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The Clp protease system is required for copper ion-dependent
turnover of the PAA2/HMA8 copper transporter in chloroplasts
Author for correspondence:
Marinus Pilon
Tel: +1 970 491 0803
Email: [email protected]
Wiebke Tapken1, Jitae Kim2, Kenji Nishimura2, Klaas J. van Wijk2 and
Marinus Pilon1
Received: 8 August 2014
Accepted: 9 September 2014
Ithaca, NY 14853, USA
1
Biology Department, Colorado State University, Fort Collins, CO 80523, USA; 2Department of Plant Biology, Cornell University,
Summary
New Phytologist (2015) 205: 511–517
doi: 10.1111/nph.13093
Key words: caseinolytic protease (Clp),
chloroplast, copper (Cu) homeostasis, copper
transport, P1B-type ATPase, proteolysis,
regulated protein turnover.
The distribution of essential metal ions over subcellular compartments for use as cofactors
requires control of membrane transporters. PAA2/HMA8 is a copper-transporting P1B-type
ATPase in the thylakoid membrane, required for the maturation of plastocyanin. When copper is
highly available to the plant this transporter is degraded, which implies the action of a protease.
In order to identify the proteolytic machinery responsible for PAA2/HMA8 turnover in
Arabidopsis, mutant lines defective in five different chloroplast protease systems were analyzed.
Plants defective in the chloroplast caseinolytic protease (Clp) system were specifically impaired
in PAA2/HMA8 protein turnover on media containing elevated copper concentrations.
However, the abundance of a core Clp component was not directly affected by copper.
Furthermore, the expression and activity of both cytosolic and chloroplast-localized superoxide
dismutases (SODs), which are known to be dependent on copper, were not altered in the clp
mutants, indicating that the loss of PAA2/HMA8 turnover in these lines was not caused by a lack
of stromal copper.
The results suggest that copper excess in the stroma triggers selection of the thylakoidlocalized PAA2 transporter for degradation by the Clp protease, but not several other chloroplast
proteases, and support a novel role for this proteolytic system in cellular copper homeostasis.
Introduction
The correct allocation of essential trace elements over subcellular
compartments that are separated by membranes presents an
important challenge for all living organisms. Copper (Cu) is
utilized as an enzyme cofactor by the vast majority of aerobic
organisms (Ridge et al., 2008). An abundant cuproprotein in green
cells of flowering plants and many algae is plastocyanin (PC), which
is localized in the thylakoid lumen of chloroplasts, where it
transports electrons between the cytochrome b6f complex and
photosystem I Ӏ (Weigel et al., 2003). The Arabidopsis thaliana
genome encodes for two PC isoforms, PC1 and PC2, which have
apparent redundant function in photosynthesis (Weigel et al.,
2003; Pesaresi et al., 2009). Studies in Arabidopsis and poplar have
indicated that Cu is preferentially allocated to PC when Cu supply
is low, which was hypothesized to allow the plant to remain
photosynthetically active (Abdel-Ghany & Pilon, 2008; Ravet
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et al., 2011). Prioritization is achieved through the expression of the
so-called Cu-microRNAs (Burkhead et al., 2009), which direct the
concerted down-regulation of transcripts that encode for seemingly
nonessential cuproproteins. Among the Cu-microRNA targets are
cytosolic and chloroplastic isoforms of copper/zinc superoxide
dismutase (CSD), CSD1 and CSD2, respectively (Sunkar et al.,
2006; Yamasaki et al., 2007). The expression of Cu-miRNAs is
up-regulated when Cu availability is low and depends on the
Cu-responsive transcription factor SPL7 (SQUAMOSA promoter
binding protein-like7). At the same time, SPL7 promotes the
expression of cell surface localized transporters and reductases,
which function to increase cellular Cu intake (Yamasaki et al.,
2009; Bernal et al., 2012).
PC is transported as an apoprotein into the thylakoid lumen by
the Sec-pathway, which translocates proteins in an unfolded state
(Cline et al., 1993). Thus, Cu has to be transported into the
chloroplast stroma and further into the thylakoid lumen for PC
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maturation to occur. In Arabidopsis, the two P1B-type ATPases
PAA1/HMA6 and PAA2/HMA8 (for P-type ATPase of
Arabidopsis 1 and 2 or Heavy Metal ATPase 6 and 8; hereafter
referred to as PAA1 and PAA2) fulfill this Cu-transport function
(Shikanai et al., 2003; Abdel-Ghany et al., 2005; Catty et al.,
2011). PAA1 and PAA2 are localized to the inner chloroplast
envelope and the thylakoid membrane respectively (Abdel-Ghany
et al., 2005). PAA2 is involved in the homeostatic regulation of Cu
allocation in the chloroplast as its abundance is c. 65% lower in
elevated Cu compared to mild deficiency (Tapken et al., 2012).
The down-regulation of PAA2 by Cu was demonstrated to be
mediated by protein turnover and independent of SPL7 (Tapken
et al., 2012). A number of protease systems have been identified in
the plastids, many of which are likely derived from the prokaryotic
ancestor of the chloroplast (for a comprehensive review see: Kato &
Sakamoto, 2010). Mutations in protease subunits often lead to
severe defects in chloroplast development and pleiotropic phenotypes, indicating that plastid proteases are crucial for the homeostatic control of organellar proteins (Olinares et al., 2011a).
However, despite their pivotal physiological functions, very few
specific substrates are known for the various protease systems in the
chloroplast. Exceptions are the processing peptidases which remove
targeting signals from precursors during protein translocation
(Teixeira & Glaser, 2013). The turnover of PAA2 constitutes a
unique example of regulated protein turnover at the thylakoid
membrane, because it occurs in response to an environmental
trigger and impacts Cu homeostasis. We therefore aimed to identify
the organellar protease(s) that mediates this process.
Materials and Methods
Plant materials and growth conditions
Arabidopsis Col-0 was used as a wild-type control and all mutant
lines have been described previously: clpc1-1 (SALK_014058,
Sj€ogren et al., 2004), clpc2-2 (hsp93-III-1, SAIL_622_B05,
Kovacheva et al., 2007), clpd (SAIL_77_G05, reviewed in Olinares
et al., 2011a), clpr2-1 (SALK_046378, Rudella et al., 2006; Kim
et al., 2009), clps1 (SAIL_326B_G12, Nishimura et al., 2013),
sppA-1 (GABI-Kat 142B03, Wetzel et al., 2009), var2-5/ftsH2-5
(Sakamoto et al., 2004), prep1xprep2 (Nilsson Cederholm et al.,
2009), deg2, deg5, deg7, deg8 (Sun et al., 2010), pc2 (Weigel et al.,
2003), paa1-3 (Shikanai et al., 2003) and paa2-1 (Abdel-Ghany
et al., 2005). The clpr2-1/R2:His line used for complementation
has been described (Rudella et al., 2006). For in vitro growth,
Arabidopsis seeds were surface sterilized according to Tapken et al.
(2012). Plants were grown on half-strength Murashige and Skoog
(MS) medium (Murashige & Skoog, 1962; Caisson Laboratories,
North Logan, UT, USA) solidified with 0.6% PhytagelTM (SigmaAldrich) and supplemented with 1% sucrose, at pH 5.8. Plants were
grown in a temperature-controlled growth chamber at a photon
density of 120 lmol m 2 s 1, in a 12 h:12 h, light:dark cycle at
23°C. Copper (Cu) was added as copper sulfate (CuSO4) and
concentrations in the media are indicated for each experiment.
Cycloheximide treatments were performed in liquid culture as
described (Tapken et al., 2012).
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Protein extraction, immunoblot analysis, enzyme activity
assay, elemental analysis and statistics
Proteins for denaturing sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS-PAGE) analysis were extracted as described
(Tapken et al., 2012). After fractionation by SDS-PAGE, proteins
were electroblotted onto a nitrocellulose membrane (TransBlot®Pure Nitrocellulose, 0.2 lm; Bio-Rad, Hercules, CA, USA).
The antisera for cFBPase and CSD2 were obtained from Agrisera
(V€ann€as, Sweden). Affinity purified antibodies for PAA1, PAA2
(Tapken et al., 2012) and ClpR2 (Asakura et al., 2012) have been
described. For the enzymatic activity of SODs, total leaf protein
was extracted under nondenaturing conditions and in-gel activity
staining was performed as described (Beauchamp & Fridovich,
1971; Abdel-Ghany et al., 2005). Elemental analysis of leaf
samples was conducted after digestion with concentrated nitric
acid of washed and dried samples, essentially as described (Ravet
et al., 2011). All assays have been performed at a minimum for
three biological replicates. Representative results for immunoblots
are shown. The software ImageJ (NIH, Bethesda, MD, USA) was
used on digitized immunoblots to quantify protein abundance
(Tapken et al., 2012). Statistical analysis was performed by
Student’s t-test using the software JMP (version 9.0.2; SAS
Institute, Cary, NC, USA). Significant differences are reported
where appropriate and represent values of P < 0.05.
Results
We tested 11 Arabidopsis mutant lines with defects in five different
chloroplast protease systems for their PAA2 abundance: stromal
and thylakoid lumen Deg (deg2,5,7,8), stromal caseinolytic
protease (Clp) (clpc1-1, clpc2-2, clpd, clpr2-1), thylakoid SppA
(sppA-1), stromal Prep (prep1xprep2) and thylakoid FtsH (var2-5).
The presence of sucrose in the in vitro media had the advantage that
the most severe phenotypes of these protease mutants were
suppressed. In addition, Cu levels were easily controlled. PAA2
protein abundance responded to Cu availability and was comparable to the wild-type in all lines except for clpc1-1 and clpr2-1
(Fig. 1a). When compared to the Col-0 wild-type, PAA2 protein
abundance was increased over four-fold in the presence of elevated
Cu in clpc1-1 and clpr2-1 and Cu addition no longer significantly
affected PAA2 (Fig. 1b). ClpC1 is a AAA+/HSP93 chaperone and
ClpR2 is a subunit of the Clp core complex of the stromal localized
Clp system (Olinares et al., 2011a). We tested if the regulatory
mechanism can be restored in a complemented line of the clpr2-1
allele (Fig. 1c). Indeed, PAA2 abundance was again reduced and the
effect of Cu was restored, indicating that ClpR2 is required for
PAA2 regulation by Cu (Fig. 1c).
PAA2 is most similar in sequence to PAA1, which is located in
the inner envelope and thus at least partially exposed to the stroma.
Therefore we tested if PAA1 is also a Clp substrate (Fig. 2a). In
clpc1-1 and clpr2-1, PAA1 protein abundance was comparable to
the wild-type (Fig. 2a). Copper (Cu) addition had no effect on
PAA1 protein abundance in either wild-type or the two mutants.
These observations indicate that Cu-mediated regulation by Clp is
specific to PAA2. Protein accumulation is the result of the balance
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Fig. 1 PAA2 protein accumulates in Arabidopsis thaliana plants that are deficient in subunits of the chloroplastic caseinolytic protease (Clp) system. (a)
Immunoblots of total leaf protein in wild-type (Col-0) and multiple mutants. Equal loading was ensured by probing for the constitutively expressed protein
cytosolic-1,6-bisphosphatase (cFBPase). (b) Quantification of PAA2 protein abundance in Col-0, clpc1-1 and clpr2-1. The signal intensity for Col-0 grown in
the presence of 0.05 lM copper sulfate (CuSO4) was set to 100%. Values are means standard deviation (SD) with n = 3 and two technical replicates. Small
letters indicate significant differences as determined by analysis of variance (ANOVA), followed by Tukey–Kramer HSD (honest significant difference) post hoc
analysis. (c) PAA2 protein abundance in complemented clpr2-1 lines. Immunoblot of total leaf extract. CSD2 antibody was used as a control of the copper (Cu)status of these plants. In all experiments, ( ) and (+) indicate 0.05 and 5 lM total CuSO4 in the growth media, respectively. All plants were 18 d old at time of
analysis.
of synthesis and degradation. Therefore, increased PAA2 abundance in Clp mutants might be explained either by decreased PAA2
turnover or by a more indirect effect on translation of either PAA2
itself or of a stabilizing factor. To analyze if the Clp system is
directly required for PAA2 turnover, we interrupted protein
synthesis in 10 d-old seedlings through a 24 h treatment with
100 lM cycloheximide (CHX) in the presence or absence of
additional Cu (Tapken et al., 2012). The clpr2-1 allele could not
produce enough biomass in this condition and therefore we limited
the analysis to a comparison of clpc1-1 to the wild-type. PAA2
protein accumulated to high levels and was not significantly
reduced by CHX treatment in clpc1-1, independent of the presence
of Cu. By contrast, CHX strongly affected PAA2 abundance in the
wild-type, which is especially evident when 5 lM CuSO4 was
present (Fig. 2b). Quantification of three biological repeats showed
that the PAA2 protein levels were 5.4-times higher in the clpc1-1
mutant when compared to the wild-type in the presence of Cu and
CHX (P < 0.05). Together the results presented in Figs 1 and 2
indicate that the Clp mediates Cu-dependent PAA2 turnover and
that Clp does not act indirectly by affecting protein synthesis. We
considered that Cu availability in the chloroplast could affect the
abundance of the Clp system; however immunoblot analysis of the
Clp core component ClpR2 indicated that this was not the case (see
Supporting Information Fig. S1).
The decreased PAA2 turnover in clp mutants might also be
explained by an effect on Cu homeostasis if leaf tissue or chloroplast
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Cu levels were lower in clpc1-1 and clpr2-1. We therefore compared
tissue Cu levels for wild-type plants and the two clp mutants grown
at low or high Cu concentrations. As expected, Cu feeding increases
its content in the leaves, but there is no difference between the wildtype and mutant lines (Fig. 3a). Similarly, no significant differences
were observed between the lines for the leaf content of other
elements tested (Supporting Information Fig. S2). The abundance
and activity of chloroplastic CSD2 is strongly affected by Cu
availability in the chloroplast (Shikanai et al., 2003; Abdel-Ghany
et al., 2005) and can therefore be used as a reliable indicator for its
Cu status. If there is less Cu in chloroplasts of clpc1-1 and clpr2-1,
then we would expect reduced CSD2 abundance and activity.
Clearly, CSD2 is not affected in clpc1-1 and clpr2-1. By contrast, its
activity is even slightly elevated in the two clp mutants grown in low
Cu conditions (Fig. 3b). Together the observations in Fig. 3
strongly suggest that the effect of the clpc1-1 and clpr2-1 mutations
cannot be explained by a lack of Cu in the chloroplasts.
Discussion
The turnover of PAA2 can be observed for plants grown in vitro in
the presence of sucrose. These growth conditions further allowed us
to reveal that the Cu-induced turnover of PAA2 is dependent on the
Clp system. ClpC1 and ClpR2 are both nuclear encoded proteins
and subunits of Clp, which functions within the chloroplast
stroma. The Clp system is of prokaryotic origin and composed of a
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Fig. 2 Specificity of the caseinolytic protease (Clp) system in mediating PAA2
turnover in Arabidopsis thaliana. (a) Loss of Clp function affects PAA2, but
not PAA1. Immunoblot of total leaf protein of 18 d-old plants. ( ) and (+)
indicate 0.05 and 5 lM copper sulfate (CuSO4) in the growth media,
respectively. (b) Clp mutants are defective in copper (Cu)-dependent PAA2
turnover. Seedlings were grown in liquid Murashige and Skoog (MS)
medium for 10 d in either the presence of 0.05 lM ( Cu) or 5 lM CuSO4
(+Cu). After 10 d, half of the plants were treated with 100 lM cycloheximide
(CHX) and then collected for immunoblot analysis 24 h later. Ten microgram
total protein was analyzed in immunoblots using PAA2, PAA1 or cFBPase
(loading control) antibodies as indicated. paa1-1 and paa2-1 were used as
controls where appropriate.
barrel-shaped proteolytic core with associated chaperones
(Olinares et al., 2011a). The core is comprised of five different
proteolytic ClpP, and four different nonproteolytic ClpR subunits,
which together organize into two asymmetric heptameric rings.
The resulting core structure harbors the proteolytically active sites
occluded within its center (Olinares et al., 2011b). The opening of
the Clp core is thought to be too narrow for large folded proteins to
enter and thus substrates need to be initially recognized for
degradation, unfolded and then actively threaded through the axial
pores (Adam et al., 2006; Clarke, 2012). In plants this is thought to
be dependent on the three putative ATP-dependent AAA+/
HSP93-type Clp chaperones ClpC1, ClpC2 and perhaps ClpD
(Sj€ogren et al., 2014). The strongest change in PAA2 abundance
was observed in the clpr2-1 mutant allele. It carries a T-DNA
insertion 7 base pairs upstream of the translational start codon,
which results in a 50% reduction of ClpR2 mRNA levels (Rudella
et al., 2006). The clpc1-1 allele has a T-DNA insertion in its fourth
exon and is a knock-out line (Sj€ogren et al., 2004). Nonetheless,
clpc1-1 is viable, probably because of functional redundancy of
ClpC1 with ClpC2 and perhaps ClpD, which might also explain
the lack of a noticeable effect on PAA2 turnover in the clpc2-2 and
clpd mutants.
Strong growth and developmental phenotypes hamper efforts to
pair chloroplast proteolytic systems with specific physiological
substrates by means of reverse genetics. Thus far only a very limited
set of proteins has been identified as potential substrates for the Clp
system in plants, and none within the thylakoids (Stanne et al.,
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Fig. 3 Loss of function in caseinolytic protease (Clp) subunits in Arabidopsis
thaliana does not lower copper (Cu) levels or the activity of the Cudependent plastid protein CSD2. (a) Copper (Cu) concentration in shoots of
18 d-old plants grown in the presence of 0.05 lM ( ) and 5 lM (+) copper
sulfate (CuSO4) as measured by inductively coupled plasma mass
spectrometry (ICP-MS). Values are means standard deviation (SD), n = 3.
Small letters indicate significant differences as determined by analysis of
variance (ANOVA), followed by Tukey–Kramer HSD post hoc analysis. (b,
upper) Superoxide dismutase (SOD) activity gel with 40 lg total leaf protein
from the indicated plant lines. SOD activity is visible as bright bands in a dark
background. The different isoforms can be identified by their relative
mobility: MnSOD, constitutive manganese superoxide dismutase; FeSOD
iron superoxide dismutase; CSD copper/zinc superoxide dismutase;
*indicates an unidentified band (b, lower). Immunoblot using CSD2 and
cFBPase (loading control) antibodies.
2009; Kim et al., 2013; Nishimura et al., 2013). In Chlamydomonas
reinhardtti, Clp has been implicated in the specific degradation of
the thylakoid-located cytochrome b6f complex under nitrogen (N)starvation or when biogenesis of the Rieske subunit is blocked
(Majeran et al., 2000). However, in plants which lack core Clp
subunits, protein abundance of all photosynthetic thylakoid
complexes are reduced by half, without a specific effect on the
cytochrome b6f complex (Rudella et al., 2006; Zybailov et al.,
2009; Kim et al., 2013). A previously identified candidate for a
ClpC1 target in plants is the thylakoid membrane-bound chlorophyllide a oxygenase, which converts chlorophyll a to chlorophyll b
and accumulates in a clpc1 (Clp chaperone) mutant (Nakagawara
et al., 2007). However, Clp chaperones may have additional
biological roles as these proteins can also function as part of the
chloroplast protein import machinery (see: Jarvis, 2008). Indeed
chlorophyllide a oxygenase, was not reported to over-accumulate in
clpr2-1 (Clp core) in which the ratio of chlorophyll a and b was also
similar to the wild-type (Rudella et al., 2006). Protease mutants
have furthermore the tendency to increase general chaperone
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activity (CPN60/20, HSP70, HSP90, ClpB3) within the plastids
to cope with increasing amounts of incomplete and mal-formed
proteins (see Olinares et al., 2011a). In this context, a number of
observations give confidence that PAA2 over-accumulation in clp
mutants is a reflection of the direct involvement of the Clp system.
Out of the 11 tested protease mutant lines only two, clpc1-1 and
clpr2-1, showed a significant increase of PAA2 protein accumulation, which indicates that only the Clp system is specifically
required for PAA2 turnover. Further, the Cu-dependent regulation
is restored through complementation of clpr2-1. Importantly,
clpc1-1 is deficient in a chaperone while clpr2-1 is deficient in the
Clp core, yet both affect PAA2. While most homozygous ClpP and
ClpR mutants are seedling lethal or unable to set seed, both clpc1-1
and clpr2-1 are relatively mild alleles, capable of completing a full
lifecycle and exhibiting a viridescent phenotype and reduced
growth on soil or MS medium. Furthermore, Cu levels used in this
study had no additional effect on phenotypes of any of the Clp
mutants tested here (Supporting Information Fig. S3). This
reduces the likelihood for indirect pleiotrophic phenotypes. In
addition, the thylakoid photosynthetic machinery is, on a whole
cell bases, decreased in abundance to c. 50% in various clp mutants
including clpr2-1; however the thylakoid protein PAA2 is fourtimes more (not less) abundant in this mutant compared to the
wild-type. Finally, PAA2 accumulation in the wild-type responds
to a defined physiological trigger, low Cu availability, and PAA2
turnover is induced by elevated Cu levels that are well below
toxicity. At the same time PAA2 transcript levels do not respond to
Cu (Tapken et al., 2012). Indeed, PAA2 accumulation on low Cu
in the clp mutants is not a result of transcript up-regulation or
protein synthesis. It is rather the result of reduced turnover
(Fig. 2b); at the same time loss of function of the two Clp subunits
does not affect indicators of the chloroplasts Cu status (Fig. 3b).
How chloroplast ClpC chaperones recognize their substrates is
still a largely open question. In Escherichia coli (E. coli), ClpS is
involved in substrate selection and delivery to the bacterial ClpAP;
in particular ClpS recognizes proteins with an N-terminal
degradation signal (N-degron), as well as aggregated proteins
(Sauer & Baker, 2011). Through phylogenetic and in silico
analyses, it was shown that angiosperms contain a homolog, named
ClpS1 which interacts with ClpC1,2 chaperones (Nishimura et al.,
2013). The Arabidopsis ClpS1 null mutant (clps1) lacks a visible
growth phenotype, similar to the E. coli clpS null mutant. Affinity
purification identified eight candidate ClpS1 substrates and
interaction with five substrates strictly depended on two conserved
ClpS1 residues involved in N-degron recognition (Nishimura
et al., 2013). Using the clps1 null mutant, it was clear however that
in vivo PAA2 degradation did not depend on ClpS1 (Supporting
Information Fig. S4). The mechanism of Clp-mediated PAA2
turnover could involve either an effect of Cu on the protease itself or
binding of Cu to the substrate, increasing its availability through an
unknown mechanism. PAA2 turnover is reduced when Cu levels
are lower. Given the known biochemical properties of Clp, a serine
protease, it is difficult to envision a mechanism by which Cu could
have an activating (in Cu excess) or inhibitory (on low Cu medium)
effect. We also observed that Cu does not affect ClpR2 protein
abundance (Supporting Information Fig. S1) and therefore the
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assembly of the Clp system is most likely not affected by Cu
availability. We mined publicly available RNAseq data (Bernal
et al., 2012) and found that neither ClpC1, ClpR2, nor any other
chloroplast protease subunit, is regulated at the transcript level in
response to Cu or via SPL7. This agrees with the notion that Clp is
not regulated by Cu. We therefore consider it likely that a Cudependent conformational change of PAA2 is the trigger for
substrate recognition by Clp. In the absence of sufficient amounts
of apo-PC, when Cu cannot be donated efficiently to the thylakoid
lumen or with relatively high Cu in the stoma, PAA2 would be
more likely in a Cu-bound state. Conformational changes in P1Btype ATPases can be inferred based on biochemical and biophysical
analyses of other ion pumping P-type ATPases, such as the Na, KATPase and Ca2+ATPase (Jorgensen et al., 2003). Indeed, the Cutransporting P1B-type ATPase CopA of Archaeoglobus fulgidus is
postulated to follow the E1/E2 Albers-Post model for transport
(Arg€
uello et al., 2007). Other conformational changes that are not
primarily associated with Cu transport can also be envisioned for
Cu-transporting P-type ATPases (Rosenzweig & Arg€
uello, 2012).
By analyzing fresh weight and the fluorescence parameter Fv/Fm,
which estimates photosystem II capacity and is a general indicator
for stress within the chloroplast (Maxwell & Johnson, 2000), we
did not observe significant Cu-induced phenotypes in clpc1-1 even
in the presence of up to 30 lM CuSO4 whereas only small effects of
Cu were seen for clpr2-1 (Supporting Information Fig. S5). Most
likely any effects of Cu are masked by other phenotypes in these clp
mutants. Nevertheless, we suggest that Clp-mediated turnover of
PAA2 is physiologically relevant for Cu homeostasis. In a
developing green leaf, the thylakoid lumen-localized PC is the
primary recipient of Cu. However, free Cu is potentially cytotoxic
in its ionic form and plants need an efficient system to avoid
overloading of the thylakoids to prevent damage to the photosynthetic apparatus (Yruela et al., 1996; K€
upper et al., 2003). Therefore, the chloroplast needs a reliable mechanism to decrease ion
flow to this compartment when Cu levels increase. Conformational
changes in PAA2 in a Cu-dependent manner could be a simple and
direct gauge for the Cu status of the thylakoids. Clp-mediated
PAA2 protein turnover will result in relatively increased Cu
availability for the maturation of other Cu enzymes such as CSDs,
which are transcriptionally up-regulated when Cu levels rise
(Yamasaki et al., 2007). In this way, chloroplast Cu homeostasis is
integrated with cellular and whole-plant regulation of Cu uptake
and use as mediated by the SPL7 master regulator.
Acknowledgements
This project was supported by the Agriculture and Food Research
Initiative competitive grant no. 2012-67013-19416 of the USDA
National Institute of Food and Agriculture and by the US National
Science Foundation grant no. MCB 1244142 to M.P. and MCB1021963 to K.J.V.W.
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Supporting Information
Rapid report
Research 517
Fig. S3 Phenotypes of wild-type and Clp mutants grown on 0.05
and 5 lM copper sulfate (CuSO4).
Fig. S4 ClpS1 is dispensable for PAA2 degradation in response to
Cu.
Additional supporting information may be found in the online
version of this article.
Fig. S5 Analysis of the effects of Cu on phenotypes of Clp mutants
grown in Murashige and Skoog (MS) medium.
Fig. S1 Chloroplast copper (Cu) availability does not affect ClpR2
abundance.
Please note: Wiley Blackwell are not responsible for the content or
functionality of any supporting information supplied by the
authors. Any queries (other than missing material) should be
directed to the New Phytologist Central Office.
Fig. S2 Elemental analysis of iron (Fe), sulfur (S) and zinc (Zn)
contents in wild-type and caseinolytic protease (Clp) mutants.
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