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Conditional Depletion of the Chlamydomonas Chloroplast
ClpP1 Protease Activates Nuclear Genes Involved in
Autophagy and Plastid Protein Quality Control
W
Silvia Ramundo,a,1 David Casero,b,2 Timo Mühlhaus,c Dorothea Hemme,c,3 Frederik Sommer,c,3
Michèle Crèvecoeur,a Michèle Rahire,a Michael Schroda,c,3 Jannette Rusch,d Ursula Goodenough,d
Matteo Pellegrini,e Maria Esther Perez-Perez,f,4 José Luis Crespo,f Olivier Schaad,g,h Natacha Civic,g
and Jean David Rochaixa,5
a Departments
of Molecular Biology and Plant Biology, University of Geneva, 1211 Geneva, Switzerland
for Genomics and Proteomics, University of California, Los Angeles, California 90095
c Max Planck Institute of Molecular Plant Physiology, D-14476 Potsdam-Golm Germany
d Department of Biology, Washington University, St. Louis, Missouri 63130
e Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, California 90095
f Instituto de Bioquimica Vegetal y Fotosintesis, Consejo Superior de Investigaciones Cientificas, Universidad de Sevilla, 41092
Sevilla, Spain
g Genomics Platform, University of Geneva, 1211 Geneva, Switzerland
h Department of Biochemistry, University of Geneva, 1211 Geneva, Switzerland
b Institute
Plastid protein homeostasis is critical during chloroplast biogenesis and responses to changes in environmental conditions.
Proteases and molecular chaperones involved in plastid protein quality control are encoded by the nucleus except for the
catalytic subunit of ClpP, an evolutionarily conserved serine protease. Unlike its Escherichia coli ortholog, this chloroplast
protease is essential for cell viability. To study its function, we used a recently developed system of repressible chloroplast
gene expression in the alga Chlamydomonas reinhardtii. Using this repressible system, we have shown that a selective
gradual depletion of ClpP leads to alteration of chloroplast morphology, causes formation of vesicles, and induces extensive
cytoplasmic vacuolization that is reminiscent of autophagy. Analysis of the transcriptome and proteome during ClpP depletion
revealed a set of proteins that are more abundant at the protein level, but not at the RNA level. These proteins may comprise
some of the ClpP substrates. Moreover, the specific increase in accumulation, both at the RNA and protein level, of small heat
shock proteins, chaperones, proteases, and proteins involved in thylakoid maintenance upon perturbation of plastid protein
homeostasis suggests the existence of a chloroplast-to-nucleus signaling pathway involved in organelle quality control. We
suggest that this represents a chloroplast unfolded protein response that is conceptually similar to that observed in the
endoplasmic reticulum and in mitochondria.
INTRODUCTION
Protein degradation plays a key role in chloroplast biogenesis
and maintenance. Under certain stress conditions, such as high
irradiance, nutrient starvation, or elevated temperature, the primary
1 Current
address: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158.
2 Current address: Department of Pathology and Laboratory Medicine,
University of California, Los Angeles, CA 90095.
3 Current address: Molecular Biotechnology and Systems Biology,
University of Kaiserslautern, Paul-Ehrlich Straße 23, D-67663 Kaiserslautern, Germany.
4 Current address: Centre National de la Recherche Scientifique,
UMR8226, Institut de Biologie Physico-Chimique, 75005 Paris, France.
5 Address correspondence to [email protected].
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Jean David Rochaix ([email protected]).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.114.124842
reactions of photosynthesis can lead to the formation of reactive
oxygen species (ROS) and can damage proteins by photooxidation. Damaged proteins are then degraded through an efficient
proteolytic system in the chloroplast (Adam and Clarke, 2002).
During assembly of the photosynthetic apparatus, most protein
subunits that are produced in excess are also rapidly degraded.
The plastid contains several proteases including stromal ClpP
(Olinares et al., 2011), stromal and thylakoid Deg (Schuhmann
and Adamska, 2012), thylakoid FtsH (Adam et al., 2001; Sokolenko
et al., 2002; Peltier et al., 2004), the intramembrane rhomboid
proteases (Adam, 2013), and the thylakoid-bound SppA (Lensch
et al., 2001) and Egy1 proteases (Chen et al., 2005). Moreover,
several processing proteases exist in these organelles, such as
SPP (stromal processing peptidase) and TPP (thylakoid processing protease), which are required for cleaving the transit peptide
and the thylakoid targeting domain, respectively (Oelmüller et al.,
1996; Richter and Lamppa, 1998). Among these proteases, only
the ClpP1 subunit is encoded by the chloroplast genome.
The bacterial ClpP holoenzyme complex consists of two main
homo-oligomeric parts, a proteolytic chamber formed by two
The Plant Cell Preview, www.aspb.org ã 2014 American Society of Plant Biologists. All rights reserved.
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The Plant Cell
stacked heptameric rings of the ClpP subunit and an unfolding
antechamber constituted by the hexameric ring of an ATPdependent chaperone, ClpX or ClpA. This AAA + (for ATPase
associated with various cellular activities) complex associates
coaxially to one or both external sides of the catalytic rings (Banecki
et al., 2001; Kim et al., 2001). ClpA and ClpX have different substrate specificities (Bewley et al., 2006) and can be further modulated through the association of ClpS with the hexameric ring of
ClpA. This association changes the affinity of the ClpA chaperone
for protein aggregates (Weber-Ban et al., 1999) and strongly
enhances the recognition of substrates with N-terminal degradation signals known as N-rule substrates (Tobias et al., 1991;
Schuenemann et al., 2009).
Although the plastid Clp proteolytic system shares the basic
features of its bacterial homolog, it is considerably more complex
(Peltier et al., 2004; Adam et al., 2006) as several ClpP isoforms
have evolved in photosynthetic organisms that can be incorporated
in the holoenzyme. Moreover, some isoforms, namely ClpR, have
lost the catalytic residues for peptide bond hydrolysis and are
often found in the ClpP heptameric rings. Three genes encoding
ClpP (ClpP1, ClpP2, and ClpP5) and five genes encoding ClpR
(ClpR1-4 and ClpR6) are present in Chlamydomonas reinhardtii.
With the exception of ClpP1, which is encoded by the chloroplast
genome, all the other ClpP components are encoded by nuclear
genes. In C. reinhardtii, the plastid-encoded ClpP1 protein contains a 30-kD insertion that can be removed by endoproteolytic
cleavage to give rise to a short ClpP1 isoform (Wang et al., 1997;
Majeran et al., 2005). Both the long and short isoforms are essential and can be detected within the ClpP1 core (Huang et al.,
1994; Derrien et al., 2009). Moreover, chaperones that are orthologs of the bacterial AAA+ proteins (ClpC1/C2 and ClpD) and
ClpS adaptors (ClpS1 and ClpS1-like) have been identified (Peltier
et al., 2004; Derrien et al., 2012). However, the plastid ClpP protease also possesses unique accessory subunits (ClpT proteins)
that are not found in Escherichia coli. Recent biochemical studies
suggest that the ClpT subunits have evolved in order to regulate
the assembly of the catalytic core (Sjögren and Clarke, 2011;
Derrien et al., 2012).
Because ClpP peptidase function is dispensable in bacteria, it
was possible to identify the targets of this enzyme using a protein trapping system in which the ClpP subunit is inactivated
through a mutation in its active site that prevents degradation
and release of the substrates. In E. coli, the targets are transcription
factors, metabolic enzymes, and proteins involved in starvation
and other stress responses (Dougan et al., 2002; Flynn et al., 2003).
By contrast, the plastid ClpP protease appears to have an
essential role based on the experimental failure to generate
homoplasmic knockout lines of clpP1 in the three photosynthetic
organisms: Nicotiana tabacum (Shikanai et al., 2001; Kuroda and
Maliga, 2003), Synechococcus elongatus pcc 7942 (Schelin et al.,
2002; Barker-Aström et al., 2005), and C. reinhardtii (Huang et al.,
1994). In this alga, attenuated expression of ClpP1 was achieved
by replacing the AUG start codon of ClpP1 with AUU, a less efficient initiation codon (Majeran et al., 2000). Although the expression
of ClpP1 protein was decreased by 70%, no striking phenotype
was observed under normal growth conditions. However, this
decrease in protein expression led to delayed degradation of the
Cytb6f complex under nitrogen starvation and was also seen in
mutants deficient in the Rieske iron-sulfur protein. Partial or
complete loss of expression of several ClpP subunits in Arabidopsis thaliana revealed that the ClpP/R protease core is essential and that its subunits exhibit little functional redundancy,
with the exception of ClpR1, whose loss can be overcome by
ClpR3 (Rudella et al., 2006; Sjögren et al., 2006; Koussevitzky
et al., 2007; Zybailov et al., 2009). Also, loss of individual Clp
chaperones led to reduced plant growth and chloroplast development (Sjögren et al., 2006; Zybailov et al., 2009).
The identification of ClpP substrates in the chloroplast remains
a challenging task because this protease is essential and there
are multiple nucleus-encoded ClpP/R catalytic isoforms that cannot
be easily inactivated. Quantitative proteomic approaches have
been employed for large-scale detection and quantification of
differences in protein abundance among wild-type and Clp mutants
(Peltier et al., 2004; Sjögren et al., 2006; Kim et al., 2009; Zybailov
et al., 2009). A consistent increase in abundance was observed for proteins involved in many different and unrelated
pathways: plastid enzymes and transporters involved in basic
metabolic pathways; proteins of plastoglobules; proteins involved
in protein import, folding, and maturation; proteins involved in
RNA metabolism; and various stromal proteases. However, it
is not clear to what extent the increased abundance of these
proteins is directly or indirectly linked to the decreased activity
of ClpP.
Here, we took advantage of a recently developed system of
repressible chloroplast gene expression (Ramundo et al., 2013)
for investigating the role of ClpP1. It is based on a vitamin- repressible riboswitch from C. reinhardtii (Croft et al., 2007).We
expected that conditional repression of ClpP1 would gradually
decrease the ClpP level, making it possible to follow the early
and late cellular events caused by its depletion. We performed
electron microscopic, proteomic, and transcriptomic analyses
to obtain time-course profiles of cell morphology and levels of
proteins and transcripts during depletion of ClpP1. Using this
approach, we identified a small group of proteins that could be
substrates of ClpP1. Moreover, we discovered a compensatory
nuclear gene expression program that may allow photosynthetic
organisms to sense and respond to perturbations of chloroplast
protein homeostasis. This chloroplast-to-nucleus retrograde
signaling pathway could involve a chloroplast unfolded protein
response.
RESULTS
Generation of a Strain in Which ClpP1 Expression Can Be
Conditionally Repressed
We previously generated the A31 strain in which expression of
Nac2, a nucleus-encoded chloroplast protein that is required for
the stability of the chloroplast psbD RNA, is driven by the MetE
upstream sequence and the TPP (thiamine pyrophosphate) riboswitch that can be repressed by addition of the vitamins B12
and thiamine to the medium (Ramundo et al., 2013). In this strain,
the chloroplast 59 untranslated region of psbD (psbD 59UTR),
which is the target of Nac2, has been replaced by the psaA 59UTR.
The expression of psbD, which encodes the photosystem II D2
Chloroplast Protein Quality Control
reaction center protein in this strain, is therefore no longer dependent on Nac2; thus, the strain grows photoautotrophically in the
presence of vitamins when Nac2 is repressed. A ClpP1-repressible
strain was produced through chloroplast transformation by replacing
the endogenous ClpP1 59UTR of the A31 strain with the psbD
59UTR, thus making clpP1 expression dependent on Nac2. The
homoplasmic state of this strain was tested by PCR (Supplemental
Figure 1).
Whereas the parental A31 strain was not affected by the presence of vitamins in the medium, the chloroplast transformants
bearing the psbD59-clpP1 transgene (DCH strains) displayed
a pale-green phenotype and died upon transfer from medium
lacking vitamins into medium supplemented with vitamins
(Figure 1A). This was expected due to the downregulation of a
gene essential for cell survival. This vitamin-mediated phenotype could not be rescued by growing the DCH strains in the
dark in the presence of a reduced carbon source (acetate and
Tris-acetate phosphate [TAP] medium), indicating that the primary cause of the lethal phenotype in the presence of vitamins is
not caused by either photooxidative damage or impairment of
photosynthesis.
To test the photosynthetic activity and growth of the DCH
transformants containing the vitamin-repressible ClpP1 gene,
the transformants were first grown in acetate-containing medium in the light and then vitamins were added. The results
obtained with one of these transformants, DCH16, are shown in
Figure 1B. During the first 24 h of vitamin treatment, we observed no significant change in growth rate or in photosynthetic
activity (as measured by FV/FM ratios that reflect the maximal
quantum efficiency of photosystem II [PSII]) (Figure 1C). After
48 h, cell growth ceased and photosynthetic activity declined.
The physiological state of the DCH16 strain changed drastically
72 and 96 h following vitamin treatment; the photosynthetic
activity decreased further and the cells became pale green.
Kinetics of ClpP1 Repression in the DCH16 Strain
To determine the time required for the down-regulation of ClpP1,
a liquid culture of DCH16 was treated with vitamins and the levels
of ClpP1 mRNA and protein were determined over a period of 72
h. RNA gel blot analysis revealed a significant decrease of ClpP1
mRNA after 12 h, and this mRNA was nearly undetectable after
36 h (Figure 1D). By contrast, in the control strain A31, the ClpP1
mRNA level was unchanged. Because clpP1 is under the control
of the strong psbD promoter and 59UTR, the amount of ClpP1
mRNA in the absence of vitamins was higher in DCH16 than in
A31, which contains the authentic clpP1 gene (Figure 1D). It is
noteworthy that the levels of several chloroplast mRNAs, in particular those of ORF1995, rpoA, and rps12 were strongly increased whereas those of others such as tufA, atpB, and psaB
did not change markedly.
Transcripts of nuclear genes that were strongly upregulated
upon ClpP1 depletion included those of Hsp70B, Hsp22A, and
Hsp22F (Figure 1E; Supplemental Figure 2). A similar pattern
was also observed for transcripts of the nuclear genes Atg8
and Atg7 involved in autophagy, Vipp1 and Alb3.2 implicated in
thylakoid biogenesis, and ubiquitin (Figure 1E; Supplemental
Figure 2).
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Immunoblot analysis showed that the steady state levels of
both the long and short forms of ClpP1 diminished gradually
over 48 h of vitamin treatment in the DCH16 strain. By contrast,
they remained constant in the A31 strain (ClpP1-H and ClpP1-L,
respectively, in Figure 2A). Although the amount of clpP1 mRNA
was considerably higher in DCH16 than in A31 in the absence of
vitamins (Figures 1D and 1E), ClpP1 accumulated to the same
level in both strains, indicating that the regulation of its accumulation occurs at the translational or posttranslational level.
The fact that the amount of ClpP1 was reduced to 30% in the
DCH16 strain 24 h after addition of vitamins and that no significant change in its growth rate and photosynthetic activity
occurred at this time point together suggest that ClpP1 is produced in excess of what is needed.
The levels of several chloroplast-encoded proteins were monitored in DCH16 cells over a 72-h period following vitamin treatment. Levels of the majority of these proteins (Rps12, Rps21,
RpoA, and Cytf) remained stable during depletion of ClpP1. The
only remarkable exception is ORF1995, a large chloroplast open
reading frame encoding a potential protein of unknown function
of more than 200 kD. Intriguingly, an antibody raised against the
middle region of this protein failed to detect the full-length protein.
Rather, it highlighted the two smaller polypeptides, ORF1995-L
(70 kD), which increased, and ORF1995-S (35 kD), which decreased, upon ClpP1 depletion (Figure 2A). This result suggests
that processing of ORF1995 might occur either at the RNA or
protein level and that ClpP1 is potentially involved in this activity.
A slightly decreased content was observed for some plastid
nucleus-encoded proteins (ClpC) and cytosolic ribosomal proteins (Rpl37), whereas the level of mitochondrial AOX protein did
not change significantly (Figure 3A). By contrast, the levels of the
plastid nucleus-encoded proteins Alb3.2, Vipp1, Vipp2, Hsp70B,
Cdj1, and FtsH1 increased upon ClpP1 depletion (Figures 2B and
3). Both Vipp1 and Alb3.2 are involved in thylakoid biogenesis in
cyanobacteria, algae, and plants (Kroll et al., 2001; Göhre et al.,
2006). The expression and localization of these two proteins is
regulated in an interdependent way; Alb3.2 is associated with
Vipp1 and Vipp1 expression is considerably enhanced when the
expression of Alb3.2 is downregulated (Göhre et al., 2006).
Moreover, Vipp1 expression is influenced by the light conditions
(Liu et al., 2005; Nordhues et al., 2012).
As these experiments were performed with cells grown in dim
light (10 mmol m22 s21), it is rather unlikely that the effect of
ClpP1 depletion is indirect and results from photooxidative damage. However, in order to eliminate this possibility, proteins were
also analyzed from dark-grown cells (Figure 2B). In the dark, these
proteins also increased in abundance, albeit to a lower extent and
with slower kinetics compared with the light conditions, indicating
that this process is light independent. Whereas a significant portion of Vipp1 and Cdj1 were in the soluble fraction under normal
conditions, they were partly relocalized to the insoluble fraction
upon depletion of ClpP1 (Figure 3B). Moreover, the immunoblot
analysis of Vipp1 revealed a smear in the large molecular weight
region after prolonged vitamin treatment (Figure 3C). This smear
was detected in light-grown but not in dark-grown cells, suggesting that it arises through photooxidative damage and could
represent protein aggregates. Indeed, treatment of the extract
with 6 M urea significantly decreased the high molecular weight
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Figure 1. Characterization of the C. reinhardtii DCH16 Strain upon ClpP1 Depletion.
(A) Growth patterns of the Rep112, A31, and DCH16 strains. Cells were spotted on HSM (minimal medium) and TAP in the absence or presence (+vit) of
vitamins B12 (20 mg/L) and thiamine (20 mM). Irradiance was 10 mmol m22 s21.
(B) Growth curves of the A31and DCH16 strains after addition of vitamins. To maintain cells in exponential growth during the entire time course, they
were diluted to 0.5 3 106 cells/mL when they reached a concentration between 2 and 4 3 106 cells/mL. The experiment was repeated three times with
similar results.
(C) Decrease of PSII activity in DCH16 after addition of vitamins. Fv/Fm was measured for the indicated strains at different times after addition of
vitamins. The experiment was repeated three times with similar results.
(D) RNA gel blot analysis of chloroplast genes in DCH16, A31, and ClpP1-AUU in the presence of vitamins or rapamycin. RNA was extracted from the
strains at different time points after addition of vitamins and hybridized with the indicated probes. ClpP1-AUU is the strain in which the AUG initiation
codon of ClpP1 was replaced by AUU. Arrows point to the different transcripts of ORF1995, a chloroplast gene of unknown function.
(E) RNA gel blot analysis of nuclear genes. Strains and conditions were as in (D). Rapamycin treated cells were transferred from 10 to 60 mmol photons
m22 s21 upon addition of the drug. vit, vitamins; Rap, rapamycin.
Chloroplast Protein Quality Control
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Figure 2. Immunoblot Analysis of Chloroplast Proteins in DCH16, A31, and ClpP1-AUU in the Presence of Vitamins or Rapamycin.
(A) Immunoblot analysis. Proteins were extracted from the three strains grown under an irradiance of 10 mmol m22 s21 at different time points after
addition of vitamins and reacted with antibodies directed against the indicated proteins as shown to the left of the immunoblot.
(B) Analysis of proteins from DCH16 grown in the light or the dark upon ClpP1 repression. Proteins were extracted at different time points and
immunoblotted with the antisera as indicated to the left of the immunoblot. Irradiance was 60 mmol m22 s21.
fraction of Vipp1 (Figure 3C). By contrast, upon ClpP1 depletion, the levels of proteins from photosynthetic complexes
such as Cytf, D2, and RbcL were decreased under standard
light conditions (60 mmol m22 s21), but maintained in the dark
or under low-light conditions (10 mmol m22 s21), indicating that
they are rather stable but sensitive to photooxidative damage
(Figure 2B).
Cellular Characterization of the ClpP1 Repressible Strain
In order to analyze the morphological changes associated
with ClpP1 depletion, samples of DCH16 were collected after
0, 48, 96, and 144 h of vitamin treatment under an irradiance
of 60 mmol m22 s21 and examined by light and thin-section
electron microscopy (Figure 4). Independently, control and
treated (0, 48, and 72 h) samples from cells grown under an
irradiance of 30 mmol m22 s21 were processed for electron
microscopy using the quick-freeze deep-etch technique that
employs no chemical fixation or dehydration (Figure 5). The
DCH16 strain grown in the absence of vitamins was used as
a control.
Thin-section electron microscopy revealed that, upon ClpP1
depletion, the integrity of the thylakoid membranes was disrupted.
The membranes were often unstacked or folded back to form
unusual whorls, and enlarged vacuolar structures were present
in the cytoplasm (Figure 4A). This vacuolization was often associated with cellular swelling and loss of flagella. Furthermore,
the starch granules became very irregular in shape and size and
in some cases they appeared to occupy a large portion of the
plastid. Although a number of vesicle-like structures could be
recognized, it was difficult to determine their origin given the
highly disorganized cell structure. These changes in cell morphology were also apparent in cells observed using light microscopy (Figure 4B).
Replicas of quick-frozen cells provided additional information.
In log-phase cells, the vacuoles were small and filled with a homogeneous flocculent material (Figures 5B and 5C, V), whereas
at 48 h, they were enlarged and filled with damaged membrane
and starch (Figure 5F, AV); by 72 h, large water-filled vacuoles
were often encountered (Figure 5G, WV). In log-phase cells, the
chloroplast envelope was smooth, with few intramembranous
particles (Figures 5B and 5C, ce), but at 48 h it carried perforations
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Figure 3. Immunoblot Analysis during ClpP1 Depletion.
(A) Immunoblot analysis. Proteins were extracted from the three indicated strains grown under an irradiance of 10 mmol m22 s21 at different time points
after addition of vitamins and reacted with antibodies directed against specific proteins.
(B) Changes in partitioning of Vipp1 during ClpP1 depletion. DCH16 cells were grown in the presence of vitamins in the dark. After cell lysis, cell extracts
of DCH16 were separated into soluble and insoluble fractions by centrifugation and subjected to gel electrophoresis and immunoblotting with the
indicated antibodies.
(C) Total cell extracts from DCH16 were subjected to gel electrophoresis in the presence of SDS without (left panel) and with urea (right panel). Cells
were grown in the dark or with an irradiance of 60 mmol m22 s21. The smear at the top of the gel marked with asterisks represents aggregated protein.
The same amount of protein was loaded in each lane.
(Figure 5E) and by 72 h it was no longer detectable and degenerating thylakoids (Figures 5D and 5E) filled the cell (Figures
5G and 5H). Therefore, many or most of the vesicles seen in
thin sections (Figure 4A) may be derived from thylakoid and
chloroplast-envelope membranes, although they could also represent autophagosomes engulfing chloroplast membranes.
When the DCH16 strain was grown in medium with vitamins
under lower irradiance (5 mmol m22 s21) or in the dark, similar
morphological changes were observed, but the time of their
appearance was clearly delayed (Figure 4A). Most likely, two
factors concur to cause this delay: the absence of photooxidative stress and the slower rate of ClpP1 depletion in the dark.
Importantly, none of these changes was detectable in the A31
strain treated with vitamins, thus confirming that the phenotype
of DCH16 is caused by the depletion of ClpP1.
Autophagy-Like Phenotype upon Depletion of ClpP1
The progressive vacuolization and swelling observed upon ClpP1
depletion (Figures 4A, 4B, and 5) is reminiscent of autophagy.
Bulk delivery of cellular components to the vacuole or to lysosomes in eukaryotic cells by autophagy is an evolutionary-
conserved and highly regulated response that functions to
degrade damaged proteins and/or organelles and to recycle
essential macronutrients under adverse conditions such as
nutrient starvation and accumulation of protein aggregates.
Moreover, upon prolonged cellular stress, massive autophagic
vacuolization has been proposed to represent an attempt of
adaptation resulting in cell death (Berry and Baehrecke, 2007;
Tasdemir et al., 2008).
One of the most widely used autophagy marker is Atg8, a
protein that is usually conjugated to phosphatidylethanolamine
and is found on the membrane of the autophagosome (Nakatogawa
et al., 2009). In C. reinhardtii, Atg8 is conserved, and a recent
study demonstrated that induction and lipidation of this protein
can be triggered by inhibition of the TOR (target of rapamycin)
protein kinase and by oxidative or endoplasmic reticulum (ER)
stresses (Pérez-Pérez et al., 2010, 2012).
In light of these findings, we performed RNA gel blot, immunoblot, and immunofluorescence analyses to follow the level
of expression of Atg8 during ClpP1 depletion in the DCH16
strain. Moreover, after we induced autophagy in the A31 strain
by rapamycin treatment, which inhibits TOR (Crespo et al., 2005),
we compared the morphological changes caused by activation
Chloroplast Protein Quality Control
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Figure 4. Increased Vacuolization of DCH16 upon Depletion of ClpP1.
(A) Analysis by transmission electron microscopy of epoxy-embedded thin sections of DCH16 and A31 cells following ClpP1 repression. Cells grown in
the light (L; 60 mmol m22 s21) or in the dark (D) were examined at different times as indicated. Dark spots represent polyphosphate granules (Komine
et al., 2000). Bars = 2 mm.
(B) Analysis by light microscopy of DCH16 grown in the absence (left panel) or presence of vitamins (right panel) for 4 d. L, light; V, vacuole; N, nucleus.
Bars = 10 mm.
of autophagy with those due to the depletion of ClpP1 by electron
microscopy (Figure 6A).
As shown in Figure 1E, the Atg8 mRNA was expressed at a
very low level in the DCH16 and A31 strains in absence of vitamins (time 0). Upon addition of vitamins, no significant change
in the abundance of the Atg8 mRNA was observed in the A31
strain. Instead, an inverse correlation between the decreasing
level of ClpP1 and the increasing amount of Atg8 protein was
apparent in the DCH16 strain (Figures 2A, 2B, and 3). A significant increase in Atg8 occurred after 36 h of vitamin treatment
when the level of the ClpP1 protein drops below a threshold
level and the Atg8 mRNA rises considerably (Figures 1E and 2).
Immunofluorescence microscopy of DCH16 treated with vitamins for 48 h revealed that Atg8 is not only strongly upregulated
but also frequently gives rise to a few speckles (Figure 6B). These
structures may reflect the phagophore assembly structure described in other organisms (Xie and Klionsky, 2007). After 72 h,
cup-shaped structures were apparent and their location coincided with that of vacuoles, suggesting the fusion of Atg8 with
large vacuoles (Figure 6C). Moreover, lipid bodies accumulated
as revealed by Nile red staining (Figure 6D). They were also
detectable after treatment of A31 cells with rapamycin (Figure
6D). Only weak signals were observed in untreated cells (Figures
6B and 6D). In the dark, enlargement and vacuolization of the
cells still occurred although more slowly (Figures 4A and 4B) and
was associated with an increase of Atg8 (Figure 2B), indicating
that the observed changes cannot be solely explained by photooxidative damage. By contrast, the increase in Atg8 induced
by rapamycin in the light was abolished in the dark (Supplemental
Figure 3A). This agrees with earlier findings, indicating that oxidative stress is involved in the regulation of autophagy and that
ROS act as potent inducers of this process (Pérez-Pérez et al.,
2012). These observations suggest that, although there are similarities between the responses induced by ClpP1 depletion and
inhibition of the TOR kinase by rapamycin, these two processes
also differ from each other. Evidence for the involvement of
a subset of these genes in the autophagocytic response to nitrogen starvation has been presented by Goodenough et al.
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Figure 5. Quick-Freeze Deep-Etch Electron Micrographs of DHC16 Cells.
Following vitamin addition in dim light (30 mmol m22 s21), images were taken after 0 ([A] to [C]), 48 ([D] to [F]), and 72 h ([G] and [H]). In (E), perforations
in the membrane are indicated by small arrows. AV, autophagocytic vacuole; C, chloroplast; ce, chloroplast envelope; cv, contractile vacuole vesicles;
G, Golgi; N, nucleus; pm, plasma membrane; S, starch granules; V, resting vacuole; WV, water-filled vacuole. Bars = 500 nm.
(2014), indicating that C. reinhardtii is endowed with a diverse
repertoire for coping with stress.
Chloroplast Transcriptome Analysis upon ClpP1 Repression
Previous studies in Arabidopsis have suggested that the chloroplast Clp protease might play a role in regulating plastid RNA
metabolism (Zybailov et al., 2009). To test this hypothesis, total
RNA was isolated from the DCH16 and A31 strains at three time
points following vitamin treatment (0, 12, and 48 h) (Supplemental
Figure 2). This RNA was subjected to quantitative hybridizations
with an array containing 58 chloroplast and 27 nuclear gene
probes using Nanostring technology as described (Geiss et al.,
2008; Ramundo et al., 2013) (Supplemental Figure 2). Independently,
the levels of several transcripts were also determined by RNA
gel blot experiments (Figure 1D). This analysis revealed that
most transcripts involved in chloroplast gene expression were
upregulated after 48 h, as seen in the heat map of the Nanostring data (Supplemental Figure 2A). This increase was particularly pronounced for the rpo genes that encode the subunits
of the chloroplast RNA polymerase and for several transcripts
of plastid genes of unknown function (ORF2971, ORF1995,
ORF712, and ORF59) (Figure 1D; Supplemental Figures 2A
and 2B). Whereas a similar increase was also detected for most
of the transcripts of the chloroplast-encoded subunits of the
photosynthetic complexes, the mRNA levels of nucleus-encoded
Chloroplast Protein Quality Control
9 of 22
Figure 6. Effect of Rapamycin on Cell Morphology.
(A) Electron microscope analysis of A31 cells treated for 8 h with rapamycin (RAP). Bars = 2 mm.
(B) Induction of Atg8 upon ClpP1 depletion. Immunofluorescence with an Atg8 antiserum was performed on DCH16 cells grown for 48 h in the absence
(+ClpP1) or presence of vitamins (-ClpP1).
(C) Induction of Atg8 upon ClpP1 depletion. Immunofluorescence with an Atg8 antiserum was performed on DCH16 cells grown for 72 h with vitamins.
Cells were visualized in a differential interference contrast microscope.
(D) Induction of lipids upon ClpP1 depletion. Left panels: Staining of DCH16 cells grown in the absence or presence of vitamins with Nile red. Right
panels: Staining of DCH16 cells grown in the absence or presence of rapamycin with Nile red. L, light (60 mmol m22 s21); DL, dim light (10 mmol m22 s21).
Bars = 4 mm in (B) to (D).
proteins involved in photosynthesis including antenna proteins
(CP29, Lhcbm1, Lhcbm5, and Lhca1) as well as ferredoxin, plastocyanin, PetC, Rbcs2, and Prx1 were decreased (Supplemental
Figures 2A and 2B).
In agreement with our immunoblot analyses and electron
microscopic studies, we found that ClpP1 depletion strongly
enhances the mRNA abundance of nuclear genes encoding
chloroplast chaperones (Hsp70B, Hsp22A, and Hsp22F), autophagyrelated proteins (Atg8 and Atg7), and proteins involved in
thylakoid biogenesis (Vipp1 and Alb3.2) (Figure 1E; Supplemental
Figure 2).
Comparative Nuclear Transcriptome Analysis upon
Vitamin-Mediated Depletion of ClpP1 and Induction of
Autophagy by Rapamycin
The induction of several nucleus-encoded proteins upon ClpP1
depletion prompted us to perform RNA-Seq analysis on the
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The Plant Cell
DCH16 and A31 strains upon vitamin treatment in order to obtain a global view on the remodeling of the nuclear gene expression program induced by the depletion of ClpP1. Based on
the kinetics of induction of the Vipp1 and Atg8 mRNAs previously determined by RNA gel blot analysis (Figure 1E), we first
selected three different time points at 0, 31, and 43 h following
vitamin treatment. To obtain additional information on early time
points, a second RNA-Seq analysis was performed at 0, 12, and
48 h. Because treatment of C. reinhardtii cells with rapamycin
induces an autophagy-like phenotype that is in many aspects
similar to the one caused by the depletion of the Clp protease,
we also performed a transcriptomic analysis of the A31 strain
treated with rapamycin for 2 and 8 h. For each time point, three
biological replicates were used. Only changes >2-fold and statistically significant were considered (see Methods). The numbers of up- and downregulated transcripts identified in this way
for the different comparisons are indicated in Table 1. The gene
lists corresponding to the different time points are shown in
Supplemental Data Sets 1 to 3. Numerous transcripts could be
aligned unambiguously on the C. reinhardtii genome, and 5085
of these are differentially expressed in at least one condition.
Supplemental Data Set 4 shows a transcriptomic analysis upon
ClpP1 depletion and rapamycin treatment.
In order to group genes with similar expression profiles along
the time courses for the DCH16 and A31 strains, we performed
a gene clustering analysis. A total of 5085 target genes in the
DCH16/A31 time courses (0, 12, 31, 43, and 48 h) were pooled
and the analysis was performed with 12 clusters (Supplemental
Data Set 5). This number was chosen to provide a satisfactory
balance between removing redundancies and gaining specificity. A heat map showing the time course of expression of these
target genes grouped by similarity in their expression profile
from 0 to 48 h for the A31 and DCH16 strains is shown in Figure
7A. Figure 7B displays the same information as kernel-density
line plots for each individual cluster (from 1 to 12). The clusters
were examined with the algal pathways functional annotation
tool (http://pathways.mcdb.ucla.edu/chlamy/) (Lopez et al., 2011)
for obtaining Gene Ontology information. Genes involved in DNA
replication and repair and the cell cycle were mostly represented
in clusters 1 and 2 and display a marked decrease of their transcripts after 43 and 48 h of vitamin treatment, whereas no major
changes in the transcript levels of these genes occurred in A31.
A similar trend was also observed for cluster 3, which contains
genes involved in photosynthesis. Clusters 6 and 7 include genes
implicated in amino acid metabolism whose transcripts were
significantly increased after 43 and 48 h. This increase in RNA
levels was more pronounced for clusters 11 and 12, which include genes involved in autophagy, programmed cell death, protein folding, and proteolysis as well as genes of proteins involved
in sulfate and phosphate transport (Supplemental Data Set 4B
and Supplemental Figure 4).
Proteomic Analysis in DCH16 upon Vitamin-Mediated
Depletion of ClpP1
To examine the changes in protein levels associated with the
depletion of ClpP1, a quantitative mass spectrometry–based
proteomics approach was used based on 15N metabolic labeling.
To generate a high-resolution map of proteomic dynamics in
the DCH16 strain, soluble protein extracts were prepared from
samples harvested at 0, 12, 24, 36, 48, 72, and 96 h after addition of vitamins. As control, samples were also taken from
a culture of the A31 strain at 0, 24, and 48 h in order to identify
the responses due to vitamin addition alone. Only proteins with
Table 1. Number of Up- and Downregulated Genes upon Vitamin or Rapamycin Treatment
Time
Strain 1
up_1
12 h
31 h
43 h
48 h
Union
Time rap
2h
2h
2h
2h
8h
8h
8h
8h
Union
A31
A31
A31
A31
A31
527
323
355
847
1495
A31
A31
A31
A31
A31
A31
A31
A31
A31 rap
Union
A31 rap
890
890
890
890
688
688
688
688
up_1
1112
1112
down_1
269
50
46
502
735
1049
1049
1049
1049
1014
1014
1014
1014
down_1
1546
1546
Time
Strain 2
up_2
down_2
up_1&2
up %_1
up %_2
down_1&2
down %_1
down %_2
12
31
43
48
h
h
h
h
DCH16
DCH16
DCH16
DCH16
DCH16
572
616
1282
1575
2288
593
376
1409
1740
2078
220
76
51
369
601
42
24
14
44
40
38
12
4
23
26
107
36
39
407
486
40
72
85
81
66
18
10
3
23
23
12 h
31 h
12 h
31 h
43 h
48 h
43 h
48 h
rap
DCH16
DCH16
A31
A31
DCH16
DCH16
A31
A31
DCH16
rap
A31
572
616
269
323
1282
1575
355
847
up_2
2288
1495
593
376
527
50
1409
1740
46
502
down_2
2078
735
97
276
105
43
362
380
17
226
up_1&2
643
408
11
31
12
5
53
55
2
33
up %_1
58
37
17
45
39
13
28
24
5
27
up %_2
28
27
366
310
30
22
546
546
9
231
down_1&2
997
420
35
30
3
2
54
54
1
23
down %_1
64
27
62
82
6
44
39
31
20
46
down %_2
48
57
Genes differentially expressed upon ClpP1 depletion and rapamycin treatment of A31 (strain 1) and DCH16 (strain 2). Number of genes up- or
downregulated for these two strains are indicated in the columns up_1, up_2, down_1, and down_2. Number of genes up- and down-regulated in both
strains 1 and 2 are listed in columns up_1&2 and down_1&2, respectively. Union refers to the total number of genes differentially expressed at the time
points indicated. Genes listed include those with (1) log2 moderate fold change >1, (2) an adjusted P value < 0.01 (false discovery rate < 0.01). The gene
lists with expression values are given in Supplemental Data Sets 1 to 3.
Figure 7. Model-Based Clustering of RNA-Seq Data.
Groups of genes with similar expression profiles across all samples were obtained using a negative binomial model as described (Si et al., 2014).
(A) Heat map of expression levels for significantly regulated genes (a total of 5085; see main text). The map shows the fold change at 0, 12, 31, 43, and
48 h following vitamin addition for the A31 and DCH16 strains (from bottom to top of the heat map), using the gene’s average expression as a reference.
The color scale ranges from dark red (positive fold changes) to illustrate significant overexpression, to light yellow (negative fold changes). The tree
shows the result of the hybrid-hierarchical clustering, which groups clusters based on their similarity (as computed from the likelihood of the negative
binomial model). The lower level of the tree and labels on the x axis represent the maximum number of clusters used in this work (12).
(B) Kernel-density line plots for individual clusters (from 1 to 12). Each plot compiles the expression profiles for both strains (A31 and DCH16) across the
time course (0, 12, 31, 43, and 48 h). For each cluster, the red line shows the cluster’s trend (average expression profile of all genes in the cluster), and
the light-blue density map is a histogram of all the individual gene profiles. Also shown are the most significant functional ontologies as described in the
main text.
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2-fold changes or higher were considered to have a reproducible
and statistically significant change in accumulation during the time
course.
A list of 348 proteins was obtained whose change in abundance appears to be specifically linked to the depletion of ClpP1
in the DCH16 strain (Table 2; Supplemental Data Set 6). Of these
proteins 27 and 11% are predicted to be targeted to the chloroplast and mitochondria, respectively, by PredAlgo, an algorithm specifically designed for predicting the cellular localization
of C. reinhardtii proteins that is based on a neural network trained
to recognize transit peptide sequences (Tardif et al., 2012). The
number of proteins differentially expressed increased at each time
point (Supplemental Figure 5). The Venn diagram comparing these
proteins reveals a set of proteins whose level changes transiently
at each time point. Some of these proteins might also be differentially expressed at the other points but below the threshold of
significance, whereas others might be linked to some short-term
time-specific cellular events.
Supplemental Data Set 7 provides, for each time point, the list
of genes encoding proteins showing at least a 2-fold difference
in protein level upon ClpP1 depletion in the DCH16 strain. See
Supplemental Data Set 8 for a proteomic analysis of A31 upon
vitamin addition. Sorting of these proteins with different abundance in functional categories in Table 3 reveals that upon ClpP1
repression many different basal metabolic functions are affected
such as amino acid synthesis and metabolism, RNA metabolism,
fatty acid synthesis, lipid metabolism, and cell cycle. Moreover,
changes in the abundance of proteins involved in translation and
signaling occur. Many of these changes are also seen at the RNA
level (Supplemental Data Set 4B); in some cases, they are likely to
represent compensatory responses, e.g., stimulation of amino
acid synthesis when proteolysis by ClpP1 is impaired.
It is noteworthy that after 36 h of vitamin treatment, proteins
with known or predicted ROS scavenger activity accumulate
(such as Gshr1 and Cre16.g676600.t1.2), suggesting that the
reduced activity of the Clp protease seriously compromises the
functionality of the plastid and leads to a response induced by
oxidative damage. Unfortunately, the majority of the proteins differentially regulated at this time point have unknown functions and
localizations.
At later time points (72 and 96 h) the number of proteins differentially present became quite large and the occurrence of
Table 2. Number of Proteins of A31 and DCH16 with >2-Fold Changes
upon Vitamin Treatment
Comparison
Strain
Genes
24
48
12
24
36
48
72
96
A31
A31
DCH16
DCH16
DCH16
DCH16
DCH16
DCH16
5
54
5
34
39
97
208
272
h/0
h/0
h/0
h/0
h/0
h/0
h/0
h/0
h
h
h
h
h
h
h
h
vitamin
vitamin
vitamin
vitamin
vitamin
vitamin
vitamin
vitamin
The listed proteins are satisfying the following conditions: significant fold
change (>2) and significant adjusted P value (<0.05).
compensatory responses is well illustrated by the increased abundance of many additional proteins that are known to be involved in
protein folding and degradation (Hsp22F, ClpB3, Rpn8, UspA,
Cpn11, Deg11, FKB-16.2, and Asp1), vesicular trafficking (Arl3 and
Arl1), autophagy (Atg10), DNA replication and repair (RecA and
PolE), and tetrapyrrole metabolism (ChlH1).
Comparison between Transcriptomic and Proteomic Data:
Potential ClpP1 Substrates
Having identified many mRNAs and proteins that change significantly during the time course of ClpP1 depletion, we next
determined how the transcriptomic and proteomic data compare with each other. Among the 348 identified proteins, we
tested to what extent the changes in protein and corresponding
mRNA levels correlate with each other during ClpP1 depletion.
Of these, 148 and 53 are increased and decreased, respectively,
both at the protein and mRNA level, whereas in 33 cases, the
proteins decrease and their mRNAs increase and, for 78 cases,
the opposite pattern occurs. Comparison of the RNA and protein changes for individual genes at 48 h (Supplemental Data Set
6 and Supplemental Figure 6) revealed that they differ widely.
Because in the absence of ClpP1, the levels of its chloroplast
protein substrates would be expected to increase presumably
with only modest changes in the corresponding RNA levels, we
examined the proteins that follow such a pattern. We chose
cutoffs of <2-fold for the RNA changes and >2-fold for protein
changes due to ClpP1 depletion. In this way, 78 genes were
identified, of which 16 were predicted to encode chloroplast
proteins that are involved in a variety of cellular processes
(Supplemental Data Set 6C and Supplemental Figure 6). This
includes several unknown proteins with possible functions in
lipid and pyrimidine metabolism and signaling, proteins involved
in amino acid metabolism, RecA, which is involved in chloroplast
DNA recombination, pyruvate dehydrogenase, a putative aspartate kinase, ClpP4 (part of the ClpP protein complex), the
chaperonin Cpn11, the plastid ribosomal protein Psrp6, and Tic
110, a component of the chloroplast inner membrane translocon. These proteins could be potential substrates of ClpP1.
Specificity of the Response Induced through
ClpP1 Depletion
The transcriptomic and proteomic data clearly indicate specific
changes in both RNA and protein accumulation upon depletion
of the ClpP1 protease that differ at least to some extent from
those induced by other stress conditions. To further study the
specificity of this response, we first compared the transcriptomic
responses induced by ClpP1 depletion and rapamycin treatment.
Table 1 shows that among the up- and downregulated genes of
DCH16 upon ClpP1 depletion, significant fractions, 58 and 64%,
respectively, are similarly affected in the A31 strain treated with
rapamycin. It should be noted, however, that the irradiance was
changed from 10 to 60 mmol m22 s21 when rapamycin was
added because induction of autophagy by rapamycin requires
this light intensity (Pérez-Pérez et al., 2010). We noticed that, in
some cases, this increased irradiance alone caused a change in
transcript level that was larger than that induced by rapamycin
Chloroplast Protein Quality Control
13 of 22
Table 3. Functional Groups of Proteins with >2-Fold Changes in the DCH16 (Plus Vitamins) or A31 (Plus Rapamycin) Strains
Upregulated Genes
DCH16 + vit
No. of Genes
Upregulated genes
2243
Common pathways
Amino acid metabolism
83
Lyosome
20
Glycerolipid metabolism
11
Glutatione metabolism
13
Porphyrin and chlorophyll metal
15
ABC transporters
29
Regulation of autophagy
10
Heme biosynthesis
6
Different pathways
tRNA charging pathway
24
Sulfur metabolism
10
Ubiquitin proteasome pathway
18
Abiotic stress
19
Downregulated Genes
A31 + rap
No. of Genes
1353
74
14
9
11
17
11
3
6
Photorespiration
Folate metabolism
(Supplemental Figure 4). Therefore, the changes observed from
the set of samples profiled by RNA-Seq are most likely not
entirely specific to rapamycin.
Two genes, Vipp2 and Deg11, whose expression is strongly
upregulated by ClpP1 depletion were further examined. The
promoter and 59UTR regions of these genes were fused to the
luciferase gene of Gaussia princeps (Shao and Bock, 2008),
giving rise to proVipp2-gLUX and proDeg11-gLUX, which produced the Vipp2gLuc and Deg11gLuc fusion proteins. These
constructs were introduced into the DCH16 strain and the transformants were tested by immunoblotting under different stress
conditions such as that resulting from ClpP1 depletion (vitamins),
inhibition of TOR (rapamycin), inhibition of protein glycosylation in
the ER (tunicamycin), high light, and nitrogen starvation (Figure 8).
Vipp2-gLUX was specifically induced upon ClpP1 depletion and
gave rise to a doublet of ;23 kD and a band of ;17 kD (Figure
8A). Control experiments with protein extracts from a control strain
lacking luciferase revealed that only the 17 kD band is specific for
the luciferase. The identity of the 23-kD band is still unknown
(Supplemental Figure 7). The luciferase protein was weakly expressed under other conditions except in cells subjected to high
light and followed essentially the same pattern as the endogenous Vipp2 protein. Induction of Vipp2 protein and mRNA during
high-light exposure was previously reported (Nordhues et al.,
2012). Importantly, the induction of Vipp2-gLUX also occurred
during ClpP1 inactivation in the dark. These data suggest that
upregulation of the Vipp2 protein is mediated by its promoter
and/or 59UTR but does not require a light signal. In these experiments, performed either under constant light or in the dark,
rapamycin did not upregulate Vipp2 RNA. An increase of this
RNA only occurred when the irradiance was changed after addition of the drug (Supplemental Figure 8). Similar behavior was
observed for Hsp22F.
Deg11-gLUX was strongly induced upon ClpP1 depletion and
high light (Figure 8B) and similarly to the case of Vipp2-gLUX, the
induction of this response also occurred in the dark, indicating
DCH16 + vit A31 + rap
No. of Genes No. of Genes
Downregulated genes
1555
Common pathways
DNA replication
26
DNA repair
18
Homologous recombination
6
Cell cycle
36
Calcium signaling
9
Pyrimidine metabolism
14
MAPK signaling
16
Photosynthesis
35
1607
28
19
6
38
8
13
16
6
5
5
that it is not caused by photooxidative damage alone. However,
contrary to the Vipp2-gLUX reporter, the Deg11-gLUX reporter
was also activated by tunicamycin.
DISCUSSION
The major goal of this study was to investigate the role of the
chloroplast ATP-dependent ClpP protease in cellular metabolism
and growth. Because it is an essential protein, we took advantage
of our recently developed repressible chloroplast gene expression system that is based on the vitamin-sensitive Thi4 riboswitch
(Ramundo et al., 2013). This allowed us to examine the fate of
C. reinhardtii cells in which ClpP1 is progressively depleted. Our
results demonstrate that ClpP1 is essential for cell survival and
show that ClpP1 depletion leads to the arrest of cell growth and
protein aggregation associated with a massive upregulation of
chaperones and proteases, a hallmark of an unfolded protein
response, and metabolic adaptation. Moreover, the cellular membrane system is considerably perturbed with a striking increase in
the number of vacuoles in the cytosol and vesicles in the chloroplast which presumably are derived from the thylakoid membrane
(Figures 4 and 5). Our comparative study of the stress response
induced by ClpP1 depletion and of rapamycin reveals that these
processes share common features but also differ in many respects
from one other.
It is particularly striking that many of the proteins with increased abundance are involved in sulfur transport and recycling (Sul2, Slt3, Met16, and Aot4), iron-sulfur cluster biogenesis
(SufD), and in trans-sulfuration pathways (DhqS). Because glutathione is a key factor for buffering the cellular redox state, the
induction of sulfur metabolism upon ClpP1 depletion may be
linked to an increased demand of cysteine, the limiting reagent
for glutathione production. Alternatively, it is possible that depletion of ClpP protease impairs a key component of the sulfur
metabolic pathway, thus activating a sulfur starvation response.
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The Plant Cell
Figure 8. Specific Expression of Nuclear Genes upon ClpP1 Depletion in DCH16.
(A) A construct with the VIPP2 promoter and 59 UTR fused to the Gaussia luciferase gene (proVipp2-gLUX) was introduced into DCH16 (CS191 strain).
Immunoblots were performed with total cell extracts from CS191 grown for the times indicated in the presence of vitamins in the light (60 mmol m22 s21)
or in the dark as indicated using antibodies against ClpP1, luciferase (gLUX), Vipp2, Vipp1, and RbcL. These cells were also treated with rapamycin
(Rap), tunicamycin (Tm), and high light (HL; 600 mmol m22 s21) or grown in the absence of nitrogen (-N) . The times of each treatment are indicated.
(B) A construct with the Deg11 promoter and 59 UTR fused to the Gaussia luciferase gene (proDeg11-gLUX) was introduced into DCH16 (CS 174 strain).
Immunoblot analysis of extracts from CS174 was performed as in (A). Rapamycin-treated cells were grown under constant light (60 mmol m22 s21).
Interestingly, among the proteins with increased accumulation
involved in protein homeostasis, some are known to have isoforms with different subcellular localizations. Examples are the
heat shock proteins Hsp22 and Hsp70, the Deg proteases, the
ClpB protein disaggregases, and the GroES-type chaperonins.
In our proteomic analysis, the isoforms identified are mainly the
ones targeted to the chloroplast compartment. Another protein
whose level increases is ClpP4, one of the other two paralogous
active catalytic subunits of the Clp protease encoded by the
nuclear genome. It is possible that the cell senses the impaired
stoichiometry during assembly of the Clp catalytic chamber and
may attempt to overcome this limitation by overexpressing another catalytic subunit of the complex.
Genes whose transcripts are downregulated upon both ClpP1
depletion and rapamycin treatment are involved in the cell cycle
and in DNA replication, repair, and recombination as well as in
various signaling pathways and photosynthesis (Supplemental
Data Sets 4A and 4B). Genes that are upregulated under both
conditions are implicated in amino acid metabolism, vacuolar
function, tetrapyrrole metabolism, autophagy, and transport of
metabolites. Among the most highly upregulated transcripts were
those of several small HSP proteins and chaperones, proteases,
proteins involved in autophagy and thylakoid membrane biogenesis, protein kinases and transporters, and ion channels
(Supplemental Data Set 4B). Among the latter, one gene of particular interest is Msc1, which encodes a mechanosensitive ion
channel suggested to be localized both in the cytosol and chloroplast (Nakayama et al., 2007). It could be involved in sensing
osmotic stress, allowing the cell to counteract changes in osmotic pressure during the cellular swelling that accompanies
autophagy.
Depletion of ClpP Affects Chloroplast and Cytosolic
RNA Metabolism
A striking effect of ClpP depletion is the increased accumulation
of many chloroplast mRNAs (Figure 1; Supplemental Figure 2).
In this respect, one chloroplast gene of particular interest is
ORF1995. It gives rise to a full-length transcript of 6600 nucleotides and to several smaller transcripts, which all increase upon
vitamin treatment (indicated by arrows in Figure 1D), suggesting
the existence of a plastid negative feedback control. However, it
is not clear whether the smaller transcripts represent functional
processed transcripts or degradation products of the full-length
Chloroplast Protein Quality Control
transcript. It is particularly noteworthy that in most of the cases,
chloroplast and nuclear genes involved in photosynthesis respond in opposite ways with chloroplast transcripts increasing
while the cytosolic transcripts decrease.
The transcriptomic and proteomic analyses indicate that prolonged ClpP1 depletion causes a drastic upregulation of several
ribonucleases, such as Rbn1 and Rbn2, which act as 39-59 exoribonucleases in the exosome complex and actively participate
in RNA maturation and quality control (Supplemental Data Set 4),
and Xrn1, a putative organelle-localized homolog of the cytosolic
59-39 exoribonuclease and a potential ribonuclease Z. Importantly,
RNases Z are mainly known for their role in tRNA processing, but
recently an additional function in mRNA decay has been suggested, especially in organisms lacking RNase E (Perwez and
Kushner, 2006). The upregulation of these genes and the unexpected upregulation of many chloroplast mRNAs may indicate
a restructuring of intracellular RNA populations and/or changes
in an RNA quality control mechanism. Consistent with this hypothesis, we found that, among the most upregulated transcripts
in this functional category, three of them encode uncharacterized
proteins (Cre17.g725750.t1.1, Cre17.g726850.t1.2, and Cre10.
g447800.t1.2) that show remarkable sequence similarity to the
bacterial Rsr and to the human Ro 60-kD autoantigen but are
not predicted to be targeted to the chloroplast (Stein et al., 2005)
(Supplemental Data Set 4 and Supplemental Figure 4F). Intriguingly, both in bacteria and in humans, these proteins are
known to act as cofactors to increase the association of exonucleases with misfolded RNA substrates during stress (Chen et al.,
2005; Stein et al., 2005; Fuchs et al., 2006; Wolin and Wurtmann,
2006). We found that the Cre17.g725750.t1.1 transcript is also
upregulated under high light (Supplemental Figure 4F). These
results raise the question whether an unfolded RNA response
exists in C. reinhardtii as part of an integrated stress response.
Potential of ClpP1
Our comparative transcriptomic and proteomic analysis of the
348 proteins that are differentially expressed upon ClpP1 depletion revealed that 137 (39%) also display changes in their
corresponding mRNAs. For the remaining 211 proteins (61%), no
significant changes in transcript accumulation could be detected,
indicating that the changes in protein levels are mainly due to
translational or posttranslational processes. In the absence of
ClpP1, its chloroplast protein substrates would be expected to
accumulate, presumably with only small changes in the corresponding RNA levels. At 48 h following vitamin treatment, we
identified 15 chloroplast proteins with this property (Supplemental
Data Set 6C and Supplemental Figure 8). These proteins are
involved in plastid chloroplast metabolism, protein synthesis,
turnover, and folding, and DNA recombination. Some of the proteins in this list were also found to increase in the ClpR2 mutant
of Arabidopsis (Zybailov et al., 2009). Moreover, RecA has been
proposed to be a ClpP substrate in Staphylococcus aureus
(Feng et al., 2013). All of these proteins are soluble, with the
exception of Tic110, which is located in the inner chloroplast
envelope membrane. This broad range of potential substrates is
compatible with the fact that ClpP1 is the most abundant protease in the chloroplast stroma. However, we cannot rule out
15 of 22
that the increased abundance of these proteins is due to an
indirect effect of ClpP1 depletion or is caused by increased translation rather than reduced proteolysis. Alternatively, removal of
posttranslational modifications from quantified peptides as a
consequence of ClpP1 depletion would also lead to an apparent
increase of these peptides as phosphorylated peptides would
have escaped detection.
Loss of ClpP1 Leads to Increased Accumulation of the
Thylakoid FtsH Protease
Chloroplasts are known to contain a number of proteases; some
are localized in the stroma, whereas others, such as the FtsH
and the Deg proteases, are associated with the thylakoid membranes
and involved in the PSII photorepair cycle (Kato and Sakamoto,
2013). Recent results indicate that in the Arabidopsis var2 mutant
deficient in FtsH2, ClpP1 is upregulated and a portion of this
protease is relocalized from the stroma to the thylakoid membranes, suggesting compensation for the limitation in FtsH (Kato
et al., 2012). Similarly, upon exposure to excess light, ClpP1 is
also upregulated, implying cooperation between different chloroplast proteases under stress conditions (Kim et al., 2009). In this
study, we showed that the reverse is true when ClpP1 is limiting.
Upon gradual depletion of ClpP1 induced by vitamin treatment
of DCH16, the level of FtsH increased significantly, suggesting a
compensatory proteolytic response to mitigate the loss of ClpP1
(Figures 2A and 3B). Importantly, this compensation effect was
only observed when ClpP1-depleted cells are grown in the dark
or dim light (10 mmol m22 s21), suggesting that that this process
is sensitive to photooxidative damage (Figure 2B).
Autophagy-Like Phenotype and Membrane Trafficking upon
ClpP1 Depletion
Direct comparison of the mRNAs differentially expressed as the
consequence of ClpP1 depletion or rapamycin treatment revealed
that the genes that are upregulated under both conditions comprise 28 and 58% of the total number of the upregulated genes in
the vitamin-treated DCH16 and A31 strains, respectively. The
corresponding numbers for the downregulated genes are 48 and
64% (Table 1). These results suggest common regulatory mechanisms used by the cells to orchestrate these stress responses.
The list of the most upregulated genes (Supplemental Data Set
4B) includes a group of autophagy-related genes including Atg8,
Atg3, Atg6, Atg7, and Atg12. The induction of these genes is a
typical hallmark of autophagy. Moreover, the set of highly upregulated mRNAs encode proteins involved in membrane biogenesis and vesicular trafficking, including two SNARE proteins
(Vmpl2 and Vamp74) that catalyze the homotypic membrane
fusion during the late stages of autophagosome formation both
in mammalian cells and yeast (Nair et al., 2011; Renna et al.,
2011; Stroupe, 2011).
The strong upregulation of the related chloroplast Vipp1 and
Vipp2 genes raises the possibility that they are involved in the
breakdown/disassembly of the thylakoids and in chloroplast lipid
trafficking and membrane remodeling. Whether the released polar
lipids serve as a cellular membrane source during autophagy
remains an open question. Vipp1 and Vipp2 are partially redundant
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and have been proposed to be involved in the assembly and
biogenesis of thylakoid core complexes, perhaps by providing
structural lipids (Nordhues et al., 2012). The absence of induction of Lhcsr3, Hsp70B, and Hsp90C in Vipp1-RNAi strains
upon high-light exposure suggests that Vipp1 might play a role
in the high-light retrograde signaling pathway. As the expression
of Vipp2 is not markedly affected by changes in the redox state
of the photosynthetic electron transport chain, it was suggested
that specific photoreceptors may be involved (Im et al., 2006).
However, in this work, we show that the amount of Vipp2 protein
can also be increased in the dark upon depletion of the chloroplast ClpP1 protease.
Marked changes in protein levels were already detected at
early time points. A decrease of 50% in ClpP1 expression after
12 h of vitamin treatment (Figure 1D) was sufficient to trigger significant changes in accumulation of a few proteins (Supplemental
Figure 6 and Supplemental Data Set 7). In particular, the levels of
two proteins, Vipp2 (Cre11.g468050.t1.1) and a protein encoded
by Cre16.g683350.t1.1, were strongly increased. The other protein
(Cre16.g683350.t1.1), which is not predicted to be targeted to the
chloroplast, is a member of the thioesterase superfamily. These
enzymes catalyze the hydrolysis of long-chain fatty acyl-CoA thioesters (acyl-CoAs) to free fatty acids and CoA (CoASH). Importantly,
acyl-CoA is not only a building block in lipid metabolism, it can also
serve as a signaling molecule in a wide range of biological activities such as development, stress responses, vesicular trafficking,
membrane biogenesis, and other signaling pathways (Xiao and
Chye, 2011). Therefore, the presence of this protein in the early
phase of ClpP1 depletion raises the possibility that retrograde
signaling may be mediated in part by lipid molecules.
Genes Regulated by ClpP1 Depletion but Not by Rapamycin
Treatment May Be Involved in a Chloroplast Unfolded
Protein Response
Together with the proteomic analysis, our transcriptomic analysis
indicates that depletion of the ClpP1 plastid protease subunit
causes induction of genes coding for factors involved in chloroplast and cytosolic protein folding and proteolysis (Supplemental
Data Sets 4 and 7). In particular, the chloroplast small heat shock
proteins Hsp22E and Hsp22F and the HtrA protease Deg11 are
three of the most highly upregulated genes identified in our study.
Other upregulated mRNAs of protein quality control factors include the ubiquitin conjugating enzyme E2 (Cre01.g027200.t1.1),
the chaperones ClpB3, DnaJ5, and DnaJ34, and a set of poorly
characterized proteases. Taken together, our data suggest the
existence of an unfolded protein response.
Genes that are downregulated both by ClpP1 depletion and
rapamycin treatment include many genes involved in nuclear
DNA replication and repair, plastid division, centriole and basal
body duplication, and in response to a chloroplast dysfunction
(Supplemental Data Set 4 and Supplemental Figure 4B). Under
these stress conditions it appears that cells devote their resources
to maintenance and survival at the expense of cell growth and
division.
Although the stress responses elicited through depletion of
the plastid ClpP1 protease and by the inhibition of the TOR kinase by rapamycin show similarities, there are also significant
differences as revealed by the global comparative transcriptional
analysis (Table 1). The list of the most upregulated genes with an
annotated function includes chaperones, proteases, and ubiquitin-related factors in addition to those already identified in the list
of the genes commonly regulated by ClpP1 depletion and rapamycin treatment. Intriguingly, some of these transcripts encode
different isoforms of proteins belonging to the same family, in
particular in the case of the small heat shock proteins. These
proteins form dimers with a hydrophobic surface at their N-terminal
end for interaction with denatured proteins with which they form
a large complex to prevent their irreversible aggregation (Lee et al.,
1997; Haslbeck et al., 2004). In C. reinhardtii, this family contains
eight members (Schroda and Vallon, 2009). Of these, isoforms A
and B are predicted to be in the cytosol and isoforms C, E, and
F in the chloroplast, all of which are upregulated upon ClpP1
depletion. Several transcriptomic analyses have revealed that
Hsp22C, Hsp22E, and Hsp22F can be induced by a variety of
stresses, such as heat shock, oxidative stress, and phosphorus
and sulfur starvation (Gonzales-Ballester and Grossman, 2009;
Moseley and Grossman, 2009; Schroda and Vallon, 2009). We
found that Hsp22B, Hsp22C, and Hsp22F are upregulated by
ClpP1 depletion but not by rapamycin treatment under constant
dark or light (Supplemental Figures 4D and 8 and Supplemental
Data Set 4). Moreover, the loss of the ClpP1 protease caused
a specific upregulation of Cgl41, an uncharacterized protein
containing an RbcX domain, usually found in an assembly chaperone for hexadecameric Rubisco (Saschenbrecker et al., 2007)
(Supplemental Figure 4 and Supplemental Data Set 4). Taken together, these findings suggest that the disruption of protein homeostasis in the chloroplast compartment can be sensed and
transduced to the nucleus to induce the expression of a specific
set of protein quality control factors. Since in Arabidopsis constitutive reduction of the ClpPR protease also results in upregulation of chloroplast chaperones and protein sorting components,
it is possible that this retrograde signaling pathway is evolutionarily conserved (Zybailov et al., 2009).
In mammalian cells, such a homeostatic pathway is well known
to operate when functional perturbations occur in the ER in which
most proteins are folded, modified, and assembled before being
secreted or displayed on its surface. The discovery of this signaling network, named erUPR, arose from a pioneering study in
which the pharmacological inhibition of folding led to the transcriptional up-regulation of several key ER chaperones (Cox et al.,
1993). More recently, a similar pathway named mitochondrial
unfolded protein response (mtUPR) was proposed to counteract
the accumulation of unfolded proteins within the mitochondrial
matrix through the transcriptional upregulation of nuclear genes
encoding mitochondrial stress proteins such as chaperones Hsp60,
Hsp70, mtDNAJ, and ClpP1, but not those encoding stress proteins of the ER or the cytosol (Aldridge et al., 2007).
The observed changes in gene expression caused by ClpP1
depletion raise the possibility that a specific chloroplast unfolded
protein response exists. It should be noted that all of the upregulated chaperones are not exclusively localized in the chloroplast, although the majority of these proteins are predicted to be
in this organelle. Moreover, because the vast majority of the
chloroplast proteins are encoded by the nuclear genome, are
synthesized in the cytoplasm, and are imported in an unfolded
Chloroplast Protein Quality Control
state into the organelle via specialized translocases, impairment
of the stromal protein folding environment is expected to limit the
availability of the stromal chaperones and ATP required for protein
import and the refolding of the mature proteins upon translocation.
In this case, the protein import capacity of the translocon complex
is likely to be compromised at the chloroplast envelope, which
would impact protein homeostasis in the cytosolic compartment
and necessitate the upregulation not only of chloroplast but also
of cytosolic chaperones. In this respect, it is interesting to note
the appearance of perforations in the chloroplast envelope upon
ClpP1 depletion (Figure 5E). They could reflect changes in the
envelope structure that may affect protein import.
In the list of the transcripts specifically upregulated by ClpP1,
we identified some mRNAs of poorly characterized and potentially organelle-associated proteins that are related to E2 ubiquitin-conjugating enzymes or contain UBX and UFD domains
(Meyer et al., 2012) (Cre10.g429001.t1.1, Cre03.g179100.t1.1,
and Cre12.g510300.t1.2) and to the mitochondrial Vms1 protein
(Cre26.g772100.t1.2) (Supplemental Figure 4E). Others contain
the DUB/PPDE domain in Cre16.g662450.t1.1 proposed to act
as deubiquitinating and desumoylating peptidase. Importantly,
in yeast, Vms1 was recently shown to be induced upon oxidative
stress and to interact with Cdc48/p97, a conserved chaperonelike ATPase that forms a homohexameric complex and regulates
a large array of cellular functions, including protein degradation,
cell division, membrane fusion, autophagosome biogenesis,
retrotranslocation of unfolded proteins from the ER to the cytosol, and segregation and delivery of ubiquitylated proteins to
the proteasome for degradation (Meyer et al., 2012). Vms1 recruits Cdc48/p97 to the outer mitochondrial membrane and
thereby mediates the extraction and ubiquitination and proteasomemediated degradation of mitochondrial proteins in the cytosol (Heo
et al., 2010).
A pathway for protein degradation through the extrusion of
chloroplast proteins to the vacuoles was proposed for C. reinhardtii
(White et al., 1996; Park et al., 1999). Major pulse-labeled polypeptides synthesized on chloroplast ribosomes were recovered in
small granules in cytoplasmic vacuoles. Moreover, protrusions of
the outer membrane of the chloroplast envelope were detected
that enclosed stroma, suggesting that chloroplast proteins from
the stromal phase were extruded from the chloroplast in membrane-bound structures to the vacuoles. Similarly, greening of the
C. reinhardtii y1 mutant in the light at 38°C revealed the presence
of light-harvesting proteins inside the chloroplast near the envelope
and also in granules within vacuoles in the cytosol. This suggests
that this pathway may operate when excess light-harvesting proteins are synthesized with respect to integration of these proteins
into thylakoid membranes (White et al., 1996; Park et al., 1999).
The observed upregulation of Vms1 and Cdc48 upon ClpP1
depletion raises the possibility that several chloroplast protein
extrusion processes may operate in C. reinhardtii under specific
stress conditions to degrade excess proteins or to provide free
amino acids when amino acid supply is limiting.
The use of the reporter constructs Vipp2-gLUX and Deg11gLUX provided several new important insights into the regulation of the expression of these genes. First, both Vipp2 and
Vipp2-gLUX are specifically induced by the depletion of ClpP1
or by high-light treatment, indicating that upregulation of the
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Vipp2 gene is mediated by its promoter and/or 59UTR (Figure 8).
Second, this induction also occurs in the dark and does not
require a light signal. Instead, it may be triggered by accumulation
of unfolded proteins and/or damaged thylakoid membranes,
which is also likely to occur upon photooxidative damage induced under high light (Figure 8). However, VIPP2 is not induced
by heat shock, a treatment that is also expected to unfold
proteins (Nordhues et al., 2012). Possibly, depletion of ClpP1
may induce a stronger and more chloroplast-specific response
to which Vipp2 is reactive. Third, in these experiments performed either under constant light or in the dark, rapamycin did
not upregulate Vipp2 RNA in contrast to the experiments profiled by RNA-Seq in which the light irradiance was changed after
addition of the drug (for more details, see Supplemental Data
Sets 3 and 4 online). Vipp2 therefore appears to be well suited
for genetic screens aimed at identifying the components of the
retrograde signaling chain, which links ClpP1 depletion with
nuclear gene expression. The Deg11-gLUX reporter responds in
a similar manner as Vipp2-gLUX except that it is also induced by
tunicamycin, which inhibits glycosylation in the ER. This raises
the question of possible interactions between ER and chloroplasts. The observation of contact sites between these two
organelles suggests that they interact physically (Andersson
et al., 2007). Moreover, it is interesting to note that a few nucleusencoded plastid proteins use the secretory pathway and are
N-glycosylated in the ER before they enter the chloroplast (Villarejo
et al., 2005).
In conclusion, we took advantage of the conditional repressible chloroplast gene expression system in C. reinhardtii to
conduct a detailed study of the role of the essential plastid clpP1
gene through a comparative cell biology, transcriptomic, and
proteomic analysis. This approach has revealed that ClpP1 is
involved, directly or indirectly, in a variety of molecular events
that underlie protein homeostasis and retrograde signaling.
Thus, this work opens new avenues for identifying new molecular components involved in these processes and pathways.
METHODS
Strains, Growth Conditions, and Media
The Chlamydomonas reinhardtii strains generated in this study were
maintained on TAP or minimal (HSM) medium plates supplemented with
1.5% Bacto-agar (Gorman and Levine, 1965; Harris, 1989) at 25°C under
constant light (60 to 40 mmol m22 s21)/dim light (10 mmol m22 s21) or in
the dark. Growth medium containing vitamins was prepared as previously
described (Ramundo et al., 2013). See Supplemental Methods for more
details on growth conditions.
Construction of the Strains DCH16, CS174, CS193, and CS365
Nuclear and chloroplast transformations were performed as previously
described (Ramundo et al., 2013). Plasmids used for generating the
strains DCH16, CS174, CS193, and CS365 are described in Supplemental
Methods.
Fluorescence Measurements
Maximum quantum efficiency of photosystem II (Fv/Fm) was measured as
described (Ramundo et al., 2013).
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The Plant Cell
Protein Extraction and Immunoblot Analysis
Total protein extraction and immunoblot analysis was performed as
described (Ramundo et al., 2013). For details, see Supplemental Methods.
sample. Nanostring nCounter Expression analysis was performed as
previously described (Ramundo et al., 2013).
RNA-Seq
Nucleic Acid Extraction
Isolation of total RNA and DNA from C. reinhardtii strains was performed
as described (Ramundo et al., 2013).
RNA Gel Blotting
Hybridizations of RNA gel blots were performed as described (Ramundo
et al., 2013). The probe used for ORF1995 corresponds to the region
between amino acids 1082 and 1463.
Immunofluorescence
C. reinhardtii cells were fixed and stained for Apg8 immunofluorescence
as described (Díaz-Troya et al., 2008).Time and light conditions are indicated in the text.
Nile Red Staining
C. reinhardtii cells were stained with Nile Red and images were acquired
through a Leica TCS-SP2 confocal microscope as described (Wang et al.,
2009). Time and light conditions are indicated in the text.
Optical and Transmission Electron Microscopy
A total of 4 3 107 cells/mL were pelleted by centrifugation (5 min, 1000g,
room temperature), resuspended very gently in 2.5% glutaraldehyde in
0.1 M cacodylate buffer, pH 7, and incubated at room temperature for 2 h.
The cells were then pelleted by centrifugation (2 to 3 min, 500g), washed in
cacodylate, and postfixed in 1% OsO4 in cacodylate for 1 h at room
temperature. They were rinsed in buffer and further fixed for 1 h in 1%
aqueous uranyl acetate. Samples were washed in water, dehydrated in
a graded ethanol series, and embedded in Epon 812. Semithin sections,
1 mm in thickness, were cut with a histoDiatome diamond knife (Diatome).
Ultrathin sections 80-nm thick were also cut with an ultra Diatome knife.
The semithin sections were first stained for 20 min at 65°C with a 1:20
dilution of a methylene blue solution (0.15% in 0.1 M phosphate buffer
containing glycerol 1% and methanol 1%). They were dried and restained
with a 1:20 solution of basic fuscin (0.1% in 50% ethanol) for 10 min at
room temperature. The sections were dried, mounted in Eukitt, and
observed using a Nikon eclipse 80i microscope. They were photographed
with a Nikon Digital Color Camera Sight DS-Fi-1. The ultrathin sections
were stained with 2.5% uranyl acetate and subsequently with Reynolds
lead citrate. They were viewed in a FEI Tecnai G2 Sphera transmission
electron microscope at 120 kV. Surface areas were measured using Leica
Twin Image analysis software. For quick-freeze deep-etch electron microscopy, cells were pelleted, placed on a cushioning material, and
dropped onto a liquid-He–cooled copper block; the frozen material was
transferred to liquid nitrogen and fractured, etched at 280°C for 20 min,
and Pt/C rotary replicated as described (Heuser, 2011). Replicas were
examined with a JEOL electron microscope, model JEM 1400, equipped
with an AMTV601 digital camera. The images are photographic negatives;
hence, protuberant elements of the fractured/etched surface are most
heavily coated with platinum and appear white.
Nanostring nCounter Expression Analysis
RNA was isolated from the A31 and DCH16 strains after 0, 12, and 48 h
of vitamin treatment. Three biological replicas were prepared for each
RNA was isolated from the A31 and DCH16 strains grown under
60 mmol m22 s21 after 0, 31, and 43 h of vitamin treatment and after 2 and
8 h of rapamycin treatment of A31 as described (Ramundo et al., 2013). RNASeq libraries were essentially built as previously described (Castruita et al.
(2011) and sequenced on a HiSequation 2500 from Illumina. Raw sequence
files were obtained using Illumina’s proprietary software and are available at
NCBI’s Gene Expression Omnibus (accession number GSE56295).
RNA-Seq reads were aligned using STAR v2.3.0 (Dobin et al., 2013).
The v4 assembly of the C. reinhardtii genome and the Augustus v10.2
gene models were used as reference for STAR. The counts-per-gene
matrix was generated with HTSeq-count v0.5.4p3 (Simon Anders; http://
www-huber.embl.de/users/anders/HTSeq/) using strict intersection and
unique alignments to the genome only. For each gene, expression estimates were obtained after normalizing by sequencing depth and transcript
mappable length and reported in units of reads per kilobase of transcript
mappable length and millions of mapped reads. Log2-fold changes relative
to the 0 time point were calculated for each strain and experiment using
average expression estimates from three biological replicates. For each
pairwise comparison, moderate fold changes, P values, and adjusted
P values were computed with DESeq v1.12.1 (Anders and Huber, 2010),
using a negative binomial model and per-condition dispersion estimates.
For each comparison, a gene was deemed differentially expressed if it met
the following three criteria: (1) significant fold change (>2); (2) significant
expression value (bigger than the median for the condition with highest
expression); and (3) significant adjusted P value (padj<0.01). Altogether,
5038 genes passed these criteria in at least one comparison. Modelbased gene clustering (Figure 6) was performed in R using the MBCluster.
Seq package (Si et al., 2013). RNA-Seq data quality control was performed with Partek Genomics Suite 6.6 beta.
Proteomics
Quantitative shotgun proteomics using a uniform 15N-labeled standard
was performed as described by Mühlhaus et al. (2011) with some modifications. In brief, one culture of the A31 strain and two cultures of the
DCH16 strain were grown for ;10 generations in TAP medium containing
14N in the case of A31 and 14N and 15N-ammonium in the case of DCH16. At
time point 0 h, vitamins were added to the three cultures that were
maintained in exponential phase through daily dilutions. Samples were
taken from A31 (14N) after 12 and 48 h of vitamin treatment and from DCH16
(14N and 15N) after 0, 12, 24, 36, 48, 72, and 96 h of vitamin treatment.
The seven time point samples from DCH16 grown in 15N-containing
medium were pooled to generate a 15N -labeled reference standard that
included all induced and repressed proteins during ClpP1 depletion. Prior
to protein extraction, each 14N sample from the A31 and DCH16 time
course was spiked with the 15N-labeled reference sample at a ratio of
0.8 15N/14N based on protein content determined by the BCA assay
(Thermo Scientific). Mixed cells were ruptured by freeze/thawing cycles
and afterwards centrifuged in a tabletop centrifuge at 4°C and 21,500g for
35 min. The supernatant, containing soluble proteins, was recovered. The
soluble proteins were precipitated with acetone, digested with trypsin and
LysC, and desalted. For each sample, extracted peptides of three biological
replicates were subjected in triplicates to reverse-phase separation by
nanoACQUITY UPLC (Waters). Separated peptides were directed by ESI
to an LTQ-Orbitrap XL mass spectrometer (Thermo Scientific), which
was operated in a cycle of one full-scan mass spectrum (Orbitrap; 300 to
1800 m/z) at a set resolution of 60,000 at 400 m/z followed by serial data–
dependent tandem mass spectrometry scans of the five most intense
peaks. Four different search engines were employed to achieve spectra-
Chloroplast Protein Quality Control
to-peptide mappings, and a total of 7342 peptides could be identified. Of
these, 5038 were mapped with high confidence, i.e., could be recognized
by all four search engines. Protein abundance was quantified using
14N/15N ratios from three biological replicas for a total number of 11,702
protein models. Of these 5035 proteins with values for at least three time
points were retained. From these data, the following comparisons were
done: (1) A31(12 h)/A31(0 h), (2) A31(48 h)/A31(0 h), (3) DCH16(12 h)/
DCH16(0 h), (4) DCH16(24 h)/DCH16(0 h), (5) DCH16(36 h)/DCH16(0 h), (6)
DCH16(48 h)/DCH16(0 h), (7) DCH16(72 h)/DCH16(0 h), and (8) DCH16(96
h/DCH16(0 h). In total, 57 proteins from the A31 comparisons and 348
proteins from the DCH16 comparisons were retained with a fold change
>2. Protein data evaluation was performed using Partek Genomics Suite
6.6 beta.
Accession Numbers
Sequence data from this article can be found in at NCBI’s Gene Expression
Omnibus under accession number GSE56295. Accession numbers for the
genes used in making constructs for this work are clpP1 (NC_005353.1),
psbD (NC_005353.1), Atg8 (Cre16.g689650.t1.1), Deg11 (Cre12.g498500.
t1.2), Hsp22B (EDP07084.1), Hsp22F (EDP10019.1), Nac2 (5,723,926),
Vipp1 (EDP09084.1), and Vipp2 (Cre11.g468050.t1.1).
19 of 22
Supplemental Data Set 6. Global Proteomic Analysis.
Supplemental Data Set 7. Proteomic Analysis during ClpP1 Depletion
in DCH16.
Supplemental Data Set 8. Proteomic Analysis of A31 upon Vitamin
Addition.
ACKNOWLEDGMENTS
We thank Emine Dinc for her valuable help during the preparation of the
RNA samples and generation of the reporter strains; Mylene Docquier,
the Genomic Platform team of the University of Geneva, and Sabeeha
Merchant for performing the RNA-Seq analysis; Martin Croft and Alison
Smith (University of Cambridge, UK) for the plasmid containing the
Thi4riboswitch fused to the upstream region of METE; Teresa Fitzpatrick
for the ThiC antibody; Olivier Vallon for the ClpP1 antibody and the
ClpP1-AUU strain; Nicolas Roggli for drawings and photography; and
Michel Goldschmidt-Clermont and Geoffrey Fucile for critical reading of
the article. This work was supported by Grant 31003A_133089/1 from
the Swiss National Foundation to J.D.R. and National Institutes of Health
Grant R24GM092473 to S. Merchant for RNA sequencing.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Homoplasmicity of the DCH16 Strain.
Supplemental Figure 2. Comparative Chloroplast RNA Analysis in
A31 (Wild-Type) and DCH16 upon ClpP1 Depletion.
Supplemental Figure 3. Stress-Induced Responses.
Supplemental Figure 4. Quantitative RT-PCR of Selected Nuclear
Transcripts.
Supplemental Figure 5. Venn Diagram for Proteins Differentially
Expressed in DCH16 upon Vitamin Treatment.
Supplemental Figure 6. Comparison of Protein and RNA Accumulation in DCH16 after 48 h of Vitamin Treatment.
Supplemental Figure 7. Immunoblot Analysis of CS191, CS174, and
DCH16.
Supplemental Figure 8. Quantitative RT-PCR of Deg11, Hsp22B,
Hsp22F, and Vipp2 in A31.
Supplemental Figure 9. Comparison of Changes in Gene Expression
between Cells Shifted from Low Light/Dark to Higher Light in the
Presence or Absence of Rapamycin.
Supplemental Table 1. List of Primers.
Supplemental Methods.
Supplemental References.
Supplemental Data Set 1. List of Genes with Significant Changes in
RNA Levels in A31 after 12, 31, 43, and 48 h of Vitamin Treatment.
Supplemental Data Set 2. List of Genes with Significant Changes in
RNA Levels in DCH16 after 12, 31, 43, and 48 h of Vitamin Treatment.
Supplemental Data Set 3. List of Genes with Significant Changes in
RNA Levels in A31 after 2 and 8 h of Rapamycin Treatment.
Supplemental Data Set 4. Transcriptomic Analysis upon ClpP1
Depletion and Rapamycin Treatment.
Supplemental Data Set 5. Clustering of 5085 Genes According to
Their Expression Patterns after Vitamin Depletion in DCH16 (0, 12, 31,
43, and 48 h).
AUTHOR CONTRIBUTIONS
S.R. designed and performed the research, analyzed the data, compiled
the Methods section, and contributed to the drafting of the article. D.C.,
M.P., and N.C. performed the bioinformatics analysis of the RNAseq
data. O.S. performed the statistical analysis of the Nanostring nCounter
expression data. M.R. performed the cloning of the reporter constructs.
T.M., D.H., F.S., and M.S. performed the proteomics analysis. M.C.,
U.G., and J.R. performed the electron microscopy. M.E.P.-P. and J.L.C.
performed the immunofluorescence analysis. J.D.R. designed the research, analyzed the data, and wrote the article.
Received March 2, 2014; revised April 16, 2014; accepted May 9, 2014;
published May 30, 2014.
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Involved in Autophagy and Plastid Protein Quality Control
Silvia Ramundo, David Casero, Timo Mühlhaus, Dorothea Hemme, Frederik Sommer, Michèle
Crèvecoeur, Michèle Rahire, Michael Schroda, Jannette Rusch, Ursula Goodenough, Matteo Pellegrini,
Maria Esther Perez-Perez, José Luis Crespo, Olivier Schaad, Natacha Civic and Jean David Rochaix
Plant Cell; originally published online May 30, 2014;
DOI 10.1105/tpc.114.124842
This information is current as of June 14, 2017
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