thylakoid membrane extracts according to . (C) The representative mass spectra for identification of D1 and CP43 proteins in samples extracted
from wild type and rps1 mutant leaves pulse-labeled with heavy (H) and medium heavy (M) stable isotope amino acids, respectively. The ratio of
peak intensities of H versus M peptides reflects difference between wild type and rps1 mutant in translation of the corresponding proteins since the
newly synthesized proteins incorporate either the H or M amino acids.
complete proteome of chloroplast ribosomes from higher plants
[76,77], a majority of the protein components of chloroplast
ribosomes have clear homologs in bacterial 70S ribosomes. In this
study, we have cloned Arabidopsis RPS1 that contains three S1
domains and shares high sequence similarity with CS1 in spinach
, but a much less similarity with CreS1 in C. reinhardtii  and
RPS1 in cyanobacteria  (Figures S3 and S12). Up to the
present, the studies on CS1 and CreS1, the orthologues of RPS1 in
spinach and C. reinhardtii, have been limited to their expression and
RNA binding properties [62,65,78]. Considered that RPS1, a
chloroplast-localized protein, has previously not been associated
with heat stress responses, we have highlighted RPS1 as a possible
candidate protein for further functional analysis of its role in heat
With reference to the roles of RPS1 orthologues in prokaryotic
and eukaryotic organisms, we had expected RPS1 to be essential
in synthesis of photosynthetic proteins encoded by chloroplast
genome in Arabidopsis. As expected, we have provided strong
evidence of a functional role for RPS1 in synthesis of thylakoid
membrane proteins that are needed for maintaining the stability of
thylakoid membrane system in chloroplasts of plants under normal
growth conditions. Importantly, TEM examination of chloroplast
structures in the green, transition and white sectors sampled from
a representative variegated leaf detached from cosuppressed
transgenic line CS1 reveals that the protein level of RPS1
positively correlates with the de-organization degree of thylakoid
membrane systems in chloroplasts (Figure 7). Knockdown of RPS1
impairs the integrity of chloroplast as evidenced by alterations in
the microscopic ultrastructures of chloroplast in the rps1 mutant
with the reduced Fv/Fm value under control condition (Figure 8).
Throughout the molecular, biochemical and microscopic analysis,
we conclude that RPS1 plays a critical role in maintaining the
chloroplast integrity. Such a conclusion was drawn on the basis of
reverse genetics analysis using the knockdown mutant of rps1,
RPS1-RNAi transgenic plants and 35S:RPS1 cosuppression lines.
In addition to identification of RPS1 as a heat-responsive
protein, it truly surprised us to find that knockdown of RPS1
expression almost eliminates the transcriptional activation of
Figure 7. Disturbance of RPS1 expression destabilizes thylakoid membranes. (A) Estrogen-induced variegated phenotypes of transgenic
seedlings harboring estrogen-inducible RPS1-RNAi constructs. (B) RPS1 mRNA levels in leaves of the transgenic plants as described in (C) were
examined by RT-PCR (left) and qRT-PCR (right). For qRT-PCR analysis, Actin2 was used as the internal standard. Error bars indicate standard deviations
of three technical replicates, and the results were consistent in three biological replicates. (C) 35S:RPS1 cosuppressed lines showing a variegated
phenotype. (D) RPS1 protein levels in green and white sectors excised from leaves of cosuppressed lines were examined by western blot analysis with
an RPS1 polyclonal antibody. Equal protein loading was confirmed with antiserum against a-Tublin. (E) Cross-sectional analysis of thylakoid
membranes in chloroplasts from the green sector, transition section, and white sector of a leaf excised from 35S:RPS1 cosuppressed line CS1 by
transmission electron microscopy (TEM). Bars = 1 mm.
HsfA2 and its target genes in the rps1 mutant in response to heat
stress. It is known that translation impairment in plastids leads to
downregulation of nuclear photosynthetic genes in higher plants
. Given that RPS1 functions in the translation initiation of
chloroplast proteins and determines the integrity of chloroplast, we
have proposed that the maintenance of chloroplast integrity is
required for initiating the many molecular processes that signal the
transcriptional activation of HsfA2 and its target genes required for
establishing cellular heat tolerance. Accordingly, the exchange of
signals is required for coordination between the activities of
organelles and the nucleus. However, the mechanisms that
generate the retrograde signal(s) to activate the expression of
heat-responsive genes in plants remain to be characterized.
Numerous studies suggest the existence of plastid signals passing
from the chloroplast to the nucleus . Mg-protoporphyrin IX
was identified as a negative signal generated from defective plastids
to repress the expression of photosynthetic genes in the nucleus
[82,83]. In C. reinhardtii, it was reported that Mg-protoporphyrin
IX could induce the expression of nuclear chaperone genes
HSP70A and HSP70B . However, recent reports have shown
that the repression of photosynthetic gene expression caused by
defective plastids has no correlation with the steady-state levels of
Mg-protoporphyrin IX [85,86]. Instead of the tetrapyrrole
pathway, it is proposed that plastid signals could derive from
various sources, including protein synthesis, reactive oxygen
species, or the redox state of the organelle, but the identity of
the putative organellar signaling molecules remains elusive .
Since the components of the photosynthetic apparatus housed
in the chloroplasts, including the oxygen evolving complex along
with the associated cofactors in PSII, carbon fixation by Rubisco
and the ATP generating system, are the primary susceptible
targets of thermal damage in plants, the chloroplasts were
proposed as sensors to changes in the growth environment,
especially to a shift up in temperature [2,88]. In this study, we
have demonstrated that the retrograde activation of HsfA2
expression is required for maintaining the integrity of chloroplasts
indicated as the stability of thylakoid membrane systems under
heat stress. We have drawn this conclusion based on several lines
of evidence. Firstly, knockdown of RPS1 inhibits the
heatresponsive activation of HsfA2 and its target genes and leads to a
heat-sensitive phenotype of the rps1 mutant plants. Secondly, the
overexpression of HsfA2 is sufficient to reestablish the lost heat
tolerance in the rps1 mutant plants (Figure 4). Thirdly, the
overexpression of HsfA2 and its target genes in the rps1 mutant
dramatically improves the stability of thylakoid membranes under
heat stress, which contributes to the restoration of heat tolerance in
the rps1 mutant plants (Figure 5 and Figure 8). Importantly, it
should be noted that the transcriptional and protein levels of
HSP21, encoded by the HsfA2 target gene Hsp25.3-P, are
enhanced dramatically in 35S:HsfA2 rps1 plants in comparison
with wild type and the rps1 mutant plants (Figure 5). These data
may explain why the heat-sensitive integrity of the rps1 mutant
chloroplasts is reverted by overexpressing HsfA2 since previous
studies indicate that HSP21 is targeted to chloroplast and
functions in protecting chloroplasts from heat or oxidative stresses
in higher plants [34,35].
In general, the central message of our study is that the
translation defects caused by downregulation of RPS1 in
chloroplast negatively modulate nuclear heat-responsive gene
expression under heat stress, leading to a loss of heat tolerance
in the mutant plants, which reveals the existence of a retrograde
activation pathway for cellular heat response in Arabidopsis.
Consistent with the model we propose for the chloroplast
regulation of the cellular heat stress responses, we found that
knockdown of RPS17, an essential subunit of chloroplast ribosome
since the knock-out mutant of its maize orthologue hcf60 is
seedling-lethal , also led to a significant reduction in the
heatresponsive expression of HsfA2 and a heat-sensitive phenotype in
the rps17 mutant plants (Figure S13). In addition, the treatment
with lincomycin, an inhibitor of the chloroplast protein synthesis,
severely inhibited the expression of HsfA2 in response to heat stress
(Figure S14). These additional data have further confirmed that
cellular heat responses are modulated by a retrograde activation
pathway in Arabidopsis.
Our findings have revealed that RPS1 is a key genetic
connection between chloroplast translation capacity and the
heat-responsive transcriptional activation of HsfA2, which helps
in better understanding of the complex regulatory network of
HsfA2 with a new angle. As a key component of the HSF signaling
network involved in cellular heat stress responses, the regulatory
mechanisms of HsfA2 have been intensely studied. In Arabidopsis,
recent studies indicate that HsfA1 transcription factors, including
HsfA1a, HsfA1b, HsfA1d, HsfA1e, function as the main regulators
in heat-responsive gene expression such as HsfA2 .
Interestingly, we found no significant difference in the expression
levels of HsfA1s between wild type and the rps1 mutant in response
to heat stress (Figure S10).
According to existing literatures and our data presented in this
study, we favor the model that the capacity of protein translation
in chloroplasts plays a critical role in generating the retrograde
signal (s) to activate the heat-responsive expressions of HsfA2 and
its target genes. We have demonstrated that RPS1 determines the
stability of thylakoid membranes (Figure 7) by modulating the
translational efficiency of thylakoid proteins encoded by the
chloroplast genes (Figure 6). It is well known that the chloroplasts
are major sites of the production of reactive oxygen species (ROS)
. ROS are proposed to diffuse away from their sites of
production and consequently elicit a different set of signaling
events under a wide range of biotic and abiotic stress conditions
[91,92]. We speculate that the alterations of thylakoid membranes
caused by the downregulation of RPS1 may affect the generation
of ROS such as H2O2 under heat stress by inhibiting thylakoid
membrane-associated physiological processes, which could repress
the activation of ROS-mediated retrograde signal transduction.
Indeed, H2O2 is thought to be a signaling molecule to activate the
core transcription regulators in response to heat stress.
Interestingly, the exogenous application of H2O2 induces the expression of
HSP genes in plant cells [93,94]. It has been suggested that the
HsfA4a acts as a H2O2 sensor in controlling the homeostasis of
reactive oxygen species in higher plants . In Class A HSFs,
HsfA2 is shown to have the highest level of expression in response
to heat stress and the treatments with H2O2 and ozone .
Studies have pointed to a critical role of the mitogen-activated
protein kinase (MAPK) in H2O2-mediated expression of HSFs,
including HsfA2 under heat stress [16,9699]. In this view, it has
been suggested that H2O2 diffuses freely across the chloroplast
envelope to activate a cytosolic MAPK cascade . It is
assumed that H2O2 may regulate the activity of HsfA1s through
the mitogen-activated protein kinase [97,98] or Calmodulin
(CaM)-binding protein kinase 3 (CBK3)  pathways.
Consequently, the activated HsfA1s regulate the heat-responsive
expression of HsfA2 and its target genes, which is required for
heat tolerance. Although H2O2 is proposed as a possible
retrograde signal molecule, the difficulty with this model lies in
that how H2O2 could specifically communicate information on the
state of chloroplasts to the nucleus because H2O2 is produced at
different sites in the cell and in response to various different stresses
and stimuli in higher plants . Therefore, these proposed
pathways remain to be further explored. The future studies should
turn towards the identification of interconnecting components
between the capacity of plastid protein translation and the nuclear
heat-responsive gene expression of HsfA2 and its target genes.
In summary, by integrating a variety of approaches, including
proteomics, reverse genetics and microscopic analysis (TEM and
AFM), we have identified RPS1 as a previously unrecognized
determinant regulator involving in plastid protein translation
control and retrograde activation of heat-responsive genes. Our
findings demonstrate the existence of a retrograde pathway in the
regulation of the cellular heat stress responses. In this view, the
maintenance of chloroplast integrity under heat stress is a highly
coordinated process in which RPS1 is a previously unrecognized
regulator that optimizes the adaptive value of the cellular heat
stress response in correspondence to capacity of plastid protein
Materials and Methods
Plant Material and Growth Conditions
Arabidopsis thaliana plants used in all experiments were of the
ecotype Columbia (Col-0). Growth chambers were used for
controlled temperature experiments. All seeds were
surfacesterilized, plated on half-strength MS medium, and stratified at
4uC for 3 days or grown in soil-culture without sterilization. Plants
were grown under long-day conditions, 16 h of white light
(80 mmol m22 s21) and 8 h of dark, with 60% relative air
humidity at 21uC. The rps1 mutant was isolated from the
TDNA insertion line (CS874869) obtained from the Arabidopsis
Biological Resource Center (Ohio State University, USA). The
rps1 mutant was backcrossed to wild type twice for removing
background mutations and the heat sensitive phenotype of F2
backcrossed lines co-segregated with T-DNA insertion as a single
Plasmid Constructions and Plant Transformations
For subcellular localization analysis, a cDNA clone containing
the full-length RPS1 open reading frame was amplified by PCR
with RGFP+ and RGFP- primers and inserted into XhoI and SpeI
cloning sites of the 35S CaMV expression cassette of the
p35SGFP-JFH1 vector , yielding a C-terminal GFP fusion
construct. For the genomic complementation assay, a genomic
fragment of RPS1 (4,597 bp in size), starting at 2,139 bp upstream of
the ATG codon and ending at 496 bp after the stop codon, was
amplified from genomic DNA by PCR with RComp+ and RComp2
primers and cloned into KpnI and XbaI sites of the pCAMBIA1300
binary vector. For the expression pattern analysis, a promoter
fragment extending 2139 bp upstream to 201 bp downstream of the
translation initiation ATG codon of RPS1 was amplified using the
primers RGUS+ and RGUS2 and the resulting fragment was cloned
into HindIII and BamHI sites of the binary vector pBI101.1. To
generate the constitutive RPS1-dsRNAi construct and
estrogeninductive RPS1-dsRNAi construct, a 120 nucleotide intron of the
AtRTM1 gene  was subcloned into XbaI and NotI sites of the
pBluescript SK+ (Stratagene, La Jolla, CA) vector to create
pBSRTM. A 485-bp sense fragment, starting at 25 bp upstream and
ending at 457 bp downstream of the ATG codon of RPS1, was
amplified with FRNAi+ and FRNAi2 primers, and the antisense
fragment was amplified with RRNAi+ and RRNAi2 primers. The
amplified sense and antisense fragments were subcloned into
pBSRTM to yield the pBS-RPS1-RTM-1SPR vector. To generate the
constitutive RPS1-dsRNAi construct, the RPS1-RTM-1SPR
fragment was released from the pBS-RPS1-RTM-1SPR vector by
digestion with SmaI and SacI and cloned into pCAMBIA1300s to
generate pCAMBIA1300s-dsRPS1. For the estrogen-inductive
RPS1dsRNAi construct, the RPS1-RTM-1SPR fragment was obtained by
digesting the pBS-RPS1-RTM-1SPR vector with XhoI and cloned
into pER8 to generate pER8-dsRPS1. The pER8 vector  was
kindly provided by N.-H. Chua (Rockefeller University, New York,
USA). For overexpressing HsfA2 in the rps1 mutant, a cDNA clone
containing the full-length HsfA2 open reading frame was amplified by
PCR with 121A2-F and 121A2-R primers and inserted into XbaI and
SacI cloning sites in the 35S CaMV expression cassette of pBI121. For
overexpression of RPS1 in transgenic plants, a cDNA clone
containing the full-length RPS1 open reading frame was amplified
by PCR with ROX+ and ROX2 primers, digested with BamHI and
KpnI and inserted into BglII and KpnI cloning sites in the 35S CaMV
expression cassette of pMON530. The co-suppression line CS1 and
CS2 were isolated from the resulting overexpression lines described
above. The primer sequences for generating the indicated constructs
are listed in Table S1.
Binary vectors harboring the desired constructs were transferred
into Agrobacterium tumefaciens strain GV3101. Transgenic plants
were generated by a floral dip method and screened on solid plates
containing 50 mg/L kanamycin or 25 mg/L hygromycin.
Heat Stress Treatments
Heat tolerance assays of seedlings, mature plants and detached
leaves were performed as described previously [30,105] with
modifications. All heat treatments were performed in dark. For the
acquired heat tolerance test of seedlings, 2.5-d-old seedlings,
grown on 1/2 MS medium, were initially acclimated to heat at
38uC for 1 h, returned to 22uC for 2 h, and then challenged at
45uC for 3 h. Challenged Seedlings recovered in a growth
chamber at 22uC for 7 d under 16/8 h light-dark cycles. To
evaluate the effect of continuous moderate heat stress on mature
plants, 21-d-old plants grown in peat soil pots were heated in the
incubator at 38uC for 9 h and the challenged plants were allowed
to recover at 22uC for 3 d under continuous light conditions. For
the heat tolerance assay of detached leaves, fully extended leaves
detached from 21-d-old plants were placed on plastic square Petri
dishes with three-layer Whatman filter paper at the bottom
immersed in 20 ml of deionized water, incubated at 38uC for 6 h,
and the challenged leaves were allowed to recover at 22uC for 3 d
under continuous light conditions. For all heat treatments, plants
were photographed following the recovery processes.
Quantitative Real-Time RTPCR
Total RNA was isolated from leaf samples frozen in liquid
nitrogen using the TRIzol reagent (Takara) according to
manufacturers protocol. For quantitative real-time RT-PCR
analysis, DNA contaminated in total RNA samples was digested
with RNase-free DNase (Takara). Complementary DNA was
produced using 1 mg total RNA and an oligo (dT) 18 primer.
Quantitative real-time PCR was performed with SYBR Premix Ex
TaqII (Takara) using a MyiQ5 single color Real-Time PCR
Detection System (Bio-Rad). The comparative threshold cycle (Ct)
method was used for determining relative transcript levels (iQ5
admin, Bio-Rad) using ACTIN2 as an internal control. Three
biological and three technical repeats were performed in the
experiments. Primer names and sequences are listed in Table S2.
Thylakoid Membrane Preparation and Western Blot
Thylakoid membranes were prepared as described previously
. Immunodetection of thylakoid membrane proteins was
performed using the indicated primary antibodies against
thylakoid membrane proteins (Agrisera) and
Alkaline-phosphataseconjugated goat anti-rabbit IgG (Chemicon) as a secondary
antibody and reaction was revealed using an ECL kit (Amersham).
Transmission Electron Microscopy
Samples were fixed with 2.5% (v/v) glutaraldehyde and 2% (v/
v) paraformaldehyde for approximately 4 h at 4uC. Thin sections
were examined by a transmission electron microscope (H7650,
Hitachi) using a voltage of 120 kV.
Protein Extraction and 2-DE Analysis
Proteins were extracted from detached, fully-extended wild type
leaves treated with control or heat treatment (38uC, 2 h) in dark.
Proteins were extracted and 2-DE analysis was performed as
described previously . Second dimension SDS-PAGE was
performed using a 12% acrylamide gel in a PROTEAN II xi Cell
system (Bio-Rad). Silver-stained gels were digitized by an
FLA7000 imaging analyzer (Fujifilm) and analyzed using Multi Gauge
MALDI-TOF/TOF MS Analysis
Mass spectra were measured using a 4700 proteomic analyzer
MALDI-TOF/TOF tandem system (Applied Biosystems,
Framingham, MA, USA). Gel pieces were detained with a solution of
15 mM potassium ferricyanide and 50 mM sodium thiosulfate
(1:1) for 20 min at room temperature, washed twice with
deionized water, and shrunk by dehydration in ACN. The
samples were swollen in a digestion buffer containing 20 mM
ammonium bicarbonate and 12.5 ng/L trypsin at 4uC for 30 min
and then digested more than 12 h at 37uC. Peptides in the samples
were extracted twice using 0.1% TFA in 50% ACN. The extracts
were dried under the protection of N2. For MALDI-TOF-MS, the
peptides were eluted onto the target with 0.7 ml matrix solution
(acyano-4-hydroxy-cinnamic acid in 0.1%TFA, 50%ACN). Samples
were allowed to dry in air before subjected to the mass
spectrometer. Data from MALDI-TOF MS/MS were searched
by GPS Explorer using MASCOT as a search engine.
SDS-PAGE and Western Blots
Total protein from plants was extracted as described previously
. Western blotting was performed with equal amounts of
protein extracts (5 mg), separated by SDSpolyacrylamide-gel
electrophoresis, immunoblotted to polyvinylidene difluoride
membranes (Millipore) and probed with affinity-purified RPS1
antibodies at a dilution of 1:1,000 (v/v) in PBS buffer (pH 7.4)
containing 0.05% Tween 20. Alkaline-phosphatase-conjugated
goat anti-rabbit IgG (Chemicon) was used as a secondary antibody
and reaction was revealed using an ECL kit (Amasham).
AntiRPS1 rabbit polyclonal antisera were generated against the
peptide of RPS1 (from Ser17 to Leu248) by Abmart biomedical
company (Shanghai, China). Anti-HSP21 rabbit polyclonal
antisera were purchased from Agrisera (Sweden).
In Vivo Chloroplast Protein Translation Assay
In vivo analysis of chloroplast translation was examined
according to . Briefly, wild type and the rps1 mutant primary
leaves detached from 15-d-old young seedlings grown in
greenhouse, were vacuum-infiltrated with cycloheximide (20 mg/ml) in
the incubation buffer (10 mM Tris-HCl, pH 6.8, 5 mM MgCl2,
20 mM KCl, and 0.1% (v/v) Tween 20) in 9 cm petri dishes with
three-layer Waterman filter paper at bottom, and incubated in dark
for 30 min for blocking cytosolic translation. Next, wild type and the
rps1 mutant leaves were pulse-labeled with 100 mg/L 13C615N4
Larginine (heavy, H) and 100 mg/L 15N4 L-arginine (medium
heavy, M) in the incubation buffer, respectively, and then
transferred into light condition (80 mmolNquanta m22Ns21) at room
temperature for 4 h. Thylakoid membranes were prepared from the
labeled leaves as described . The extracted thylakoid
membranes were separated by BN-PAGE as described . The
thylakiod membranes samples were washed with washing buffer
(50 mM BisTris-HCl, pH 7.0, 330 mM sorbitol) and then
suspended in suspension buffer (25 mM BisTris-HCl, pH 7.0, 20% glycerol)
at 2.0 mg chlorophyll/ml. The samples of wild type and the rps1
mutant were mixed with equal chlorophyll quantity and the mixed
sample was diluted with the equal volume of suspension buffer
containing 2% (w/v) DM in a dropwise manner. After incubation at
4uC for 30 min, the insoluble material in the thylakoid samples was
removed by centrifugation at 10,000 g for 30 min. The supernatant
(5 ug chlorophyll) was mixed with one-tenth volume of 5% Serva
blue G solution (100 mM BisTris-HCl, pH 7.0, 0.5 M
6-amino-ncaproic acid and 30% (w/v) glycerol) and then applied to
0.75-mmthick 5 to 13.5% acrylamide gradient gels in a Hoefer Mighty Small
vertical electrophoresis unit connected to a cooling circulator.
Gel slices in the expected molecular weight range of thylikiod
complex proteins were excised, reduced, alkylated, and
trypsindigested. Extracted peptides were analyzed by LC-MS/MS on a
high performance mass spectrometer (LTQ-Orbitrap XL,Thermo
Finnigan, San Jose, CA). Raw data were processed using the
MaxQuant 1.1.36 software package for protein identification and
GFP images were visualized by a LSM510 laser scanning
confocal microscope (Zeiss, Jena, Germany) with argon laser
excitation at 488 nm and a 505- to 550-nm emission filter set for
Preparation of De-Enveloped Chloroplasts
Envelope-free chloroplasts were prepared according to the assay
described previously . Briefly, fully expanded leaves detached
from 3-week-old plants were challenged with heat treatment
(38uC) in the indicated time, floated on icecold water for 30 min in
dark and then blotted dry. The following experimental procedures
were carried out in dark on ice. Next, the blotted leaves were
blended in grinding buffer containing 0.4 M sorbitol, 5 mM
EDTA, 5 mM EGTA, 5 mM MgCl2, 10 mM NaHCO3, 20 mM
Tricine, pH 8.4, and 0.5% (w/v) fatty acidfree BSA. The
resulting slurry was filtered and centrifuged for 3 min at 2600 g
(4uC). The half of the resulting pellet (topmost) was suspended in
resuspension buffer (RB; 2 mL) containing 0.3 M sorbitol,
2.5 mM EDTA, 5 mM MgCl2, 10 mM NaHCO3, 20 mM
HEPES, pH 7.6, and 0.5% (w/v) fatty acidfree BSA. The
suspension was centrifuged for 3 min at 200 g, (4uC) and the
collected supernatant was then centrifuged (2600 g for 3 min, at
4uC) to form pellets that contained the de-enveloped chloroplasts.
The resulting pellets were resuspended in RB buffer for
Atomic Force Microscopy (AFM)
Samples were attached to glass cover slips (10 mm of diameter)
coated with 0.01% (w/v) poly-L-lysine by gentle centrifugation
(5 min at 1,000 g) and fixed for overnight at 4uC in RB containing
2% (v/v) glutaraldehyde and 3% (v/v) paraformaldehyde. Signals
were recorded in contact mode with the MultiMode-SPM (Veeco
Co.) equipped with a 30-mm scanner, using oxide-sharpened Si3N4
cantilevered tips (k = 0.12 N/m). Images were acquired with forces
set minimally above lift-off values, at 1 to 2 Hz.
Chlorophyll Fluorescence Measurements
Chlorophyll fluorescence emissions were detected with an
LI6400XT Portable Photosynthesis System (LI-COR Biosciences,
Lincoln, Nebraska USA). The fifth leaves were excised from
21-dold plants, and challenged with heat treatment (38uC) for the
indicated time in dark. The maximum photochemical efficiency of
PSII was determined from the ratio of variable (Fv) to maximum
(Fm) fluorescence (Fv/Fm).
Assays for Sensitivity to Salt and Osmotic Stresses
Measurements were performed by root-bending assay as
described previously [111,112] and by seedling-growth assay as
described previously .
Tissues were immersed into the staining solution (50 mM Na
phosphate buffer, pH 7.0, 10 mM EDTA, 0.5 mM potassium
ferricyanide, 0.5 mM potassium ferrocyanide, 0.1% Triton X-100,
and 2 mM 5-bromo-4-chloro-3-indolyl--D-glucuronide),
vacuum- infiltrated for 5 min and incubated at 37uC overnight.
Stained tissues were decolorized with 70% ethanol and examined
with an Olympus BX51 microscopy or Olympus SZX7
stereomicroscopy (Olympus, Japan).
Seedlings were treated with lincomycin according to .
Sequence data from this article can be found in the Arabidopsis
Genome Initiative or GenBank databases under the following
accession numbers: RPS1 (At5g30510), Hsp101 (At1g74310), Hsp70
(At3g12580), Hsp25.3-P (At4g27670), Hsp18.1-CI (At5g59720),
Hsp17.7-CII (At5g12030), APX2 (At3g09640), GolS1 (At2g47180),
Actin2 (At3g18780). The National Center for Biotechnology
Information accession numbers of the proteins used in Alignment
analysis under the following accession numbers: CS1 in spinach
(M82923) and CreS1 in Chlamydomonas reinhardtii (AJ585191).
Figure S1 Silver stains of 2D-PAGE electrophoresis separation
of proteins in response to heat treatment. (A) Control leaves. (B)
Figure S2 Representative tandem mass spectra for identification
of RPS1 by MALDI-TOF/TOF MS analysis. Representative
tandem mass spectra were showed according to precursor ions
with m/z value (A) 1083.6990, and (B) 1321.7111, corresponding
respectively to: (A), peptide GGLVALVEGLR, spanning residues
G200 to R210 of protein RPS1 (At5g30510); (B), peptide
NIQYELAWER, spanning residues N170 to R179 of protein
RPS1 (At5g30510). The b ions, y ions and the resulting peptide
sequences were displayed.
Figure S3 Alignments of derived amino acid sequences of
AtRPS1, CS1 and CreS1. Amino acid sequences of AtRPS1
(At5g30510), CS1 in spinach (GenBank accession number:
M82923) and CreS1 in Chlamydomonas reinhardtii (GenBank
accession number: AJ585191) were aligned using ClustalW
(http://www.ebi.ac.uk/clustalw). Alignment was shaded using
html). Identical amino acid residues and conservative changes
were depicted in black and grey background, respectively. Three
S1 domains were labeled.
Figure S4 Transcriptional and protein levels of RPS1 in
response to heat stress. (A) Western blot analysis showing RPS1
protein levels in wild type leaves in response to heat treatment
(38uC) in dark for the indicated time with an RPS1 polyclonal
antibody. Equal protein loading was confirmed with antiserum
against a-Tubulin. (B) qRT-PCR analysis of mRNA levels of RPS1
in detached, fully-extended WT leaves challenged with heat
treatment (38uC) for the indicated time in dark. Actin2 was used as
the internal standard. Error bars indicate standard deviations of
three technical replicates, and the results were consistent in three
Figure S5 Schematic diagram of RPS1 gene showing the
TDNA insertion site. Open box indicates 59 or 39 UTR; Closed box
indicates ORF. Exons (boxes) and introns (lines) were determined
by a comparison of the genomic and cDNA sequences. The
TDNA insertion site and positions of the start and stop codons are
Figure S6 Analysis of pRPS1:GUS expression in transgenic
plants. Transgenic Arabidopsis plants harboring pRPS1:GUS
constructs were analyzed by GUS-staining assay. GUS-staining
patterns of the representative 5-d-old (A) and 15-d-old (B)
transgenic seedlings grown on half-strength MS medium.
Figure S7 Characterization of primary root growth of wild type
and rps1 mutant plants under salt or osmotic stress. 5-d-old
wildtype and rps1 mutant seedlings grown under normal growth
conditions were transferred to MS medium containing NaCl or
mannitol respectively. Phenotypes of wild type and rps1 mutant
seedlings treated with NaCl (A) or mannitol (C) were
photographed and bending-growth of primary roots under salt (B) or
osmotic stress (D) was measured at day 7 after transfer. Error bars
represent standard deviations (n = 24 plants). Results from one of
two independent experiments are shown.
Figure S8 Growth characterization of wild type and rps1 mutant
seedlings under salt or osmotic stress. Phenotypes of wild type and
rps1 mutant seedlings grown on MS medium with NaCl or
mannitol were photographed at day 14 after germination.
Figure S9 Overexpression of RPS1 improves heat tolerance in
the genomic complementation lines. (A) Heat-tolerant phenotypes
of the transgenic lines of rps1 complemented with RPS1 genomic
DNA in comparison with WT seedlings challenged the heat
treatment as described in Methods. (B) qRT-PCR analysis
showing expression levels of RPS1 in the genomic
complementation lines as described in (A). For qRT-PCR analysis, Actin2 was
used as the internal standard. Error bars indicate standard
deviations of three technical replicates, and the results were
consistent in three biological replicates. (C) The ratio of peak
intensities of M versus H peptides reflects difference between the
genomic complementation line Comp rps1-3 and wild type in
translation of the corresponding proteins since the newly
synthesized proteins incorporate either the M or H amino acids.
Samples were extracted from 7-d-old seedlings of wild type and the
transgenic line of Comp rps1-3 pulse-labeled with heavy (H) and
medium heavy (M) stable isotope amino acids, respectively, as
described in Methods.
Figure S10 Heat-responsive expression analysis of HSF members
in class A in wild type and rps1 mutant plants. qRT-PCR analysis of
mRNA levels of 15 class A HSF members in detached,
fullyextended WT and rps1 leaves challenged with heat treatment (38uC)
for 1 h in dark. Actin2 was used as the internal standard. Error bars
indicate standard deviations of three technical replicates, and the
results were consistent in three biological replicates.
Figure S11 Chloroplast localization patterns of RPS1-GFP in
guard cells of epidermal peels. Chloroplast localization patterns
were inspected with a confocal microscope. Epidermal peels were
excised from the transgenic plants expressing the indicated
Figure S12 Phylogenetic relationships of RPS1s in Arabidopsis
thaliana (At5g30510), Spinacia oleracea (accession number: M82923),
Synechocystis sp. PCC 6803 (accession number: gi|1652650),
Marchantia polymorpha (accession number: gi|786212), Chlamydophia
felis Fe/C-56 (accession number: AJ585191) and Plasmodium
chabaudi (accession number: gi|70945988). A rooted phylogenetic
tree was constructed using TreeView version 1.6.6 software with
the neighbor-joining method based on ClustalW multiple
alignments of the possible RPS1s. Bar = 0.1 amino acid
substitutions per site.
Figure S13 Knockdown of RPS17 expression in rps17 mutant
plants leads to heat susceptibility. (A) Schematic diagram of RPS17
gene (At1g79850) showing the T-DNA insertion site. Open box
indicates 59or 39UTR; Closed box indicates ORF. The T-DNA
insertion site and positions of the start and stop codons are
indicated (SALK_066943). (B) RPS17 mRNA levels in leaves of
wild type and rps17 mutant plants were analyzed by qRT-PCR.
Actin2 was used as the internal standard. (C) Western blot analysis
of thylakoid membrane proteins extracted from WT and rps17
leaves. Equal protein loading was determined by contents (2 mg) of
chlorophyll in thylakoid membrane extracts according to (Peng et
al., 2006). (D) to (E) Heat-challenged phenotypes of wild type and
rps17 mutant as examined with detached leaf (D) and whole plant
(E) assays performed as described in Methods. (F) qRT-PCR
analysis of mRNA levels of HsfA2 in detached, fully-extended WT
and rps1 leaves challenged with heat treatment (38uC) for the
indicated time in dark. For qRT-PCR analysis, Actin2 was used as
the internal standard. Error bars indicate standard deviations of
three technical replicates, and the results were consistent in three
Figure S14 Heat-responsive expression of HsfA2 is severely
inhibited in wild type seedlings by Lincomycin treatment.
qRTPCR analysis of mRNA levels of HsfA2 in 6-d-old seedlings of WT
challenged with heat treatment (38uC) for 2 h in dark under
control or lincomycin treatment condition. Actin2 was used as the
internal standard. Error bars indicate standard deviations of three
technical replicates, and the results were consistent in three
Sequences of the primers for constructs.
Sequences of the primers for qRTPCR and RT
We are grateful to Drs. X.-Y. Chen, H. Huang, H. Xiao (Shanghai
Institutes for Biological Sciences, Chinese Academy of Sciences), H.-Q.
Yang (Shanghai Jiao Tong University, China), and S. Gan (Cornell
University, USA) for suggestions and critically reading the manuscript;
X.Y. Gao for assistance with electron microscopy; and the Arabidopsis
Biological Resource Center for providing Arabidopsis mutant seeds. We
also thank Shanghai Applied Protein Technology for the technical support.
Conceived and designed the experiments: F-QG H-DY X-FY. Performed
the experiments: H-DY X-FY S-TC Y-TW J-KL QS X-LL. Analyzed the
data: H-DY X-FY. Contributed reagents/materials/analysis tools: J-KL
QS X-LL. Wrote the paper: F-QG H-DY.
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