Fig. 3: Proteomic analyses demonstrating the increased abundance of components of the cytosolic ribosome and the PQC in response to cPOS.

a Volcano plot illustrating the differentially expressed proteins between the WT and ClpD-GFP line #5 on day 3 (left) or day 6 (right) of seedling development. Proteins with a Student’s t-test (two-tailed) p < 0.01 and fold change >1.5 between genotypes were identified as differentially accumulated proteins. PS photosynthesis, Ribo ribosome, 26S 26S proteasome. b Histogram showing intensity-based fractions of peptides mapped to transit peptide regions (P-TPs, indicating precursor accumulation in the cytosol) relative to the total peptide intensities of the proteins from which the P-TP was derived. Data are means ± s.e. (n = 3 biologically independent samples). c Immunoblot analyses showing the preClpD-GFP and preGluTR accumulated in 6-d-old ClpD-GFP #5 seedlings. The whole seedlings were harvested for analyses. The numbers below the immunoblots represent the accumulation of precursors relative to mature proteins (in %). d Interaction network of increase accumulated proteins in ClpD-GFP line #5 on day 6 showing proteins related to the proteasome, ribosome, translation regulation, and chaperone system. The interaction network was generated by STRING³¹ (including physical and functional interactions) and visualized by Cytoscape⁸⁷. e Immunoblot analyses showing the increased abundance of 26S proteasomal subunits (RPT2a and RPT5a), the cytosolic chaperones HSP70 (cy70) and HSP90.1 (cy90.1), a 40S ribosomal subunit (US3Z), and polyubiquitinated proteins in 6-d-old ClpD-GFP line #5 seedlings. cp70, chloroplast HSC70. Actin served as loading control. f Quantification of selected proteins from the immunoblots shown in (e). Data are means ± s.e. (n = 3 biological replicates). In (b, f), two-tailed Student’s t-tests were performed to determine the significance of differences between the WT and ClpD-GFP lines #5 or #6. g Polysome profiles of 6-d-old WT and ClpD-GFP line #5 seedlings. h Genome-wide aggregated normalized P-site counts in the WT and ClpD-GFP line #5 from Ribo-seq data. The regions 9 nt upstream and 18 nt downstream of the start codon, and 9 nt downstream and 18 nt upstream of the stop codon are shown. Source data are provided as a Source Data file.