{'fraction_non_alphanumeric': 0.04020858403529884, 'fraction_numerical': 0.032170076213397514, 'mean_word_length': 4.959648116274622, 'pattern_counts': {'":': 0, '<': 3, '<?xml version=': 0, '>': 2, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Modified Clp protease complex in the ClpP3 null mutant and consequences for chloroplast development and function in Arabidopsis thaliana

The plastid ClpPRT protease consists of two heptameric rings of ClpP1/R1/2/3/4 (the R-ring) and ClpP3/P4/P5/P6 (the P-ring) in different molar ratios and peripherally-associated ClpT1/2 subunits . Here we address the contributions of ClpP3 and ClpP4 to ClpPRT core organization and function . ClpP4 is strictly required for embryogenesis, similar to ClpP5. In contrast, loss of ClpP3 ( clpp3-1 ) leads to arrest at the hypocotyl stage; this developmental arrest can be removed by supplementation with sucrose or glucose. Heterotrophically grown clpp3-1 can be transferred to soil and generate viable seed, which is surprising since we previously showed that CLPR2 and CLPR4 null alleles are always sterile and die on soil. Based on native gels and mass spectrometry-based quantification, we show that despite the loss of ClpP3, modified ClpPR core(s) could be formed albeit at strongly reduced levels. A large portion of ClpPR subunits accumulated in heptameric rings, with overaccumulation of ClpP1/P5/P6 and ClpR3. Remarkably, ClpT1 association to the modified Clp core was unchanged. Large scale quantitative proteomics of clpp3-1 showed a 50% loss of photosynthetic capacity, upregulation of plastoglobules and all chloroplast stromal chaperone systems. Specific chloroplast proteases were significantly up-regulated whereas the major thylakoid protease (FtsH1/2/5/8) was clearly unchanged, indicating a controlled protease network response. clpp3-1 showed a systematic decrease of chloroplast-encoded proteins that are part of the photosynthetic apparatus, but not of chloroplast-encoded proteins with other functions. Candidate substrates and an explanation for the differential phenotype between the CLPP3, CLPP4 and CLPP5 null mutants are discussed. SCO2 protein disulphide isomerase is required for thylakoid biogenesis and interacts with LHCB1 chlorophyll a/b binding proteins which affects chlorophyll biosynthesis


INTRODUCTION
Intracellular proteolysis is essential for proteome homeostasis, regulation of metabolic and signaling pathways and ultimately, the maintenance of organellar and cellular viability. Chloroplasts contain multiple soluble and membrane-bound proteases and processing peptidases (Kato and Sakamoto, 2010;Olinares et al., 2011), that operate in parallel as well as in series, presumably with partially overlapping substrates. The Clp protease system is the most abundant and complex stromal protease family in the plastid. It consists of five serine-type Clp proteases (P1, P3-P6) and four non-proteolytic ClpR subunits Under autotrophic conditions on agar plates, homozygous clpp3-1 seedlings were identified, but their development was arrested at the cotyledon stage and seedlings died after several weeks ( Fig. 2A) similar to null mutants in CLPR2 and CLPR4 (Kim et al., 2009). We do note that the hypocotyls of clpp3-1 seedlings were clearly greener than clpr2-2 and clpr4-1 null alleles ( Fig. 2A). Under heterotrophic conditions on agar plates supplemented with 1-2% sucrose, homozygous clpp3-1 seedlings, developed beyond the cotyledon stage, even if growth and development was slow and with pale-green serrated leaves (Fig. 2B), similar to previously observed for CLPR2 and CLPR4 null alleles. The clpp3-1 allele did neither accumulate CLPP3 mRNA nor ClpP3 protein (Fig. 2C). clpp3-1 plants grown on agar were greener on 2% sucrose than on 1% sucrose, and the youngest leaves were paler than older leaves (Fig.  2D). The chlorophyll and carotenoid concentrations in clpp3-1 on a fresh weight basis of 7 week old seedlings grown on agar medium supplemented with 2% sucrose were reduced by respectively 77% and 65% compared to wt (Supplemental Table S1). The total chlorophyll : total carotenoid ratio in clpp3-1 was reduced by 33%.
In many plant studies, glucose is considered primarily a signaling molecule, whereas sucrose is primarily considered a source of energy (and used for long distance transport) (Hanson and Smeekens, 2009;Eveland and Jackson, 2012). We therefore compared the effect of sucrose (1-3-5%) and glucose (1-3-5%) on seedling development in clpp3-1. For comparison, we also included the leaky clpr2-1 allele which does not show seedling arrest but has strongly delayed development; this is the Clp core mutant with the strongest phenotype that can still produce seeds (Rudella et al., 2006). Both sucrose (1-5%) and glucose (1-5%) broke clpp3-1 seedling arrest for up to 80%, with glucose being somewhat more effective (Fig. 2E). Both sugars strongly stimulated leaf development of clpp3-1 seedlings as measured by the number of leaves, with an optimum concentration of 3% for both sugars, but leaf formation was faster on sucrose (Fig. 2F). In case of wt and clpr2-1, both sugars repressed leaf formation at the higher concentrations (3 and 5%). Glucose and sucrose showed differential effects on leaf expansion of wt, clpp3-1 and clpr2-1, as measured by the rosette diameter (Fig. 2G). For all three genotypes, sucrose stimulated rosette diameter, with the largest diameter at 1% sucrose for wt (16 cm) and clpr2-1 (11 cm), but at 3% sucrose for clpp3-1 (10 cm). Rosette diameter was repressed at 3% and 5% sucrose for wt and clpr2-1. In contrast, glucose showed a concentration-dependent repression of rosette diameter for wt and clpr2-1. In case of clpp3-1, 1% glucose facilitated seedling development by breaking developmental arrest, but higher concentrations repressed leaf growth and expansion, similar to wt and clpr2-Increased concentrations of glucose, but not sucrose, slightly increased chlorophyll and total carotenoid levels on a fresh weight basis in clpp3-1. Increase of either glucose or sucrose levels increased pigment levels on fresh weight basis in wt and clpr2-1, presumably by reduced cell expansion and unaffected pigment accumulation (Supplemental Fig. S1).
The clpp3-1 null allele can flower and produce viable seeds, unlike the other seedling lethal ClpPR core mutants After several weeks on sucrose, clpp3-1 seedlings started to green and accumulate chlorophyll, and surprisingly, they could be transferred to soil, eventually flowered and generated viable seeds (Fig. 3), unlike the CLPR2 and CLPR4 null alleles which died soon after transfer to soil (Kim et al., 2009). Chlorophyll and carotenoid levels of these clpp3-1 plants were about 50% lower than in wt plants (Supplemental Table S1). Thus CLPP3 is different from CLPR2, CLPR4, CLPP4 and CLPP5, indicating that CLPP3 is not strictly essential once chloroplasts and leaves have reached the mature stage. This suggested that other CLP-P/R subunits partially compensate for loss of ClpP3 (even if very poorly and most of ClpT1, but not ClpT2, showed a ~1 kD upward mass shift due to an unknown post-translational modification or modified processing (Fig. 5).
To determine the consequences of the loss of ClpP3 on the assembly state of the ClpPR complex and the association of ClpT, chloroplast soluble proteomes (stroma) of wt and clpp3-1, were extracted under non-denaturing conditions and proteins were separated by one-dimensional native gel electrophoresis. For comparison, we also included the clpr2-1 mutant in the analysis. In case of wt, ClpR2, ClpP4 and ClpP6 were mostly observed in the expected 350 kD tetradecameric ClpPRT complex (Fig.  6A). However, some signal (in particular for ClpR2) was also detected in a band around 200 kD, corresponding with single heptameric rings; this reflects destabilization of the tetradecamer at the heptamer interface, similar to our previous observations (Olinares et al., 2011) (Fig. 6). In the case of clpr2-1, the total signal for ClpR2 was reduced, which is consistent with the 5-fold reduced expression in clpr2-1 previously reported (Rudella et al., 2006). Similar to wt, ClpR2 in clpr2-1 was mostly found in the 350 kD complex with lower amounts accumulating in the 200 kD heptamers. In contrast, in clpp3-1 nearly all of ClpR2 and ClpP4 were found around 200 kD, with only small amounts accumulating between 350-400 kD. It also appears that this complex was about 50 kD larger than the 350 kD band observed in wt and clpr2-1. ClpP4, but not ClpR2, was found in two bands in the 200 kD region. For both clpr2-1 and clpp3-1, about 50% of ClpP6 accumulated in 350 kD complexes, with the remainder of ClpP6 found in two separate bands in the 200 kD region. In the case of ClpT1, essentially all protein was found in the 350 kD complex for wt, clpr2-1 and clpp3-1. A very small amount (<5%) of ClpT1 was also found in a single band around 200 kD in case of clpr2-1 and clpp3-1. The signals for ClpT2 in the 200-400 kD were very weak and therefore inconclusive, perhaps because the vast majority of ClpT2 accumulated as free monomers or dimers (not shown).
Together these results show that in wt, a single ClpPRT complex accumulates as expected, with some destabilization occurring resulting in accumulation of ~200 kD heptameric rings. Previously, we showed by affinity tagging and MS analysis that these heptameric rings contained either ClpP1, R1,2,3,4 or ClpP3,4,5,6 (Olinares et al., 2011). In case of clpp3-1, only ClpP6 and ClpT1, but not ClpP4 and ClpR2, accumulated at high levels in 350-400 kD Clp core complexes. In clpp3-1 and clpr2-1, ClpP4 and ClpP6, were found in two complexes in the 200 kD region. The nature of this heterogeneity in the 200 kD region is not clear.

Stoichiometry determination of Clp assemblies in clpp3-1
To further determine the composition and assembly state of the ClpPR complex in clpp3-1, and possibly determine if one or more Clp subunits compensate for ClpP3 in the assembled core, we implemented the QConCat approach on chloroplast stroma, but now without StrepII-affinity purification of the Clp assemblies (Fig. 7). This is more challenging because of the higher complexity of the samples, but it was not practically feasible to create such StrepII-tagged Clp lines in the clpp3-1 background. Chloroplast stromal samples from wt and clpp3-1 were separated by native gel electrophoresis and the region between 150 and 400 kD was cut into 4 gel slices, such that it would capture the intact ClpPRT cores (250-400 kD range) and the individual heptameric rings (150-250 kD) (Fig. 7). Proteins in each gel slice were digested with trypsin and the resulting peptide mixtures were spiked with the in-gel digested stable isotope-labeled Clp-QconCAT standard followed by LC-MS analysis using an LTQ-Orbitrap. The absolute amount of each Clp subunit was determined from the measured sample-to-standard peak area ratios of the representative peptides. The MS analysis was carried out in triplicate. This workflow is summarized in Fig. 7.
ClpP5, ClpP6, ClpR2 and ClpR4 could be quantified by two distinct peptides each, whereas the amounts of ClpP1, ClpR1 and ClpR3 could be measured by one peptide each (Supplemental Table S2). ClpP4 could not be quantified since the standard peptides could not be detected due to the relatively high sample complexity. However, we detected ClpP4 from large-scale proteomic analyses of total leaf extracts from clpp3-1 (see next section) and we determined accumulation levels and assembly state from immunoblotting (Figs. 5 and 6); this allowed us to consider ClpP4 as a candidate for compensation of the loss of ClpP3 (see Discussion). From the molar amounts of each Clp subunit per gel band, the relative distribution of the Clp subunits across various assembly states could be determined (Fig. 8). To simplify the calculations and outcomes, and because the stoichiometries between the Clp subunits were similar between the two gel slices within the 250-400 kD range (bands 1 and 2; the Clp core complexes) and also within the 150-250 kD range (bands 3 and 4; the heptameric rings) we pooled the data within each of these mass ranges (Supplemental Table S2). Figures 8A,B show the molar distribution of the Clp subunits across the Clp cores (panel A), and P-and R-rings (panel B) whereas Figure 8C shows the overall distribution.
In wt, 54% of the Clp subunits were detected in the Clp core complex with the remaining 46% accumulating in heptamers. In contrast, in clpp3-1 only 12% of ClpPR subunits assembled in the Clp cores, with the remaining 88% in Clp heptameric rings (150-250 kD). This indicates that Clp subunits in clpp3-1 could still associate into Clp rings, but Clp core assembly formation was inefficient, in agreement with the immunoblotting results (Fig. 6).
The asymmetric Clp core complex is composed of the P-ring and R-ring in  (Table I), which was identical to what we observed with the affinity purified Clp core (Olinares et al., 2011), except that we could not quantify ClpP4. Moreover, the ClpPR stoichiometries in the mixture of individual heptameric rings (between 150-250 kD) in wt was similar in the core (Table I). In contrast, the ClpPR stoichiometries in the Clp core of clpp3-1 were altered with increased numbers of ClpP5, ClpP1 and ClpR3 (and complete loss of ClpP3), resulting in an approximate ratio of [0:4:1]:[5:1:1:2:1] for [P3:P5:P6]:[P1:R1:R2:R3:R4], which suggest mixing of the subunits of the R-and P-rings. Similarly, also in the mixture of individual heptameric rings (between 150-250 kD) the ratio was changed, now with even stronger overrepresentation of ClpP5 (Table I). Summation of the data for the core and the rings resulted in the expected stoichiometry for the wt, but increased copy numbers for ClpP5, ClpP1, ClpR3 and to a lesser degree ClpP6 for clpp3-1 ( Fig. 8C and Table I). Absolute amounts of ClpR1, ClpR2 and ClpR4 (normalized to total stromal proteins) were unchanged in clpp3-1 as compared to wt, whereas there was 2-2.5 fold more ClpP1,ClpP5, ClpP6, and ClpR3 (Fig. 8C).

Phenotypic analysis of clpp3-1 by comparative quantitative proteomics
To gain insight in the consequences of the loss of CLPP3, we compared the total denatured leaf proteomes of clpp3-1 and wt rosettes (Fig. 9). Leaf rosettes were obtained from pale-green clpp3-1 plants (initially grown on heterotrophic conditions and then transferred to soil) and soil-grown wt plants (Fig. 9A), both with 20 leaves. Total leaf proteomes were extracted with SDS and each proteome was separated by SDS-PAGE and visualized by Coomassie staining (Fig. 9B). Each gel lane was excised in 20 slices, followed by ingel trypsin digestion and protein identification by nanoLC-MS/MS. Three biological replicates per genotype were analyzed resulting in 120 MS/MS runs and 799,375 acquired MS/MS spectra; proteins were identified and quantified MS data were searched and filtered (Supplemental Fig. S2 and Supplemental Table S3). This resulted in the identification of 2313 proteins, quantified as 1993 individual proteins and 123 protein groups. These groups contained closely related proteins which were identified with peptides most of which also matched to homologues -see (Friso et al., 2011). Based on our recent reference Arabidopsis chloroplast proteome (Huang et al., 2013), 918 proteins were chloroplast-localized, representing 63% of the protein mass in clpp3-1 and 69% in wt. This reduced chloroplast mass was due to a 50% reduction in lumenal mass and 26% in thylakoid protein mass (Supplemental Table S4). The average pair-wise correlation coefficients among the three biological replicates within the wt and clpp3-1 datasets were 0.990 and 0.983, respectively, indicating high reproducibility between the independent replicates for each genotype. Principle Component Analysis (PCA) also showed that the variation between genotypes was larger than between replicates within each genotype (Fig. 9C). Together this shows that the quantitative proteome data are of high quality with little noise, and that clpp3-1 has a measurable proteome phenotype.

Significance analysis for determining differentially expressed proteins
To determine which proteins/protein groups were differentially expressed in clpp3-1 relative to wt, two statistical tools namely QSpec (Choi et al., 2008) and GLEE (Poliakov et al., 2013) were employed. These tools were specifically developed for significance analysis of large-scale spectral counting experiments and both have their merits (see Supplemental text for discussion). Figure 9D compares the results of the significance analyses of the total leaf datasets using QSpec (Bayes Factor >10) and GLEE (p-value <0.01). 148 of the plastidlocalized proteins and 64 extra-plastidic proteins passed both statistical tests (Supplemental Table S3). These extra-plastidic proteins were located in diverse subcellular compartments and did not show any functional trends; this indicates the lack a specific extra-plastidic phenotype in clpp3-1. In the remainder of the paper, we will therefore focus on the effects on the plastid proteome and the differentially accumulating plastid proteins (Table II). Immunoblot analysis for a number of thylakoid and stromal proteins were consistent with the results of the MS-based quantification providing further support for the reliability of MS-based quantification and statistics (Fig. 10). To evaluate general effects of loss of ClpP3 on the plastid, we compared the chloroplast protein mass investments (based on the NadjSPC of assigned chloroplast proteins) across 35 functions between clpp3-1 and wt (Fig. 11).
In the next sections, we discuss the results of the proteome analysis in an effort to i) determine the molecular chloroplast phenotype of clpp3-1, ii) identify potential substrates among the up-regulated proteins, and iii) compare the results with previous quantitative proteome analysis clpr2-1 (Zybailov et al., 2009) andclpr4-1 (Kim et al., 2009) to look for consistency and differences between ClpPR core mutants. We note that due to the improved experimental workflow and MS acquisition settings, we quantified substantially more proteins in clpp3-1 than in clpr2-1 and clpr4-1 (for more information on how this improvement was accomplished -see Supplemental text, Supplemental Fig. S3 and Supplemental Table  S5).
Reduced Photosynthetic capacity in clpp3-1 About 100 proteins involved in the dark and light reactions of photosynthesis were quantified. The overall protein mass of the thylakoid bound photosynthetic apparatus was decreased by 30% (Fig. 11A). Nearly 40 proteins in the thylakoid photosynthetic apparatus were significantly down-regulated (Table II). These included proteins of each of the five thylakoid complexes (Photosystem I and II, cytb 6 f, ATP-synthase and NDH). An interesting exception was LHC1-5 which was nearly five-fold up-regulated. LHCI-5 is a low abundant protein shown to loosely associate with PSI (Ganeteg et al., 2004) and interacts with the light harvesting complex I at the Lhca2/Lhca3 site (Lucinski et al., 2006). The NDH-PSI supercomplex was absent in knockout lines of LHCI-5 indicating a potential role for LHCI-5 in mediating NDH and PSI association (Peng et al., 2010). Related, stromal CRR6 (chlororespiratory reduction 6) involved in assembly of the NDH complex (Munshi et al., 2006) was ~4-fold up-regulated (Table II). The overall mass of the Rubisco complex was decreased by 40% ( Fig. 11A), consistent with the visible loss of Rubisco subunits in the Coomassie stained gel (Fig. 9B). Indeed, down-regulation of the small and large subunit of Rubisco was statistically significant (Table II). Surprisingly, the mass of the rest of the Calvin cycle enzymes was not affected in clpp3-1 (Fig. 11A) and none of the individual proteins was significantly affected, with the exception for one of the isoforms of fructose-biphosphate aldolase (SFBA-1; At2g21330) which was 50% up-regulated. SFBA-1 was found associated in significant amounts with plastoglobules (Kessler and Schnell, 2006;Ytterberg et al., 2006), which may explain its up-regulation.

Upregulation of the plastoglobule proteome indicates a stressed chloroplast in clpp3-1
Plastoglobules (PGs) are thylakoid-associated lipoprotein particles that serve as compartments for synthesis, storage and degradation of prenyl-lipids and thylakoid membrane remodeling (Brehelin et al., 2007;Lundquist et al., 2012). The total mass of PG proteins increased 2.6 fold (Supplemental Table S4). As structural proteins, fibrillins maintain the PG coat and likely control PG size, but they may have metabolic functions (Singh and McNellis, 2011). Out of the seven PG-localized fibrillins, three (FBN1a, FBN1b and FBN8) were significantly 3-15 fold upregulated in clpp3-1. PG-localized metabolic enzymes NADH dehydrogenase (NDC1), carotenoid dioxygenase (CCD4) and ABC1 kinase3 (ABC1K3) were also strongly upregulated (Table II). This is in striking agreement with co-expression analysis based on mRNA abundance, which showed that these three PG enzymes were part of the same co-expression module as the ClpPR core proteins (Lundquist et al., 2012). Figure 11B summarizes the effects on plastid gene expression and protein homeostasis. Proteins (in total 268) involved in these processes were grouped in 11 functional classes. This showed that overall protein mass investments in plastid ribosomes, the Clp system, protein assembly and DNA related functions were unchanged in clpp3-1. In contrast, overall protein mass investments in protein (un)folding, tRNA synthases, protein synthesis and other proteolytic systems were 2-fold increased. Furthermore, protein targeting machinery and proteins involved in RNA metabolism also overaccumulated in clpp3-1 (Fig. 11B).

Effects on plastid gene expression and protein homeostasis
This general pattern was also reflected in the significantly affected proteins across these functions (Table II). More than 40 proteins were significantly affected, all of which were up-regulated, except for a lumenal isomerase (consistent with the reduction of the lumenal proteome) and three 50S ribosomal proteins which were down-regulated. The upregulated proteins include the chloroplast chaperones CPN60α,ß (see also Fig. 10), CPN10,20, cpHSP70-1,2 and cpHSP90 which play crucial roles in protein folding and maturation (Boston et al., 1996;Wandinger et al., 2008). ClpB3, the chloroplast homologue of the bacterial ClpB protein (Myouga et al., 2006) was five-fold upregulated. ClpB3 is involved in unfolding aggregated proteins together the cpHSP70 system (Goloubinoff et al., 1999;Haslberger et al., 2008). The upregulation of these chaperone systems was also observed in other Clp mutants (Rudella et al., 2006;Kim et al., 2009;Zybailov et al., 2009). ROC4, an abundant stromal peptidylprolyl isomerase (Peltier et al., 2006;Zybailov et al., 2008) with in vitro rotamase activity (Lippuner et al., 1994) also overaccumulated. In summary, loss of ClpP3 accumulation clearly resulted in major protein folding stress, perhaps due the accumulation of unwanted proteins.
Out of the 25 other detected proteases, just three stromal proteases/peptidases and two thylakoid protease were significantly affected in clpp3-1. Stromal Zn 2+ -protease PreP1, stromal processing peptidase (SPP) and leucyl aminopeptidase 2 (LAP2) were 2.4-5.5 fold upregulated (Table II). Prep1 was suggested to be involved in degradation of cleaved chloroplast transit peptides (Bhushan et al., 2005;Stahl et al., 2005;Glaser et al., 2006), whereas stromal SPP is involved in transit peptide removal of most nuclear-encoded, chloroplast-targeted proteins (Richter andLamppa, 1998, 1999). LAP2 belongs to the family of soluble aminopeptidases (Walling, 2006) and it was recently suggested that LAP2 also moonlights as a chaperone in the chloroplast (Scranton et al., 2012). The two affected thylakoid proteases are EGY2 and SPPA, respectively 3-and 6-fold increased in clpp3-1. EGY2 is an ATP-independent intramembrane protease (RIP) in the Site-2 protease (S2P) family (Chen et al., 2012) and is a paralogue of the better characterized EGY1 thylakoid-bound metalloprotease that is crucial for thylakoid development and accumulation of chlorophyll-containing proteins (Chen et al., 2005). SppA is a light stress-induced, thylakoid-bound ATP-independent serine-type protease with unknown substrates (Lensch et al., 2001). Interestingly, the very abundant thylakoid FtsH protease complex (FtsH1,2,5,8) (Liu et al., 2010;Kato et al., 2012), robustly quantified with many MS/MS spectra (114, 499, 528, 146 for FtsH1,2,5,8 respectively; Supplemental Table S3), was not significantly affected (between 0.8-and 1.2-fold). Immunoblot analyses confirmed the fold-changes of SppA, whereas the abundance levels of ClpR2 and the thylakoid protease FtsH2 were unchanged consistent with the MS-based quantifications (Fig. 10). Thus, loss of Clp protease capacity specifically affects a subset of chloroplast proteases, indicative of a controlled protease network. pTAC11 (WHY3) and pTAC17, initially identified in transcriptionally active chromosomes (TACs) fractions (Pfalz et al., 2006), were 7-fold and 3-fold upregulated in clpp3-1, respectively. In particular pTAC11, but not pTAC17, is strongly enriched in nucleoids (Majeran et al., 2012;Huang et al., 2013). The function of pTAC17 is unknown but sequence analysis suggests that it maybe be involved in DNA repair; importantly we recently identified it as a candidate substrate for the substrate selector ClpS (Nishimura et al., 2013). Plastid pTAC11 is a member of the Whirly family of multifunctional RNA-and DNA-binding proteins located in mitochondria and plastids (Krause et al., 2005). Members of the Whirly family are involved in organelle genome stability and quality control and perhaps RNA metabolism (Prikryl et al., 2008;Marechal et al., 2009;Cappadocia et al., 2012). A SET domain-containing protein (At5g14260) was also significantly over-accumulating in the clpp3-1; the SET domain is frequently found in DNA interacting proteins. Finally, a DEADbox DNA helicase likely involved in DNA folding and YMLG1 were upregulated. YMLG1 (Kabeya et al., 2010) is involved in the distribution of nucleoids in chloroplast. Together these results suggest that the loss Clp protease function has an impact on the plastid chromosome.
Nine proteins (likely) involved in RNA metabolism were significantly upregulated in clpp3-1 (Table II). These include PPR and RRM proteins, ribonuclease Rnase J and an uncharacterized ribonuclease, as well as three DEAD/DEAH RNA helicases, including RH22, RH3 and uridylate kinase (DPT1). Bacterial RNase J has been implicated in 16S rRNA maturation and ribosome assembly (Britton et al., 2007) and the Arabidopsis Rnase J is essential for embryogenesis (Meinke et al., 2009). Recently we characterized RNA helicase 3 (RH3) as a plastid RNA splice factor, also affecting ribosome biogenesis (Asakura et al., 2012). PPR protein SVR7 was identified as a suppressor of the FTSH2 protease VAR2 and has a chloroplast rRNA phenotype (Liu et al., 2011). Uridylate kinase is involved in post-transcriptional steps of psaA/B transcript accumulation (Hein et al., 2009). Thus loss of Clp protease function also impacts RNA metabolism.
Protein elongation factors EF-Tu, EF-G, TypA/bipA (SVR3), three tRNA synthetases and the Arabidopsis orthologue of bacterial trigger factor (TF) were several fold upregulated (Table II). E. coli TF binds to the 70S exit tunnel and prevents miss-folding and aggregation of emerging nascent proteins (Ferbitz et al., 2004). This indicates that loss of Clp protease function leads to a bottle-neck in plastid translation.

No systematic defect in accumulation of plastid-encoded proteins
To determine if clpp3-1 suffered from a systematic problem in accumulation of chloroplast-encoded proteins, we evaluated the accumulation of chloroplast-encoded proteins. In total 53 chloroplast-encoded proteins were identified and comprised 34% of the total chloroplast proteome mass in wt, but only 23% in clpp3-1. Only chloroplast-encoded proteins that are part of the photosynthetic apparatus (25 proteins) were down-regulated, likely reflecting a systematic down-regulation of the photosynthetic apparatus, including nuclear-encoded proteins, rather than a general defect in plastid gene expression. Indeed, accumulation levels of chloroplast-encoded proteins that are not involved in photosynthesis (in total 28 proteins), including ribosomal proteins, the PEP complex, YCF1 and YCF4, ClpP1 and carboxyltransferase ß (part of ACCase) were unchanged (or even upregulated) in clpp3-1. This is an important conceptual result. Figure 11C shows the mass investments in primary carbon metabolism including metabolism of starch and minor carbohydrates, photorespiration, glycolysis, the oxidative pentose phosphate (OPP) pathway and organic transformation (dominated by the abundant carbonic anhydrases and malate dehydrogenase). Likely in response to the loss of photosynthetic capacity, the OPP pathway was 2.6 fold up-regulated in clpp3-1; indeed two OPP enzymes significantly increased in clpp3-1 (Table  II). Investment in metabolism of various minor carbohydrates and well as starch metabolism were also up-regulated (Fig. 11C). Consistently, six enzymes in starch synthesis and degradation were significantly up-regulated, in agreement with observations for the other Clp core mutants, clpr2-1 and clpr4-1 (Table  II). It is not clear why starch metabolism is up-regulated, but is perhaps in response to the shortage of reduced carbohydrates produced by the chloroplast.

Strong up-regulation of ATP transporters suggest that the chloroplast is starved for ATP
Four envelope metabolite transporters were upregulated in clpp3-1 (Table II), of which the two nucleoside triphosphate transporters (NTT1,2) are involved in importing ATP (Reinhold et al., 2007). These NTT isoforms were six-to ten-fold up-regulated in clpp3-1, which is consistent with the strong loss of photosynthetic capacity. Figure 11D summarizes the investment of other functions in the chloroplast. Investments in metal homeostasis and the function plastid division and signaling were doubled in clpp3-1. Specifically, metal homeostasis included three ferritins involved in storage of iron and two Cu chaperones (CCS and CUTA). Up-regulation (4-fold) of ferritin-1 was significant (Table II). The increased abundance in plastid division was in particular due to increased levels of the FtsZ family. Investments in several other metabolic pathways (metabolism of fatty acids, isoprenoids, nucleotides, nitrogen, sulfur and amino acids) were increased by 50%. Consistently, several dozen proteins in these functions were significantly up-regulated, in particular proteins involved in fatty acid and amino acid metabolism (Table II). In contrast investment in tetrapyrrole metabolism (chlorophyll, heme and siroheme) was unchanged in clpp3-1 (Fig. 11D); indeed, only one protein in tetrapyrrole metabolism was affected in clpp3-1 (Table II). NADH-glutamate synthase (NADH-GOGAT/GLT1), mostly involved in assimilation of nitrogen, stands out for its very strong (22 fold) upregulation. Fd-GOGAT, primarily involved in the generation of glutamate to support assimilation of photorespiratory ammonium, was also significantly up-regulated in clpp3-1 (Table II). 4-hydroxy-3methylbutyl diphosphate synthase (HDS) controls the methylerythritol phosphate (MEP) pathway that generates precursors for plastid isoprenoids. HDS was upregulated clpp3-1, in agreement with the other Clp core mutants, clpr2-1 (Rudella et al., 2006) andclpp4-1 (Kim et al., 2009). Interestingly, methylerythritol cyclodiphosphate, the substrate of HDS elicits the expression of selected stressresponsive nuclear-encoded plastid proteins (Xiao et al., 2012).

Comparison of clpp3-1 with clpr2-1 and clpr4-1 showed exceptional consistency in proteome phenotypes
Previously, we carried out comparative proteome analysis of leaf extracts of the clpr2-1 and clpr4-1 core mutants. Because the ClpPR subunits assemble in a single functional complex, we postulated that it is likely that the molecular phenotypes should be very similar. Therefore, we determined if the significantly altered proteins in clpp3-1 were also affected in these core mutants. Indeed, respectively 36 and 40 proteins were also significantly affected in these mutants, and importantly, for all but one (AOS), these proteins showed similar direction of response (up or down) as in clpp3-1. This shows that the molecular plastid phenotypes of the three ClpPR core mutants are very similar and also underscores that our workflow was very robust and that our significance analysis did not produce many false positives.

Functional and structural contributions of ClpPR subunits to the chloroplast ClpPR core complex
The presence of an extended family of 4 non-catalytic ClpR proteins and 5 catalytic ClpP proteins in higher plant chloroplasts suggests specific evolutionary adaptation of the Clp protease system to higher plant plastid/chloroplast metabolism and protein homeostasis. Therefore, in an effort to unravel this adaptation, we and others have aimed to establish the functional and structural contribution of each of the nine different ClpPR subunits to the chloroplast Clp protease in Arabidopsis -reviewed in (Olinares et al., 2011). The tetradecameric Clp protease core consists of the heptameric R-ring with ClpP1,R1,R2,R3 and R4 in a 3:1:1:1 ratio and one heptameric P-ring with ClpP3, P4, P5 and P6 in a 1:2:3:1 ratio (Olinares et al., 2011). The stoichiometry and distribution of the ClpPR subunits across and within these two heptameric rings provides an excellent basis for understanding the contribution of each subunit.
Including the current study, null mutants for 6 of the 8 nuclear-encoded ClpPR subunits(R1,R2,R4,P3,P4,P5) have now been obtained and characterized (Koussevitzky et al., 2007;Kim et al., 2009). Moreover, chloroplast-encoded ClpP1 was shown to be essential for shoot development in tobacco (Shikanai et al., 2001;Kuroda and Maliga, 2003). In addition, antisense lines for ClpP4 (Zheng et al., 2006) and for ClpP6 (Sjogren et al., 2006) were investigated that could germinate, develop on soil and produce seeds -these plants still expressed ClpP4 or ClpP6 albeit at low levels. Down-regulation of ClpP4 or ClpP6 resulted in reduced greening and reduced photosynthesis and delayed development as expected. In case of antisense ClpP4, immunoblotting suggested that the subunits in the P-ring decreased more than of the R ring, but the assembly state of the subunits was not investigated (Zheng et al., 2006). In case of antisense ClpP6, using SDS-PAGE gels and immunoblotting did not show consistent changes in ClpPR accumulation levels nor systematic differences between the subunits of the R-ring and P-rings (except of course for reduced ClpP6) (Sjogren et al., 2006). Currently, ClpR3 is the only ClpPR subunit for which no mutant has been described. This subunit is interesting, as it can fully complement the ClpR1 null mutant when ClpR3 is overexpressed (Kim et al., 2009) and ClpR3 protein levels increased in the clpr1-1 mutant background (Stanne et al., 2009).
Taking this organization and all other experimental data, as well as the primary sequences for the ClpPR proteins, into account, we can describe the following emerging view of the Clp protease core: both the non-catalytic ClpR and catalytic ClpP subunits make important contributions to Clp core functions with very little structural or functional redundancy. Within the R-ring, ClpR3, but not the other ClpR proteins, can partially substitute for ClpR1. Loss of ClpP1, R2 or R4 all results in dramatic chloroplast and leaf developmental phenotypes and are strictly required for flowering whereas also embryo plastid development is negatively impacted. Within the P-ring, the subunits present in more than one copy (ClpP4 and P5) are absolutely required for embryogenesis, whereas the function of ClpP3 present in one copy is not strictly required for embryogenesis, but is strictly required for leaf development and flowering. However, supplementation with sugars (glucose or sucrose) can suppress part of the developmental phenotype, unlike in mutants for members of the R-ring (R2 and R4). Whereas antisense lines of ClpP6 show that this subunit is important for chloroplast biogenesis and leaf development, it is not known if null alleles in ClpP6 phenocopy the ClpP3 null allele or the ClpP4 and ClpP5 null alleles. However, given that ClpP6 is present in only one copy, we speculate that a null allele would resemble ClpP3 or ClpR2, R4 alleles.

The assembly state of the Clp core without ClpP3
Our results indicate that in clpp3-1 a much smaller percentage of ClpPR proteins can effectively assemble into 350-400 kD core complexes. Combining the native immunoblot and QconCAT results, the more dominant proteins in the mutant core are ClpR3, ClpP5, ClpP6, ClpP1, ClpT1 with underrepresentation of ClpP3 (null), ClpP4, ClpR1,ClpR2 and ClpR4. This suggests that a core is assembled with only a subset of ClpPR proteins. Given that ClpP3 is absent and ClpP4 is not well assembled into the Clp core, as compared to ClpP6 in clpp3-1, this suggests a significantly modified core complex in clpp3-1. Interestingly, when considering both core and individual rings, ClpP5, ClpP6, ClpP1 and ClpR3 each overaccumulated between 2-and 2.5-fold in clpp3-1 as compared to wt. For ClpP6 this could be confirmed by immunoblotting of the native gels, which also showed that ClpP4 levels increased several fold. In contrast accumulation levels of ClpR1, ClpR2 and ClpR4 were unchanged; again for ClpR2 this could be confirmed by immunoblotting of the native gels.
Comparison of the Clp assembly states in clpp3-1 with the knock-down mutant clpr2-1 shows that the two mutants have different core complexes. With lower overall levels of ClpR2 in clpr2-1, ClpP4 and ClpP6 assemble into the 350 kD complex, but not as efficiently as in wt. It appears as if all copies of ClpR2 are used to assemble a wt-like core with the excess of other ClpPR proteins accumulating mostly in heptameric rings. In contrast, in clpp3-1 only a subset of the Clp subunits assemble in a core complex with a composition substantially different from wt, with the remaining subunits accumulating in heptameric rings.
Double rings for ClpP4 and ClpP6 were observed in clpp3-1 and clpr2-1 but not in the wt. In contrast, ClpR2 assembles in a single ring. This indicates heterogeneity in the P-ring in the mutants. It is unlikely that differential association of ClpT can explain this heterogeneity because immunoblotting showed ClpT1 only in core complexes and barely in 180-200 kD rings. Interestingly, ClpT1 showed an upward shift in SDS-PAGE in case of clpp3-1 but not in clpr2-1. We do not know what this PTM represents but a PTM search based on MS/MS analysis did not show a particular modification, nor alternative processing or splicing.

Proteomes of ClpR2, ClpR4 and ClpP3 mutants show a consistent phenotype
Comparative quantitative proteomics analyses of clpp3-1 (this study) was integrated with our previous comparative proteome analyses of clpr2-1 (Zybailov et al., 2009) andclpr4-1 (Kim et al., 2009) in an effort to: i) determine if there are consistent or unique molecular phenotypes across these different Clp core mutants that may help to explain the function of the Clp protease system and its evolutionary adaptation to the chloroplast, ii) find potential Clp substrates observed as up-regulated proteins that overaccumulated because of their increased life time due to reduced Clp protease capacity, and iii) provide a basis for determination of the chloroplast protease network, through the monitoring of accumulation levels of proteases. Here we will summarize and discuss our findings in the context of these objectives.
In general, we observed very similar proteome phenotypes between clppr2-1, clpr4-1 and our current analysis of clpp3-1. Due to the much improved workflow for clpp3-1, we were able to identify many more significant changes in clpp3-1 than the other mutants. The consistent phenotype across these mutants indicates that the activities of each of the ClpPR subunits occurs through their contribution to a single ClpPR core complex and not because of the activity of individual subunits per se. Collectively, a clear chloroplast phenotype emerges from these Clp core mutants with nine key characteristics: i) a strong loss of photosynthetic capacity through systematic loss of the thylakoid-bound photosynthetic machinery and the Rubisco holocomplex; this is consistent with its pale-green phenotype and delayed growth, ii) strong differential up-regulation of plastoglubular proteins, in particular of module 2 of the plastoglobular co-expression network (Lundquist et al., 2012), indicative of a thylakoid membrane homeostasis problem, iii) upregulation of a subset of DNA/nucleoid interacting proteins most likely involved in DNA/genome quality control, iv) differential effects on RNA metabolism, v) strong upregulation of protein translation factors, but not at all of plastid ribosomes, vi) systematic up-regulation of stromal chaperone systems, vii) upregulation of the chloroplast Sec machinery suggesting a bottleneck in thylakoid protein insertion, viii) up-regulation of a narrow set of chloroplast proteases, and ix) a limited number of changes in envelope transporters and enzymes involved in primary and secondary metabolism (most can be explained by loss of ATP/NADPH production). A clear example is the up-regulation of the inner envelope ATP/ADP translocators (NTTs) which import cytosolic ATP into the chloroplast confirming the reduced ATPgenerating capacity in the chloroplast (Reinhold et al., 2007). Finally, the selective decrease in plastidencoded proteins indicates that there is no systematic defect in plastid gene expression in clpp3-1. Only chloroplast-encoded proteins that are part of the photosynthetic apparatus were down-regulated, likely reflecting a systematic down-regulation of the photosynthetic apparatus, including nuclear-encoded proteins. The mechanism for this selective loss of accumulation of chloroplast-encoded protein is unclear but could either be accelerated turnover of thylakoid proteins, or a retrograde signaling specifically resulting in down-regulating expression of thylakoid proteins. Together this proteome phenotype suggests that the Clp protease system likely has broad substrate specificity and that its general function is essential for chloroplast biogenesis and cannot be replaced by other proteases. Loss of Clp protease capacity specifically affected a subset of chloroplast proteases, indicative of a controlled protease network. Targeted crosses between Clp mutants and other chloroplast protease mutants is in progress to define this network (unpublished).
The search for substrates Additional motivation for our comparative quantitative proteomics analysis of clpr2-1, clpr4-1 and clpp3-1 was to find up-regulated proteins that over-accumulated because of their increased life-time due to reduced Clp protease capacity. Indeed, we identified 95 statistically significant up-regulated proteins, but in many cases these can be explained as compensatory responses to the loss of photosynthetic capacity, as well as destabilization of protein homeostasis resulting in increased levels of chloroplast chaperone systems and selected proteases. There are however a number of proteins for which the up-regulation is not so easily explained through pleiotropic effects, in particular those proteins involved in DNA metabolism and regulation of plastid gene expression. We have therefore shifted efforts to more direct substrate protease interactions, focusing on the putative ClpS substrate regulator (Nishimura et al., 2013).

Why is the Clp machinery needed in the embryo plastids and subsequent seedling development?
Solving the question as to why the ClpPR protease is needed for embryogenesis requires an understanding of the role of the plastid in the embryo during seed development. During seed development, embryo plastids begin to differentiate into green chloroplasts at the torpedo stage and then become colorless during seed ripening as chloroplasts dedifferentiate to non-photosynthetic plastids by losing their thylakoids and associated chlorophylls. Upon germination, these dedifferentiated plastids are converted into chloroplasts in the cotyledons (Ciamporova and Pretova, 1980) -for recent reviews on the significance and biogenesis of plastids in embryogenesis, see (Hsu et al., 2010;Bryant et al., 2011).
In the light of the recent analysis of embryogenesis mutants, it is perhaps surprising that the ClpP4/P5 proteins are required at that very early state of embryogenesis; most genes in this category are metabolic enzymes involved in eg biosynthesis of acetyl-coA, folate, etc, although others are involved in protein import, and plastid gene expression (Hsu et al., 2010). The lack of normal chloroplast development in the seeds of ClpR2, ClpR4 and ClpP3 null mutants apparently results in a developmental block at the cotyledon stage upon germination. Once this block is broken, by addition of either glucose or sucrose to the growth media, cotyledons unfold and true leaves are formed. These CLP alleles do not have white cotyledons as observed for a subset of plastid mutants, the locus white cotyledon 1 (wco1) (Yamamoto et al., 2000), cyo1 stromal elongation factor G (SCO1) (Albrecht et al., 2006;Ruppel and Hangarter, 2007) or the thylakoid protein disulfide isomerase -CYO1/SCO2 (Shimada et al., 2007;Albrecht et al., 2008;Tanz et al.) (reviewed in (Pogson and Albrecht, 2011)). These sco mutants are not affected in chloroplast development during embryogenesis in the developing silique. After initial growth on sucrose, these sco mutants can be transferred to soil and continue their life cycle under autotrophic conditions, without obvious defects in the chloroplasts of rosette leaves. A different class of mutants have a white cotyledon phenotype, but require sugars to develop green true leaves, such as plastid type I signal peptidase1 (Shipman and Inoue, 2009;Shipman-Roston et al., 2010;Ruppel et al., 2011). Once green leaves have developed, these mutants can grow autotrophically. In contrast, the Clp protease mutants have strong chloroplast phenotypes even when grown under heterotrophic conditions. Thus the role of the Clp protease system in plant development and plastid function is clearly distinct from the specific developmental program for plastid development in cotyledons. The main challenge is now to determine direct substrates and substrate recognition mechanismms for the Clp system. Based on the collective dataset, we hypothesize that proteins involved in DNA metabolism and regulation of plastid gene expression may represent Clp substrates (e.g. pTAC17). These candidates provide an excellent starting point to study Clp substrate selection.

Plant Growth, Mutant Isolation and RT-PCR analysis T-DNA insertion lines in
Columbia-0 for CLPP3 (AT1G66670) and CLPP4 (AT5g45390) are SALK_000913 and SALK_065330, respectively. The location of the T-DNA insertions was confirmed by DNA sequencing. Genotyping and RNA extraction were carried out as described previously (Rudella et al., 2006). Various growth conditions are detailed in the figure legends. For RT-PCR, total RNA was isolated with an RNeasy plant mini kit (Qiagen). First strand was synthesized from equal amounts of total RNA with Superscript III Reverse Transcriptase (Invitrogen). We tested 15-20-25 and 30 cycles for the primer pairs. 15 cycles were insufficient to visualize all transcripts, while 20 and 25 cycles best allowed us to visualize the transcripts, and we observed good linearity for 20 and 25 cycles. Primers for genomic PCR and RT-PCR analysis and various complementations are listed in Supplemental Table S6.
Complementation Full length CLPP4, CLPP5, and CLPP6 cDNA fragments and a 3424 bp genomic CLPP3 DNA fragment were amplified using Taq polymerase. Primers for complementation are also listed in Supplemental Table S6. The PCR products were subcloned into pCR8/GW/TOPO vector (Invitrogen). Using LR clonase the DNA was introduced into pMDC123 (for genomic), pEARLEYGATE100 or pMDC32 (for cDNA) Gateway destination plant binary vector (Curtis and Grossniklaus, 2003). Agrobacterium transformation, plant transformation, and selection were carried out as described previously (Rudella et al., 2006).

Microscopy of developing seeds
Seeds were removed from siliques and cleared for 1 to 24 hours in Hoyer's solution (3.75 g of gum Arabic, 50 g of chloral hydrate, and 2.5 mL of glycerol in 15 mL of water) on a microscope slide. Seeds of later developmental stages required extended clearing periods. Cleared seeds were examined using Nomarski optics on an Olympus BX51 microscope.

Pigment analysis
Chlorophyll and carotenoid content on a fresh weight basis was measured in 80% acetone as described (Lichtenthaler, 1987).

Chloroplast stroma and total leaf proteome isolation for analysis of Clp assembly states
For chloroplast stroma isolations, leaves of wt and various mutant alleles were briefly homogenized in grinding medium (50 mM HEPES-KOH, pH 8.0, 330 mm sorbitol, 2mM EDTA, 5mM ascorbic acid, 5mM cysteine and 0.03% BSA) and filtered through a nylon mesh. The crude plastids were then collected by a 2-min spin at 1100 × g and further purified on 40-85% Percoll cushions (Percoll in 0.6% Ficoll, 1.8% polyethylene glycol) by a 10-min spin at 3750 × g and one additional wash in the grinding medium without ascorbic acid, cysteine and BSA. Chloroplasts were subsequently lysed in 10 mM HEPES-KOH, pH 8.0, 5 mM MgCl 2 , 15% glycerol with a mixture of protease inhibitors under mild mechanical disruption. The lysate was then subjected to ultracentrifugation (100,000 xg) to pellet the membrane components. The supernatant (stroma) was then collected and concentrated using Amicon 4, 10 kD MWCO (Millipore). Protein amounts were determined using the Bradford reagent (Bio-rad) or the BCA protein assay kit (ThermoScientific). For total leaf proteome isolation under non-denaturing conditions, total leaf material was ground in liquid nitrogen and solubilized in 50 mM HEPES-KOH, pH 8.0, 15% glycerol and 10 mM MgCl 2 with protease inhibitor cocktail. The suspension was then filtered in miracloth and spun at 100,000xg.
Native PAGE analysis Light blue native PAGE was performed for separation of stromal and total leaf extracts under non-denaturing conditions using the NativePAGE Novex gel system (Invitrogen) with precast 4-16% acrylamide Bis-Tris gels (Invitrogen).

QconCAT analysis
The design, expression and purification of the stable isotope-labeled Clp-QconCAT protein used here for MS-based quantification was described in (Olinares et al., 2011). Prior to SDS-PAGE separation, guanidine was removed from the purified Clp-QconCAT as in (Mirzaei et al., 2008). Equal volumes of 20% trichloroacetic acid and purified Clp-QconCAT were mixed and incubated on ice for 30 min. The acidified solution was then centrifuged at 6,000g at 4°C for 15 min, and the supernatant was carefully removed. The pellet was washed twice with 1mL of 80% cold acetone, vortexed, centrifuged at 18,000g at 4 °C for 5 min and acetone was decanted. The pellet was then dried in a speed vacuum concentrator to fully remove acetone and resuspended with a brief sonication on ice in 50mM Tris pH 6.8, 1% SDS. Protein concentration was determined by the BCA Protein Assay Kit (ThermoScientific). Samples were then separated on a precast 10.5-14% gradient acrylamide Laemmli gel (Biorad) and protein bands were stained with Coomassie dye. Stromal proteomes of wt and clpp3-1 were run out on native PAGE separation and selected gel regions containing Clp assemblies and SDS-PAGE separated labeled Clp-QconCAT protein were excised and in-gel digested separately with trypsin. LC-MS analysis and data processing for absolute quantification of Clp subunits was carried out as described in (Olinares et al., 2011).

Large scale quantitative proteomics
Wild type (Col-0) plants were grown on soil for 40 days under a short day cycle (10h/14h of light/dark) at 100 µmol photons.m -2 .s -1 . Homozygous clpp3-1 plants were first grown on agar plates with 1/2 Murashige-Skoog medium and 2% sucrose under short day conditions at 40 µmol photons.m -2 .s -1 , and then transferred to soil after 70 days, and grown under a short day cycle at 100 µmol photons.m -2 .s -1 for 40 more days. Total leaf proteins were extracted by grinding 180 mg fresh leaves in liquid N 2 into a fine powder. 1 ml extraction buffer (1% SDS, 125 mM Tris-HCl (pH 8.8), 5 mM EDTA, 5 mM tributylphosphine, 2.5 mg/ml of protease inhibitor Pefablok) was added and a pestle was used to solubilize the material. Unsolubilized materials were removed by centrifugation and proteins in the resulting supernatant were precipitated in 75% acetone at -80ºC. Proteins were collected as pellets by centrifugation, followed by two additional acetone washes to remove lipids. The resulting protein pellet was solubilized in 1% SDS, 50 mM Tris-HCl (pH 8.25) and protein concentrations were determined using the BCA Protein Assay Kit (ThermoFisher). 50 μg total leaf protein of clpp3-1 and wt samples were each run out on a Biorad Criterion Tris-HCl precast gels (10.5-14% acrylamide gradient). Each of the gel lanes were cut into 20 bands followed by reduction, alkylation, and in-gel digestion with trypsin as described in (Shevchenko et al., 2006) (Friso et al., 2011). The resuspended peptide extracts were analyzed by data-dependent MS/MS using an on-line LC-LTQ-Orbitrap (Thermo Electron Corp.). Peptide samples were automatically loaded on a guard column (LC Packings MGU-30-C18PM) via an autosampler followed by separation on a PepMap C 18 reversephase nanocolumn (LC Packings nan75-15-03-C18PM) using 90-min gradients with 95% water, 5% ACN, 0.1% FA (solvent A) and 95% ACN, 5% water, 0.1% FA (solvent B) at a flow rate of 200 nl/min. Two blanks were run after every sample (see (Zybailov et al., 2009) for the gradient and sample injection scheme). The acquisition cycle consisted of a survey MS scan in the Orbitrap with a set mass range from 350 to 1800 m/z at the highest resolving power (100,000) followed by five data-dependent MS/MS scans acquired in the LTQ. Dynamic exclusion was used with the following parameters: exclusion size, 500; repeat count, 2; repeat duration, 30 s; exclusion time, 180 s; exclusion window, ±6 ppm or ±100ppm. Target values were set at 5 x 10 5 and 10 4 for the survey and tandem MS scans, respectively. MS survey scan in the Orbitrap was acquired in one microscan. Fragment ion spectra were acquired in the LTQ as an average of three microscans. Mass spectrometry data processing, data searching against TAIR8 using Mascot and subsequent filtering and quantification based on normalized and adjusted spectral counts was carried out as in (Zybailov et al., 2009)     B, Segregation analysis of green and white seeds in developing and mature siliques of heterozygous clpp3-1 and clpp4-1. Asterisks mark smaller and darker colored seeds, likely representing homozygous mutants. C, Nomarski bright field microscopy of cleared seeds of siliques of heterozygous clpp3-1 and clp4-1.
Arrows indicate the underdeveloped embryos in the colorless seeds (torpedo stage in clpp3-1 and (pre)globular stage for clpp4-1). D, Complementation of clpp3-1 (clpp3-1/P3g) and clpp4-1 (clp4-1/P4c) using respectively CLPP3 genomic and CLPP4 cDNA. Complementation was confirmed at the mRNA level by RT-PCR.   B, Dosage effect of the clpp3-1 allele in the clpr2-1 background. Seedlings, homozygous for clpr2-1 but heterozygous for clpp3-1 (right hand side), were grown on soil for 7 weeks under 18h/6h light/dark cycle at 100 µmol photons.m -2 .s -1 . For comparison parent clpr2-1 is shown on the left hand side grown together with the double mutant. C, CLPP3 and CLPR2 mRNA accumulation for wt, clpr2-1, clpp3-1 and the clpp3-1xclpr2-1 double mutants and ACTIN2 as control, using RT-PCR. Figure 5. Accumulation levels of Clp proteins in clpp3-1 as compared to wt Total soluble leaf proteins were extracted from clpp3-1 and wt. clpp3-1 plants were transferred to soil after initial growth on agar plates with ½ MS medium supplemented with 2% sucrose. Soil grown wt plants were at the same developmental stage. After SDS-PAGE, proteins were transferred to blots and probed with specific antisera against ClpP3, ClpP4, ClpP6, ClpR2, ClpT1, ClpT2, ClpC1, ClpC2 and ClpS. ClpT* indicates ClpT with ~1 kD increased mass -this is due to an unknown post-translational modification or modified processing. The amount of protein loaded in each lane is indicated. Figure 6. Assembly state of the ClpPRT proteins determined by immunoblot analysis after native gel electrophoresis. Stromal proteins obtained from isolated chloroplasts from wt, clpr2-1 and clpp3-1 were separated by native gels, transferred to blots and probed with antisera against ClpR2, ClpP4, ClpP6, ClpT1. The larger arrows indicate ClpPRT core complexes (350-400 kD), while the smaller arrows indicate Clp complexes between 180 and 240 kD, corresponding to heptameric ClpPR rings. The lower mass bands on the ClpT1 blot marked with an asterisk are unspecific. 30 µgram of stromal protein was loaded to each lane.   . Large-scale SPC-based comparative proteomics of the clpp3-1 mutant A, The clpp3-1 and wt plants used for proteome analysis. Wild type (Col-0) plants were grown on soil for 40 days under a short day cycle (10h/14h of light/dark) at 100 µmol photons.m -2 .s -1 . Homozygous clpp3-1 plants were initially grown on agar plates with 1/2 Murashige-Skoog medium and 2% sucrose under short day conditions at 40 µmol photons.m -2 .s -1 , and then transferred to soil after 70 days and grown under short day cycle at 100 µmol photons.m -2 .s -1 for 40 more days. B, 1D-SDS PAGE gel separation (biological replicate 1) of the total leaf proteomes from clpp3-1 and wt. RBCL and RBCS are indicated. C, Principle component (PCA) analysis of the quantified proteomes of wt (circles) and clpp3-1 (squares). The symbols in the PCA plot represent each of the biological replicates for each of the genotypes. PC1 and PC2 explained respectively 95.6% and 3.8% of the total variation. D, Comparison of leaf proteins determined to be significantly expressed in clpp3-1 relative to wt. This includes proteins that have BF>10 for QSpec or p-value <0.01 for GLEE. p =plastid-localized proteins and np = not plastid. Figure 10. Confirmation of quantitative proteome analysis of total leaf extracts of wt and clpp3-1 by immunoblotting. Immunoblot analysis of titrations (indicated as 1/10x, 1/2x, 1x) of total leaf protein extracts from wild type and clpp3-1 used in the proteome analysis. Membranes were probed with antibodies generated against different proteins of the photosystem I and II (PsaF: a small peripheral subunit of PSI; OEC23: oxygen evolving complex component of PSII); chaperones and proteases (ClpR2: subunit of the Clp protease complex; ClpC2; stromal chaperone; Hsp60alpha; SppA: ATP-independent light induced serine-type thylakoid protease; FtsH2: thylakoid protease of the Zn-metallo-protease family; SppA) and the stromal RNA helicase RH3. 1x = 20 µg. * indicates a precursor form of PsaF which is also observed in clpr2-1 (Rudella et al., 2006). The clpp3-1/wt ratio is indicated on the right hand side. Figure 11. Protein mass investment based on NadjSPC in specific plastid functions in wt and clpp3-1. The error bars correspond to the standard deviation across three biological replicates. The clpp3-1/wt ratios are also indicated for each function. Black bars indicate wt and grey bars clpp3-1. A, Investment in photosynthesis in the thylakoid and stroma (Calvin Cycle). B, Investments in plastid gene expression and protein homeostasis. Proteins (in total 268) involved in these processes were grouped in 11 functional classes: plastid ribosomes (53 proteins), the Clp system (12 detected proteins), protein assembly (20 proteins) and DNA related functions (10 proteins), protein (un)folding (23 proteins), amino acid activation (22 t-RNA synthases), protein synthesis (21 proteins), other proteolytic systems (25 proteins), protein targeting machinery (22 proteins) and proteins involved in RNA metabolism (51 proteins) C, Investments in primary carbon metabolism including metabolism of starch and minor carbohydrates, photorespiration, glycolysis, the oxidative pentose phosphate pathway and organic transformation (dominated by the abundant carbonic anhydrases and malate dehydrogenase). D, Investment in other functions. Abbreviations are: AA -amino acid; ptms -post-translational modifications (eg kinases); vit -vitamins; met. -metabolism SUPPLEMENTAL INFORMATION Supplemental Text. Technical comparison of the current clpp3-1 analysis with comparative proteomics studies of Clp mutants and comment on the development of the GLEE statistical package. Supplemental Table S1. Chlorophyll and carotenoid accumulation in clpp3-1 grown on agar (2% sucrose) or soil. Supplemental Table S2. Quantification of Clp subunits and determination of subunit stoichiometry in various Clp assemblies in wt and clpp3-1 using the QConCAT technique Supplemental Table S3. Large scale comparative proteomics of the clpp3-1 mutant and wt. Supplemental Table S4. Summary of the subcellular location and investment in clpp3-1 and wild-type proteins. Supplemental Table S5. Sample preparation, MS data acquisition parameters, peptide match rates and identified proteins among the Clp comparative proteomic studies. Supplemental Table S6. Primers used for genotyping, RT-PCR and complementation Supplemental Figure S1. Effect of sucrose and glucose on wt, clpr2-1 and clpp3-1 on pigment levels measured after 28 days of sowing. Standard deviations are indicated. Supplemental Figure S2. MS analysis and bioinformatics workflow for the comparative proteome analysis. adjSPC, adjusted spectral counts. Supplemental Figure S3. Comparison of LTQ-Orbitrap data acquisition cycles in comparative proteomics studies of clpr2 (Zybailov et al, 2009), clpr4 (Kim et al, 2009) and clpp3 (this study) mutants.