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. 2006 Oct;18(10):2635-49.
doi: 10.1105/tpc.106.044594. Epub 2006 Sep 15.

Structural and functional insights into the chloroplast ATP-dependent Clp protease in Arabidopsis

Lars L E Sjögren  1 , Tara M Stanne, Bo Zheng, Sirkka Sutinen, Adrian K Clarke
Affiliations

Structural and functional insights into the chloroplast ATP-dependent Clp protease in Arabidopsis

Lars L E Sjögren et al. Plant Cell. 2006 Oct.

Abstract

In contrast with the model Escherichia coli Clp protease, the ATP-dependent Clp protease in higher plants has a remarkably diverse proteolytic core consisting of multiple ClpP and ClpR paralogs, presumably arranged within a dual heptameric ring structure. Using antisense lines for the nucleus-encoded ClpP subunit, ClpP6, we show that the Arabidopsis thaliana Clp protease is vital for chloroplast development and function. Repression of ClpP6 produced a proportional decrease in the Clp proteolytic core, causing a chlorotic phenotype in young leaves that lessened upon maturity. Structural analysis of the proteolytic core revealed two distinct subcomplexes that likely correspond to single heptameric rings, one containing the ClpP1 and ClpR1-4 proteins, the other containing ClpP3-6. Proteomic analysis revealed several stromal proteins more abundant in clpP6 antisense lines, suggesting that some are substrates for the Clp protease. A proteolytic assay developed for intact chloroplasts identified potential substrates for the stromal Clp protease in higher plants, most of which were more abundant in young Arabidopsis leaves, consistent with the severity of the chlorotic phenotype observed in the clpP6 antisense lines. The identified substrates all function in more general housekeeping roles such as plastid protein synthesis, folding, and quality control, rather than in metabolic activities such as photosynthesis.

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Figures

Figure 1.
Figure 1.
Antisense Repression of clpP6 Produces a Chlorotic Phenotype. (A) Comparison between 6-week-old wild-type Arabidopsis and three independent clpP6 antisense lines (clpP6-1, clpP6-2, and clpP6-3). Plants were grown under the same standard conditions of 23/18°C day/night temperatures, 8-h photoperiod with ∼150 μmol·m−2·s−1 light, and 60 to 65% RH. (B) Shown underneath each photograph in (A) is the level of ClpP6 protein in each plant as determined by immunoblotting. Because of the more extensive chlorosis within the inner leaves of the antisense lines, the level of ClpP6 protein was examined in both inner and outer rosettes of all plants. Samples were prepared from total leaf extracts and separated by one-dimensional PAGE on the basis of equal protein content. (C) Comparison of growth rate and phenotype development in a representative clpP6 antisense line (clpP6-1) relative to the wild type over a 3- to 6-week (w) period.
Figure 2.
Figure 2.
Antisense Repression of clpP6 Reduces Chlorophyll Content and Photosynthetic Performance in Inner Leaves. (A) Chlorophyll (Chl) content in inner and outer leaves of wild-type Arabidopsis and clpP6 antisense lines (clpP6-1 to -3). Values shown are averages ± se (n = 3). FW, fresh weight. (B) Photochemical efficiency of PSII in inner and outer leaves of wild-type Arabidopsis and clpP6 antisense lines (clpP6-1 to -3). Values shown are averages ± se (n = 3). (C) Photosynthetic electron transport (ETR) rates in inner and outer leaves of wild-type Arabidopsis and clpP6 antisense lines (clpP6-1 to -3). Electron transport rate as determined by chlorophyll fluorescence was measured at different PAR levels between 0 and 400 μmol·m−2·s−1. Values shown are from three independent wild-type plants and one plant from each clpP6 antisense line (clpP6-1 to -3). In all panels, plants were compared at the same developmental stage.
Figure 3.
Figure 3.
Microscopic Analysis of Chloroplast Structure in Wild-Type Arabidopsis and clpP6 Antisense Lines. (A) to (D) Electron micrographs of inner ([C] and [D]) and outer ([A] and [B]) leaves of wild-type Arabidopsis ([A] and [C]) and clpP6 antisense lines ([B] and [D]). Plants were compared at the same developmental stage (5 weeks for the wild type, 6 weeks for clpP6), with all micrographs being representative of chloroplasts from three individual wild-type plants and one plant each from the three clpP6 antisense lines. Bars = 0.5 μm. (E) Chloroplast size as determined by area in outer and inner leaves of wild-type Arabidopsis and clpP6 antisense lines (clpP6-1 to -3). All values shown are averages ± sd (n = 6).
Figure 4.
Figure 4.
Reduced Levels of Photosynthetic Protein Complexes in Inner Leaves of the clpP6 Antisense Lines. (A) Amounts of marker proteins for different photosynthetic protein complexes were determined by immunoblotting in outer and inner leaves of 5-week-old wild-type Arabidopsis and 6-week-old clpP6 antisense lines (clpP6-1 to -3). Total leaf proteins were separated by one-dimensional PAGE on the basis of equal protein content. Antibodies were used to detect specific marker proteins for each photosynthetic protein complex: PsaL for PSI; D1 and Lhcb2 for PSII; β-subunit for ATPase; and LSU for Rubisco. (B) Quantification of the amount of each photosynthetic marker protein in the outer and inner leaves of clpP6 antisense lines (clpP6-1 to -3) relative to the wild type. Values shown are averages ± se (n = 3), with the wild-type values set to 100%.
Figure 5.
Figure 5.
Changing Levels of Chloroplast Clp Proteins in the clpP6 Antisense Lines. (A) Relative amounts of ClpP and ClpR proteins in the inner and outer leaves of wild-type Arabidopsis and clpP6 antisense lines (clpP6-1 to -3). Total cell extracts were isolated from 5-week-old wild-type Arabidopsis and 6-week-old clpP6 antisense lines and separated by one-dimensional PAGE on the basis of equal protein content. The amount of each chloroplast ClpP and ClpR paralog was determined by immunoblotting with specific polyclonal antibodies. (B) to (D) Quantification of the relative amounts of each chloroplast ClpP and ClpR paralog in wild-type Arabidopsis and clpP6 antisense lines: wild-type inner leaves relative to outer leaves (B); clpP6 antisense outer leaves (C); and clpP6 antisense inner leaves (D). Values shown are averages ± se (n = 3), with the wild-type values for outer and inner leaves ([C] and [D]) set to 100%.
Figure 6.
Figure 6.
Clp Protein Complexes in Wild-Type Arabidopsis and clpP6 Antisense Lines. Clp protein complexes in isolated stromal fractions from 3-week-old wild-type Arabidopsis and 4-week-old clpP6 antisense lines were separated by native PAGE on the basis of equal protein content. The different Clp protein complexes were visualized by immunoblotting using specific antibodies for Arabidopsis ClpP1, ClpP3-6, ClpR1-4, and ClpS1 as indicated below each panel. The size of each Clp protein complex is indicated at left.
Figure 7.
Figure 7.
Changes in Stromal Protein Composition in Wild-Type Arabidopsis and clpP6 Antisense Lines. Isolated stromal proteins from wild-type Arabidopsis and clpP6 antisense lines were separated by two-dimensional PAGE and visualized by Coomassie blue staining. Those proteins consistently more abundant in the clpP6 antisense lines relative to the wild type are circled and numbered. Shown are representative results from three replicate assays for each line. The identity of each numbered protein is detailed in Table 1.
Figure 8.
Figure 8.
Identification of Protein Substrates for the Chloroplast Clp Protease. (A) An equal number of intact chloroplasts from wild-type Arabidopsis and the clpP6 antisense lines were incubated for 3 h in the presence of light and ATP. At 1-h intervals, aliquots were taken and chloroplasts were ruptured. After fractionation, stromal proteins were separated by SDS-PAGE and visualized by Coomassie blue staining. Those proteins showing significant degradation over the 3-h time course in the wild type but not in the clpP6 antisense lines were identified by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (see Table 2 for details). Shown are results from a representative degradation assay, with the three putative protein substrates indicated by arrows at right. (B) Degradation profiles for the high molecular mass substrate proteins EF-Ts, HSP90, and RNA helicase in intact chloroplasts from wild-type Arabidopsis and the clpP6 antisense lines. Values shown are averages ± se (n = 3) plotted as a percentage of the amount of each protein at time 0, which was set to 100%. (C) Extent of degradation for the three small molecular mass protein substrates in intact chloroplasts of wild-type Arabidopsis and the clpP6 antisense lines over the 3-h time course. After fractionation, stromal proteins were separated by two-dimensional PAGE and quantified after either Coomassie blue or silver staining. The relative amount of each protein remaining after 3 h is shown as an average + se (n = 3) plotted as a percentage of the time-0 value, which was set to 100%.
Figure 9.
Figure 9.
Abundance of Putative Substrates for the Chloroplast Clp Protease during Leaf Development. (A) Isolated stromal proteins from outer and inner wild-type leaves were analyzed by SDS-PAGE based on equal protein content. Arrows indicate the three high molecular mass substrates for the Clp protease. (B) Abundance of the six putative Clp protein substrates in the inner leaves of wild-type Arabidopsis relative to that in the outer leaves. Shown are averages + se (n = 3), with the level of each protein in the outer leaves set to 100%.
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