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. 2006 Jul;18(7):1704-21.
doi: 10.1105/tpc.106.042861. Epub 2006 Jun 9.

Downregulation of ClpR2 leads to reduced accumulation of the ClpPRS protease complex and defects in chloroplast biogenesis in Arabidopsis

Andrea Rudella  1 , Giulia Friso, Jose M Alonso, Joseph R Ecker, Klaas J van Wijk
Affiliations

Downregulation of ClpR2 leads to reduced accumulation of the ClpPRS protease complex and defects in chloroplast biogenesis in Arabidopsis

Andrea Rudella et al. Plant Cell. 2006 Jul.

Abstract

Plastids contain tetradecameric Clp protease core complexes, with five ClpP Ser-type proteases, four nonproteolytic ClpR, and two associated ClpS proteins. Accumulation of total ClpPRS complex decreased twofold to threefold in an Arabidopsis thaliana T-DNA insertion mutant in CLPR2 designated clpr2-1. Differential stable isotope labeling of the ClpPRS complex with iTRAQ revealed a fivefold reduction in assembled ClpR2 accumulation and twofold to fivefold reductions in the other subunits. A ClpR2:(his)(6) fusion protein that incorporated into the chloroplast ClpPRS complex fully complemented clpr2-1. The reduced accumulation of the ClpPRS protease complex led to a pale-green phenotype with delayed shoot development, smaller chloroplasts, decreased thylakoid accumulation, and increased plastoglobule accumulation. Stromal ClpC1 and 2 were both recruited to the thylakoid surface in clpr2-1. The thylakoid membrane of clpr2-1 showed increased carotenoid content, partial inactivation of photosystem II, and upregulated thylakoid proteases and stromal chaperones, suggesting an imbalance in chloroplast protein homeostasis and a well-coordinated network of proteolysis and chaperone activities. Interestingly, a subpopulation of PsaF and several light-harvesting complex II proteins accumulated in the thylakoid with unprocessed chloroplast transit peptides. We conclude that ClpR2 cannot be functionally replaced by other ClpP/R homologues and that the ClpPRS complex is central to chloroplast biogenesis, thylakoid protein homeostasis, and plant development.

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Figures

Figure 1.
Figure 1.
Isolation and Complementation of a T-DNA Insertional Mutant for CLPR2. (A) The T-DNA insertion site is 7 bp upstream of the translational start codon of CLPR2. Exons are indicated in black. The fragment used for complementation is shown through primer annealing sites (arrows) as well as the introduced restriction sites. The T-DNA (clpr2-1) and (his)6 (R2:h) tags are not drawn to scale. Bar = 1 kb. (B) Homozygous clpr2-1 shows delayed development and a yellow/pale-green phenotype but can germinate and be maintained on soil. Younger leaves are paler with serrated leaves. The complemented line (clpr2-1/R2:h) shows a wild-type phenotype. (C) Semiquantitative RT-PCR profile for CLPR2 (top panels) and ACTIN2 (bottom panels) in leaves and roots of clpr2-1 and the wild type.
Figure 2.
Figure 2.
Incorporation of Transgenic ClpR2:(his)6 into the 325-kD ClpPRS Complex. The incorporation of transgenic ClpR2:(his)6 into the 325-kD ClpPRS complex in the complemented clpr2-1 line was analyzed. Native stroma was obtained from purified intact chloroplasts and separated in triplicate first on CN-PAGE gels, followed by SDS-PAGE in the second dimension. Two 2D gels were blotted to PVDF membranes and probed with anti-polyHis serum (A), and the third gel was stained with Sypro Ruby (B). (A) Immunoblot of the bottom part (dotted square) of a CN-PAGE separation of stromal complexes from the complemented line clpr2-1/R2:h. The region where the Clp core complex subunits migrate is enclosed by a dotted ellipse. (B) CN-PAGE as in (A) but stained with Sypro Ruby. Dotted square and ellipse are as in (A); relative position of the anti-polyHis signal is represented by white ovals. The dotted lines indicate the transition from stacking to resolving gel in the first dimension.
Figure 3.
Figure 3.
Native Stromal Proteome in clpr2-1. CN-PAGE separation and Sypro Ruby staining of 1 mg of stromal proteins from wild-type (A) and clpr2-1 (B) chloroplasts from fully developed rosettes. The identified proteins are indicated with spot numbers. Protein identities were determined by nanoLC-ESI-MS/MS in this study or for the wild type in a parallel and independent study (Peltier et al., 2006). For the complete data set, see Supplemental Table 1 online. Spot numbers 1 to 9 correspond to the ClpPRS complex. Spot numbers 10 to 31 correspond to the following proteins: 10, uridylyltransferase-related; 11, glyceraldehyde-3-phosphate dehydrogenase A-1,2 (GAPA-1,2); 12, Cpn60-α,β-1,2; 13, ferredoxin-dependent Glu synthase (Fd-GOGAT 1); 14, ClpC1,C2,D; 15, metalloproteases (M16) (AtPreP1,2); 16, lipoxygenase AtLOX2; 17, HSP90, elongation factor Tu-G (EF-G), and starch branching enzyme class II (SBEII); 18, cpHSP70-1,2 (DnaK homologues); 19, Transketolase-1 (TKL-1); 20, plastid phosphoglucomutase (PGM1); 21, fructose-bisphosphate aldolase-1,2 (SFBA-1,2); 22, fructose-bisphosphatase (FBPA); 23, monodehydroascorbate reductase (MDHAR); 24, phosphoglycerate kinase-2 (PGK-2); 25, phosphoglycerate kinase-1 (PGK-1); 26, annexin (AnnAt1); 27, sedoheptulose-bisphosphatase (SBPase); 28, GrpE-1; 29, thiazole biosynthetic enzyme (THI1); 30, 3-β-hydroxy-δ5-steroid dehydrogenase; 31, triosephosphate isomerase-1 (TPI-1); 32, Cpn21 (also Cpn20), carbonic anhydrase-1 (CA1); 33, peptidylprolyl isomerase ROC4.
Figure 4.
Figure 4.
Quantitative Comparative Analysis of ClpPRS Complex in the Wild Type and clpr2-1 Using Differential Stable Isotope Labeling with iTRAQ. (A) Schematic representation of the isobaric iTRAQ tags used for the differential labeling. The tag consists of a balance group (28 or 31 D) and a reporter group (114 or 117 D), which is released upon fragmentation in the mass spectrometer. (B) Spots containing Clp subunits and the reference spot THI1 were sliced as four gel pieces as indicated (1 to 4) from 2D gels (CN-PAGE followed by SDS-PAGE) of the stromal proteome of the wild type and clpr2-1. Gel pieces were washed and digested with trypsin. Peptides were extracted with formic acid and differentially labeled with the iTRAQ reagents (i.e., 114 for wild-type and 117 for clpr2-1 samples). After pairwise (pairs of identical spot numbers) mixing of samples from the wild type and clpr2-1, the samples were cleaned up using C18 microcolumns. The eluates for each pair were analyzed by reverse-phase nanoLC-ESI-MS/MS. After mass spectral data processing, sequence information was used for protein identification, and the reporter ions (114 and 117) were used for peptide quantification. The experiment was performed with two independent biological replicates, with a label switch between the replicates to avoid a possible systematic bias due to the label. Within one biological replicate, duplicate gels for the wild type and for clpr2-1 were used to reduce variability. Each sample was analyzed twice by MS/MS.
Figure 5.
Figure 5.
Quantitative Comparative Analysis of ClpPRS Complex in the Wild Type and clpr2-1 Using Differential Stable Isotope Labeling with iTRAQ. (A) An example of the MS/MS spectrum of a double-charged peptide (m/z = 599.84; IALQSPAGAAR; the y-ion series is indicated) belonging to the ClpR2 protein and the reporter ion pair at 114 and 117 generated in the collision cell of the mass spectrometer. Peptides from clpr2-1 were labeled with the heavy tag (117) and the wild type with the light tag (114). Inset 1 shows a close-up of the 114 to 117 region of this MS/MS spectrum, with a calculated ratio between the areas under the 114 and 117 peaks of 0.2. This shows that accumulation of ClpR2 in the mutant is fivefold lower than in the wild type (normalized to total stromal protein). Inset 2 shows a close-up of the 114 to 117 area for a peptide belonging to the THI1 protein in gel slice 4. In this case, the 114:117 ratio is 1.4, indicating that THI1 is 40% higher in the mutant than in the wild type (normalized on basis of total stromal protein). High-quality MS/MS spectra and 114:117 reporter ratios were obtained for 85 peptides belonging to the ClpP/R/S and THI1 proteins. A complete list of these peptides and reporter ion ratios is listed in Supplemental Table 2 online. (B) Summary of quantification of the different ClpP/R/S proteins accumulating in the ClpPRS complex displayed as ratios between clpr2-1 and the wild type. Standard deviations for the biological replicates are indicated. The black bars show uncorrected average ratios, and the gray bars show average ratios corrected for differences in protein loading, as determine by total spot volume per gel. Details for the peptides used for quantification are listed in Supplemental Table 2 online.
Figure 6.
Figure 6.
Light, Confocal, and Transmission Electron Microscopy Analysis of Leaves of clpr2-1 and the Wild Type. (A) to (D) Light microscopy of transverse sections from young, expanding leaf blades of the wild type (A) and clpr2-1 (B) and of mature leaves of the wild type (C) and clpr2-1 (D). (E) and (F) Laser scanning confocal microscopy images of mesophyll cells of the wild type (E) and clpr2-1 (F). Only the red channel for chlorophyll autofluorescence is shown, collected at the same excitation intensities. (G) to (L) TEM analysis of the ultrastructure of chloroplasts from young expanding leaves of wild-type (G) and clpr2-1 plants (H) and from mature leaves of wild-type (I) and clpr2-1 (J) leaves. Close-ups of plastoglobules in chloroplasts of mature leaves of the wild type (K) and clpr2-1 (L). Bars = 25 μm in (A) to (D), 5 μm in (E) and (F), 1 μm in (G) to (J), and 0.2 μm in (K) and (L). UE, upper epidermis; LE, lower epidermis; PM, palisade mesophyll; SM, sponge mesophyll; S, stomata; V, vasculature.
Figure 7.
Figure 7.
Oligomeric Soluble Cellular Proteomes of the Wild Type and clpr2-1. CN-PAGE separation and Sypro Ruby staining of 2 mg of total soluble proteins from fully developed rosettes of wild-type (A) and clpr2-1 (B) plants. The predominant chloroplast stromal components are the Rubisco large and small subunits (RbcL and RbcS, respectively). The extrachloroplastic 26S proteasome complex (ovals) and cytosolic Met synthase 1,2 (MS1, At5g17920; MS2, At3g03780) and vacuolar TGG1 (At5g20980) are indicated.
Figure 8.
Figure 8.
Starch Accumulation and Starch Breakdown in Wild-Type and clpr2-1 Leaves. To determine the net balance of reduced carbohydrate production (supply) and consumption (demand), leaves of the wild type and clpr2-1were stained (by iodine) for starch accumulation at the end of the light or dark periods. (A) shows starch staining of the wild type and clpr2-1 of 6-week-old plants, and (B) shows starch staining of the wild type and clpr2-1 ∼1 week prior to bolting.
Figure 9.
Figure 9.
Thylakoid Protein Profiles of Chloroplasts from Wild-Type and clpr2-1 Leaves and Identification by Mass Spectrometry. (A) Thylakoid proteins were purified from leaves of wild-type and clpr2-1 plants and were separated by tricine SDS-PAGE and stained with Sypro Ruby, and 100-μg proteins were loaded in each lane. Molecular markers are indicated. Bands that are numbered were excised and digested with trypsin, and peptides were extracted and analyzed by nanoLC-ESI-MS/MS. A complete listing of all identified proteins is given in Supplemental Table 3 online. (B) Sequence alignment of the N termini of LHCII1.1 and 2 (At1g29910 and At1g29920), LHCII1.3 (At1g29930), LHCII1.4 (At2g34430), LHCII1.5 (At2g34420), and LHCII.3 (At5g54270). Boxed residues indicate the peptides identified by MS/MS in clpr2-1, containing the predicted cTP cleavage site indicated by arrowheads. The most N-terminal peptides identified by MS/MS previously in extensive thylakoid proteome analyses of wild-type plants are underlined (Friso et al., 2004; Peltier et al., 2004a). N termini determined by Edman sequencing reported in the literature and in SWISS-PROT are indicated as single boxed residues (see Gomez et al., 2003). (C) MS/MS fragmentation of doubly charged precursor ion with m/z of 764.40. This is a 1526.79-D peptide AVNLSPAASEVLGSGR matching to LHCII-1.1 and 2 (At1g29910 and At1g29920) with a Mowse score of 75.
Figure 10.
Figure 10.
Immunoblot Analyses of Chloroplast Protein Populations. To evaluate the effect of reduced ClpPRS protease complex accumulation on the major thylakoid protein complexes, as well as proteases and chaperones in stroma and thylakoid, a protein gel blot analysis was performed. Titrations (indicated as 1x, 2x, 3x, and 10x) of thylakoid proteins ([A] and [B]) or total soluble protein extracts (C) from the wild type, clpr2-1, and the complemented line were separated by SDS-PAGE and blotted onto PVDF membranes. (A) Membranes were probed with antibodies generated against different proteins of PSI and II, the cytochrome b6f complex, F-type ATP-synthase (Group I), and different chloroplast chaperones and proteases (Group II). Group I proteins are as follows: D1/2, the D1 and D2 proteins of PSII; PsbS, a unique antenna protein in PSII; Cytf, the 32-kD heme binding cytochrome f protein in the cytochrome b6f complex; PSIA/B, the reaction center A and B proteins of PSI; PSIC/E, PsaC and PsaE of PSI; CF1, the peripheral α- and β-subunits of the thylakoid ATP synthase complex. The protein ratios in clpr2-1/wild type were determined by two or three independent experiments and were consistently close to one. Group II proteins are as follows: FtsH2 and 5, thylakoid members of the FtsH Zn metalloprotease family; SppA, an ATP-independent light-induced Ser-type thylakoid protease; ClpC1,2, Hsp100 chaperones in the Clp family. Protein ratios in clpr2-1/wild type were quantified in two to three independent experiments and were on average 2.7 (FtsH5), 2 (FtsH2), 10 (SppA), and >10 (ClpC1 and C2). (B) Protein gel blot of purified thylakoid membrane proteomes of the wild type, clpr2-1, and the complemented line clpr2-1/R2:h, probed with anti-PsaF. A strong cross-reacting band at ∼25 kD can be seen in clpr2-1 but not in the wild type or clpr2-1/R2:h. This band is the unprocessed PsaF protein (marked as pPsaF and indicated with a solid arrow). The bottom panel shows the response of antiserum directed against thylakoid protease SppA in the wild type, clpr2-1, and clpr2-1/R2:h. Lanes were loaded with 10, 30, and 100 μg of thylakoid protein (1x, 3x, and 10x, respectively). (C) Protein gel blot of total soluble leaf proteomes of the wild type and clpr2-1. Equal protein concentrations (1x and 2x) were loaded for the wild type and clpr2-1 and probed with antisera directed against Cpn60, Hsp70, ClpC1, and ClpC2. Protein ratios in clpr2-1/wild type were quantified in one to three independent experiments and were on average 6 (Cpn60), 2 (HSP70), 2 (ClpC1), and 7 (ClpC2).
Figure 11.
Figure 11.
Summary of the Analysis of clpr2-1. Simplified summary of the consequences of reduced CLPR2 expression for chloroplast protein homeostasis. Arrows indicate the differential response, with thickness of the arrow correlating to the fold change. Car., carotenoid/thylakoid protein ratio; PG, plastoglobule; ZnMP, Zn2+ metalloprotease.
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