Abstract
The clpr2-1 mutant is delayed in development due to reduction of the chloroplast ClpPR protease complex. To understand the role of Clp proteases in plastid biogenesis and homeostasis, leaf proteomes of young seedlings of clpr2-1 and wild type were compared using large scale mass spectrometry-based quantification using an LTQ-Orbitrap and spectral counting with significance determined by G-tests. Virtually only chloroplast-localized proteins were significantly affected, indicating that the molecular phenotype was confined to the chloroplast. A comparative chloroplast stromal proteome analysis of fully developed plants was used to complement the data set. Chloroplast unfoldase ClpB3 was strongly up-regulated in both young and mature leaves, suggesting widespread and persistent protein folding stress. The importance of ClpB3 in the clp2-1 mutant was demonstrated by the observation that a CLPR2 and CLPB3 double mutant was seedling-lethal. The observed up-regulation of chloroplast chaperones and protein sorting components further illustrated destabilization of protein homeostasis. Delayed rRNA processing and up-regulation of a chloroplast DEAD box RNA helicase and polynucleotide phosphorylase, but no significant change in accumulation of ribosomal subunits, suggested a bottleneck in ribosome assembly or RNA metabolism. Strong up-regulation of a chloroplast translational regulator TypA/BipA GTPase suggested a specific response in plastid gene expression to the distorted homeostasis. The stromal proteases PreP1,2 were up-regulated, likely constituting compensation for reduced Clp protease activity and possibly shared substrates between the ClpP and PreP protease systems. The thylakoid photosynthetic apparatus was decreased in the seedlings, whereas several structural thylakoid-associated plastoglobular proteins were strongly up-regulated. Two thylakoid-associated reductases involved in isoprenoid and chlorophyll synthesis were up-regulated reflecting feedback from rate-limiting photosynthetic electron transport. We discuss the quantitative proteomics data and the role of Clp proteolysis using a "systems view" of chloroplast homeostasis and metabolism and provide testable hypotheses and putative substrates to further determine the significance of Clp-driven proteolysis.
Highlights
The clpr2-1 mutant is delayed in development due to reduction of the chloroplast ClpPR protease complex
Large Scale Leaf Protein Quantification of wt and clpr2-1 Seedlings by Spectral Counting—To determine the effect of reduced clpr2-1 expression on the developing leaf proteome, clpr2-1 and wt plants were grown side by side, and seedlings were harvested at growth stages 1.07 and 1.14 when plants have, respectively, 7 and 14 leaves (Fig. 1A)
We showed that the clpr2-1 mutant has a 2–3-fold reduced accumulation of the 325-kDa ClpPRT complex in mature leaves [36]
Summary
Plant Growth of clpr for Proteome Analysis—A. thaliana clpr and wt (Col-0 background) plants were grown side by side on soil under short day (10/14-h light/dark) with 120 mol photons1⁄7mϪ21⁄7sϪ1 light at 23/19 °C in controlled growth chambers (Conviron). Similar to the LTQ-Orbitrap analysis of total leaf proteome, 400 g of isolated chloroplast stromal proteins (wt or clpr2-1) were separated by SDS-PAGE, and each gel lane was cut in 12 gel slices and manually processed and digested with trypsin followed by MS/MS analysis. The Plastid Proteomics Database—Mass spectrometry-based information of all identified proteins was extracted from the Mascot search pages and filtered for significance (e.g. minimum ion scores, etc.), ambiguities, and shared spectra as described previously [14] This information includes MOWSE scores, number of matching peptides, number of matched MS/MS spectra (counts), number of unique and adjusted counts, highest peptide score, highest peptide error (in ppm), lowest absolute error (ppm), sequence coverage, and tryptic peptide sequences. Information for a particular accession can be found on each “protein report page.” The MapMan bin system [50] was used for functional assignment, and proteins were reassigned to other bins if needed
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