Inactivation of the clpC1 gene encoding a chloroplast Hsp100 molecular chaperone causes growth retardation, leaf chlorosis, lower photosynthetic activity, and a specific reduction in photosystem content

Abstract

ClpC is a molecular chaperone of the Hsp100 family. In higher plants there are two chloroplast-localized paralogs (ClpC1 and ClpC2) that are approximately 93% similar in primary sequence. In this study, we have characterized two independent Arabidopsis (Arabidopsis thaliana) clpC1 T-DNA insertion mutants lacking on average 65% of total ClpC content. Both mutants display a retarded-growth phenotype, leaves with a homogenous chlorotic appearance throughout all developmental stages, and more perpendicular secondary influorescences. Photosynthetic performance was also impaired in both knockout lines, with relatively fewer photosystem I and photosystem II complexes, but no changes in ATPase and Rubisco content. However, despite the specific drop in photosystem I and photosystem II content, no changes in leaf cell anatomy or chloroplast ultrastructure were observed in the mutants compared to the wild type. Previously proposed functions for envelope-associated ClpC in chloroplast protein import and degradation of mistargeted precursors were examined and shown not to be significantly impaired in the clpC1 mutants. In the stroma, where the majority of ClpC protein is localized, marked increases of all ClpP paralogs were observed in the clpC1 mutants but less variation for the ClpR paralogs and a corresponding decrease in the other chloroplast-localized Hsp100 protein, ClpD. Increased amounts of other stromal molecular chaperones (Cpn60, Hsp70, and Hsp90) and several RNA-binding proteins were also observed. Our data suggest that overall ClpC as a stromal molecular chaperone plays a vital role in chloroplast function and leaf development and is likely involved in photosystem biogenesis.

Figure 1.

Confirmation of the T-DNA insertion lines for clpC1. A, Schematic picture of the genomic clpC1 gene in Arabidopsis. Gray boxes and black lines represent exons and introns, respectively. Arrows indicate the ATG start codon and the proposed T-DNA insertion sites for the two independent mutant lines, clpC1-1 and clpC1-2. B, RT-PCR analysis of clpC1 and clpC2 gene expression of wild type (WT), and clpC1-1 and clpC1-2 mutant lines. Reactions were performed with equal total RNA, with the resulting RT-PCR products visualized by staining with GelStar. C, Immunoblot analysis of total ClpC protein in wild type, and clpC1-1 and clpC1-2 mutant lines. Total proteins were extracted from leaves of each plant and separated by denaturing PAGE on the basis of equal fresh weight. Total ClpC protein was detected by immunoblotting with an antibody that crossreacts with both ClpC1 and ClpC2.

Figure 2.

The clpC1 mutants exhibit visible phenotypes. A, Photograph of 55-d-old wild-type (WT), clpC1-1, and clpC1-2 plants grown side by side under the standard conditions of 23°C/18°C day/night temperatures, an 8-h photoperiod with 150 μmol white light m⁻² s⁻¹, and approximately 65% humidity. B, Photographs of fully grown flowering plants showing the shorter primary and more perpendicular secondary influorescences (magnified in the insert for clpC1-2) in the clpC1 mutant lines compared to the wild type. Scale bars = 2 cm.

Figure 3.

Loss of ClpC1 reduces chlorophyll content and impairs photosynthesis. A, Table showing the reduced chlorophyll content and lower photochemical efficiency of PSII (F_v/F_m) in the clpC1 mutants. Values shown are the mean value from measurements done on replicate plants of each line ± se (n = 3). B, Light response curves of photosynthesis for wild type (WT) and clpC1 mutant lines. Photosynthetic ETRs as determined by chlorophyll fluorescence were measured at irradiances (photosynthetically active radiation [PAR]) from 0 to 460 μmol photons m⁻² s⁻¹. Measurements were done on fully grown leaves from three separate plants of each line grown under identical conditions and of identical age.

Figure 4.

Altered levels of PSI and PSII proteins in the clpC1 mutants. Relative amounts of different photosynthetic protein complexes were compared between the wild type (WT) and clpC1 mutant lines, clpC1-1 and clpC1-2. Total cell proteins were extracted from 55-d-old leaves from each plant and separated by denaturing PAGE on the basis of equal fresh weight (1 μg). Marker proteins for each complex were detected by immunoblotting using specific polyclonal antibodies (A) and quantified (B). Selected marker proteins were PsaD and PsaL for PSI, D1, and Lhcb2 for PSII, CF₁ β-subunit for ATPase, and large subunit for Rubisco. Amounts of each marker protein in the clpC1-1 (white bar) and clpC1-2 (black bar) lines are shown as a percentage of the relative wild-type level (± se, n = 4), which was set at 100%.

Figure 5.

Altered levels of various Clp proteins in the clpC1 mutants. Relative amounts of different chloroplast Clp proteins and the chaperone Cpn60 were compared between the wild type (WT) and clpC1 mutant lines, clpC1-1 and clpC1-2. Total cell proteins were extracted from 55-d-old leaves from each plant and separated by denaturing PAGE on the basis of equal fresh weight (1 μg). Various chloroplast Clp proteins and Cpn60 were then detected by immunoblotting, using specific polyclonal antibodies (A) and quantified (B). Proteins detected were ClpC (total), ClpD, ClpP1, ClpP3–6, ClpR1–4, and Cpn60. Amounts of each protein in the clpC1-1 (white bar) and clpC1-2 (black bar) lines are shown as a percentage of the relative wild-type level (± se, n = 4), which was set at 100%.

Figure 6.

Loss of ClpC1 has no effect on chloroplast import of preproteins or the degradation of mistargeted preproteins. A, ³⁵S-labeled precursor of Rubisco small subunit (pSSU) was imported into chloroplasts isolated from 21-d-old wild type (WT) and clpC1-1 mutant. Samples were taken at selected times during the import assay and separated by denaturing PAGE. The top picture shows a representative import assay, indicating the pSSU (p) and processed mature SSU (m). Rate of import, as measured by the increase in mature SSU, was plotted as a percent of the total radiolabeled pSSU at the start of the assay. Values are averages ± se (n = 3) fitted to a second-order linear regression. B, ³⁵S-labeled precursor of a chimeric OEC33 protein lacking thylakoid membrane targeting sequences was imported into isolated chloroplasts from 21-d-old wild type and clpC1-1 mutant. After preprotein import for 20 min, chloroplasts were treated with thermolysin to halt import and remove any unimported preprotein. Chloroplasts were then incubated at 25°C, with aliquots taken at different time points. Proteins were separated by denaturing PAGE. Shown is a representative assay over 120 min, with the degradation rate of the chimeric OEC33 preprotein quantified relative to the amount of chimeric OEC33 at the 0-min time point, which was set as 100%. Values shown are averages ± se (n = 3).

Figure 7.

Changes in chloroplast protein composition in clpC1 mutants. A, Stromal and thylakoid membrane proteins extracted from isolated chloroplasts of wild type (WT) and clpC1 mutants (clpC1-1 and clpC1-2) were separated by denaturing PAGE. After staining with coomassie blue, proteins showing the greatest quantitative change in the clpC1 mutants that were identifiable by MALDI-TOF MS are indicated with arrowheads. B, Table containing MALDI-TOF data from analyzed proteins. Footnote a, Molecular mass of protein determined by MS; footnote b, molecular mass calculated from PAGE size markers; footnote c, predicted size in amino acids (aa) from the MIPS Arabidopsis database (http://mips.gsf.de); footnote d, AGI gene code for identified protein; footnote e, significant protein scores >73 (P < 0.05); footnote f, peptide match at mass tolerance ± 100 ppm, allowing max one missed cleavage; footnote g, approximate up-regulation of protein based on stained gel bands; footnote h, RH3 found both in stroma and thylakoid (137/203), respectively; footnote i, no stained band could be located in wild type, therefore ∞ = interminable.