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. 2009 Jun;21(6):1669-92.
doi: 10.1105/tpc.108.063784. Epub 2009 Jun 12.

Subunits of the plastid ClpPR protease complex have differential contributions to embryogenesis, plastid biogenesis, and plant development in Arabidopsis

Jitae Kim  1 , Andrea Rudella, Verenice Ramirez Rodriguez, Boris Zybailov, Paul Dominic B Olinares, Klaas J van Wijk
Affiliations

Subunits of the plastid ClpPR protease complex have differential contributions to embryogenesis, plastid biogenesis, and plant development in Arabidopsis

Jitae Kim et al. Plant Cell. 2009 Jun.

Abstract

The plastid ClpPR protease complex in Arabidopsis thaliana consists of five catalytic ClpP and four noncatalytic ClpR subunits. An extensive analysis of the CLPR family and CLPP5 is presented to address this complexity. Null alleles for CLPR2 and CLPR4 showed delayed embryogenesis and albino embryos, with seedling development blocked in the cotyledon stage; this developmental block was overcome under heterotrophic conditions, and seedlings developed into small albino to virescent seedlings. By contrast, null alleles for CLPP5 were embryo lethal. Thus, the ClpPR proteins make different functional contributions. To further test for redundancies and functional differences between the ClpR proteins, we overexpressed full-length cDNAs for ClpR1, R2, R3, R4 in clpr1, clpr2 and clpr4 mutants. This showed that overexpression of ClpR3 can complement for the loss of ClpR1, but not for the loss of ClpR2 or ClpR4, indicating that ClpR3 can functionally substitute ClpR1. By contrast, ClpR1, R2 and R4 could not substitute each other. Double mutants of weak CLPR1 and 2 alleles were seedling lethal, showing that a minimum concentration of different ClpR proteins is essential for Clp function. Microscopy and large-scale comparative leaf proteome analyses of a CLPR4 null allele demonstrate a central role of Clp protease in chloroplast biogenesis and protein homeostasis; substrates are discussed. Lack of transcriptional and translational feedback regulation within the CLPPR gene family indicates that regulation of Clp activity occurs through Clp complex assembly and substrate delivery.

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Figures

Figure 1.
Figure 1.
Gene Models with the Location of Gene Disruptions and Transcript Levels of the 10 CLPPR Single Mutants Used in This Study and Sequence Identity Matrix for the ClpPR Family. (A) Gene model structures and position of T-DNA inserts and the EMS mutation (W299→stop) in the CLPPR mutants used in this study. Exons (black boxes for coding sequence; open boxes for 5′ and 3′ untranslated regions [UTR]) and T-DNA insertions (triangles) are indicated. (B) Transcript accumulation levels in the leaves of the seven CLPR single mutants used in this study. Transcript levels were determined by RT-PCR (25 cycles) using gene-specific primer pairs; ACTIN2 was used as internal control. At least three biological replicates were performed for each RT-PCR analysis (primers are listed in Supplemental Table 1 online). (C) Identity matrix (percentage of amino acid sequence identity) for the Arabidopsis plastid-localized ClpP (P1,3-6) and ClpR (R1-4) proteins and E. coli ClpP (1TYF). The N-terminal cleavable transit peptide was removed for the nuclear-encoded proteins.
Figure 2.
Figure 2.
Growth and Development of Wild-Type and the 10 Different clpr1, clpr2, clpr4, and clpp5 Mutant Alleles under Autotrophic and Heterotrophic Conditions. (A) Development of wild-type and homozygous clpr1-1, clpr1-2, clpr2-1, clpr2-2, clpr2-3, clpr4-1, clpr4-2, clpp5-1, clpp5-2, and clpp5-3 on agar plates with Murashige and Skoog medium (without sucrose) grown under a 10/14-h light/dark cycle at 60 μmol photons·m−2·s−1. The clpr2-2, clpr2-3, clpr4-1, and clpr4-2 seedlings are arrested in the cotyledon stage, and the cotyledons are covered by the seed coat. None of the clpp5 alleles germinated. All plants are 20 d old. Bars = 1 or 3 mm, as indicated. (B) Homozygous clpr2-2, clpr2-3, clpr4-1, and clpr4-2 plants on agar plates with Murashige and Skoog medium and 2% sucrose grown under a 10/14-h light/dark cycle at 40 μmol photons·m−2·s−1. All plants are 7 weeks old. Bar = 10 mm. (C) and (D) Homozygous clpr4-1 plants grown on 2% sucrose on agar plates under a 16-h/8-h light/dark cycle at 100 μmol photons·m−2·s−1 (C) or under a 10-h/14-h light dark cycle at 40 μmol photons·m−2·s−1 or 60 μmol photons·m−2·s−1 (D). The seedlings grown under 100 μmol photons·m−2·s−1 were initially red from anthocyanin accumulation (1 week old) and subsequently developed into white plantlets (3 months old). Plants grown at 40 μmol photons·m−2·s−1 were greener than those grown at 60 μmol photons·m−2·s−1. Bars = 5 mm.
Figure 3.
Figure 3.
Double Mutant Analysis of clpr1-2 clpr2-1, the Comparison between clpr1-1, clpr1-2, and clpr2-1 Mutants, and the Allelic Test for clpr2-1 and clpr2-2. (A) Direct comparison of wild-type, clpr1-1, clpr1-2, and clpr2-1 mutants grown on soil for 22 d under a 16/8-h light/dark cycle at 120 μmol photons·m−2·s−1. (B) Development of homozygous clpr1-2 and clpr1-2 clpr2-1 plants on agar plates with Murashige and Skoog medium without sucrose grown under a 10/14-h light/dark cycle at 40 μmol photons·m−2·s−1. The double mutant is arrested in the cotyledon stage. Plants are 21 d old. Bars = 1 or 3 mm. (C) Direct comparison of wild-type and homozygous clpr1-2, clpr2-1, and clpr1-2 clpr2-1 plants grown for 7 weeks on agar plates with 2% sucrose under a 10/14-h light/dark cycle at 40 μmol photons·m−2·s−1. Bar = 10 mm. (D) CLPR1 and CLPR2 transcript accumulation levels in the leaves of the wild type, clpr1-2 (r1-2), clpr1-2 clpr2-1 (r1r2), and clpr2-1 (r2-1). Transcript levels were determined in three biological replicates by RT-PCR (25 cycles) using gene-specific primer pairs; ACTIN2 was used as internal control (primers are listed in Supplemental Table 1 online). (E) Heteroallele of clpr2-1 clpr2-2 confirms that clpr2-1 and clpr2-2 are allelic, since the phenotype is intermediate between that of the clpr2-1 and clpr2-2 null mutants. Plants were grown for 6 weeks on Murashige and Skoog medium + 2% sucrose under a 10-h/14-h light/dark cycle at 40 μmol photons·m−2·s−1. Bar = 5 mm.
Figure 4.
Figure 4.
Microscopy of Developing Embryos in Siliques of Wild-Type and heterozygous clpr2, clpr4, and clpp5 Mutants and Homozygous clpr4-1 Embryos from Imbibed Dry Seeds. (A) and (B) Opened developing (top images) and mature (bottom images) siliques of wild-type plants (A) and heterozygous clpr4-1 mutants (B). Seeds in developing siliques of the wild type are all green (A), while the siliques of heterozygous mutant alleles show white and green seeds on average in a 1:3 ratio (see Table 3 for clpr2-2, clpr2-3, clpr4-1 clpr4-2, clpp5-1, clpp5-2, and clpp5-3). In the mature silique stage, homozygous mutant seeds are recognizable as smaller, wrinkled seeds as indicated by the asterisks (B). Bars = 1 mm. (C) to (E) Nomarski bright-field microscopy of cleared seeds of siliques in different ripening stages of a heterozygous clpr4-1 mutant. g, globular stage; h, heart stage; t, torpedo stage; c, cotyledon stage; t*, torpedo-like stage. Bars = 100 μm. (F) Nomarski bright-field microscopy of cleared seeds of siliques of a heterozygous clpp5-1 mutant. The arrow points at the homozygous clpp5-1 globular embryo stage. g, globular stage; t, torpedo stage. Bar = 100 μm. (G) Overlays of Nomarski bright-field and chlorophyll red fluorescence confocal microscopy images for wild-type and clpr4-1 embryos extracted from imbibed dry seeds. The wild-type embryo shows clear cotyledon (c) and radicle (r) formation with accumulation of chloroplasts. The mutant embryo develops a normal root tip but has thicker and stunted cotyledons. Bars = 80 μm.
Figure 5.
Figure 5.
Complementation of clpr1-1, clpr2-1, and clpr4-1 with 35S-Driven cDNAs for CLPR1, CLPR2, CLPR3, and CLPR4 as well as Genomic DNA for CLPR4. Plants were grown for 21 d on soil under a 16-h/8-h light/dark cycle at 120 μmol photons·m−2·s−1. (A) Comparative analysis of wild-type, homozygous clpr1-1, and overexpressor lines of CLPR1, CLPR2, CLPR3, and CLPR4 in a homozygous clpr1-1 background using cDNAs under the 35S promoter. Overexpression of CLPR1 and CLPR3 resulted in complete complementation. (B) Comparative analysis of wild-type, homozygous clpr2-1, and overexpressor lines of CLPR1, CLPR2, CLPR3, and CLPR4 in a homozygous clpr2-1 background using cDNAs under the 35S promoter. Only overexpression of CLPR2 resulted in a complete complementation. (C) Complementation of homozygous clpr4-1 or clpr4-2 by overexpression of the cDNA for CLPR4 (clpr4-2/R4) or genomic CLPR4 DNA (clpr4-1/R4g). We could not recover overexpressor lines for homozygous clpr4-1 with cDNA for CLPR1, CLPR2, or CLPR3, indicating complete lack of complementation and consequently display of the embryo lethal clpr4-1 phenotype. Overexpressor lines for heterozygous clpr4-1 plants did not show any distinctive phenotype. (D) Transcript levels of ClpR1, ClpR2, ClpR3, and ClpR4, and CLP3, CLP4, CLP5, and CLP 6 in wild-type, homozygous clpr1-1, and clpr1-1 overexpressing CLPR1, CLPR2, CLPR3, or CLPR4. ACTIN was used as internal control. Twenty PCR cycles were performed, and the analysis was performed in two technical replicates.
Figure 6.
Figure 6.
Light Microscopy and TEM of clpr4-1 Leaves Grown at Different Light Fluences under Heterotrophic Growth Conditions and of Roots. (A) and (B) Thick (A) and thin (B) sections at low (A) and high magnification (B) of young wild-type leaves (top left-hand panels) is compared with correspondent tissue for clpr4-1, grown under 100 μmol photons·m−2·s−1 (white plant) (top right-hand panels), at 40 μmol photons·m−2·s−1 (greener plant) (bottom left-hand panels), or shifted to very low light (<10 μmol photons·m−2·s−1) at 4°C for 3 weeks (bottom right-hand panels). Bars = 0.5 μm (A) or 1 μm (B). Insets show examples of the plants used for the microscopy analysis. Plastoglobuli are indicated by arrows. (C) and (D) TEM sections of primary root vasculature showing plastids (P) and mitochondria (M) for wild-type and clpr4-1 tissues of plants grown under a 10-h/14-h light/dark cycle at 40 μmol photons·m−2·s−2. In plastids of clpr4-1 plants, many electron-dense particles are visible, most likely representing plastoglobuli (arrows). Bars = 0.5 μm. (D) Thin section of primary root cortex showing plastids (P) for wild-type and clpr4-1 tissues. [See online article for color version of this figure.]
Figure 7.
Figure 7.
Transcript Accumulation of the Clp Gene Family in the Wild Type and clpr4-1. ACTIN2 was used as internal control. Total RNA was isolated from 40-d-old plants grown on Murashige and Skoog medium, supplemented with 2% sucrose grown under a 10-h/14-h light/dark cycle at 40 μmol photons·m−2·s−1. Twenty PCR cycles were performed. Three independent biological replicates are shown.
Figure 8.
Figure 8.
Native Protein Profiles and Protein Gel Blot Analysis of White and Green clpr4-1 Leaves. (A) CN-PAGE and Sypro Ruby staining of 2 mg of total soluble proteins from leaves of wild-type and white clpr4-1 seedlings grown as in Figure 2D. The predominant chloroplast stromal proteins are the Rubisco large (RbcL) and small (RbcS) subunits. Chaperone 60, the extrachloroplastic 26S proteasome complex, mitochondrial ATP synthase complex (ovals), cytosolic methionine synthase 1,2 (MS1 and MS2), and vacuolar thioglucoside glucohydrolase 1 (TGG1) are indicated. (B) Protein gel blot analysis of titrations (indicated as 1x, 2x, 3x, 4x, and 10x) of total leaf protein extracts from wild-type and clpr4-1 plants grown as in Figure 2B. Protein ratios in clpr4-1/wild type are indicated. Membranes were probed with antibodies generated against different proteins of the PSI and PSII (CP43,47, Core Protein 43 and 47 of PSII; PsaF, a small peripheral subunit of PSI; PsbS, a unique antenna protein in PSII), as well as chaperones and proteases (stromal chaperone Cpn60, stromal protease DegP2, thylakoid proteases FtsH2 and 5, members of the FtsH Zn-metalloprotease family; ATP-independent light-induced Ser-type thylakoid protease SppA).
Figure 9.
Figure 9.
LTQ-Orbitrap MS/MS Analysis of clpr4-1 and Wild-Type Plants. (A) Examples of clpr4-1 and wild-type plants used for proteome analysis. (B) One-dimensional SDS-PAGE gel lanes (Replicate 1) with leaf proteomes from clpr4-1 and the wild type. (C) Cross-correlation between adjusted SPC of identified proteins with at least 10 adjusted SPCs in both biological replicates. These adjusted SPCs are the raw data not normalized for the total SPC per sample. Proteins that surpass the 95% confidence by the G-test are indicated with filled symbols in black, red, or blue. The red filled symbols are those chloroplast proteins that surpass the 95% confidence level in both biological replicates. The blue filled symbols are those nonchloroplast proteins that surpass the 95% confidence level in both biological replicates. The dashed line is the linear regression line for SPC in clpr4-1 and the wild type and corresponds to a 1:1 ratio of clpr4-1:wild type based on normalized SPC. The protein identities and function of the numbered data points can be found in Table 4. (D) Cross-correlation between log(nSAF) for chloroplast-localized proteins in the wild type and clpr4-1 for each of the replicates; the nSAF values represent relative protein abundance normalized to the total chloroplast protein abundance in the respective sample. Only those proteins are displayed that were identified with at least 10 adjusted SPC in both replicates. Proteins that surpass the 95% confidence by the G-test in both biological replicates are indicated with filled red symbols, and those proteins that surpass the 95% confidence level in only that biological replicate are shown in filled black symbols. The protein identities and function of the numbered data points can be found in Table 4. (E) Relative accumulation of thylakoid photosynthetic complexes and light stress proteins in clpr4-1 leaves compared with wild-type leaves, determined by spectral counting, based on the clpr4-1:wild type ratio of average values (with sd) for all quantified subunits of each complex. The number of proteins quantified for each complex (n) is indicated. LHCII, light-harvesting complex II proteins; LHCI, light-harvesting complex I proteins; Lil3.2, light harvesting-like protein 3.2; Ohp2, One-helix protein 2.
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