Plastid ribosomal protein S5 is involved in photosynthesis, plant development, and cold stress tolerance in Arabidopsis

Abstract

Plastid ribosomal proteins are essential components of protein synthesis machinery and have diverse roles in plant growth and development. Mutations in plastid ribosomal proteins lead to a range of developmental phenotypes in plants. However, how they regulate these processes is not fully understood, and the functions of some individual plastid ribosomal proteins remain unknown. To identify genes responsible for chloroplast development, we isolated and characterized a mutant that exhibited pale yellow inner leaves with a reduced growth rate in Arabidopsis. The mutant (rps5) contained a missense mutation of plastid ribosomal protein S5 (RPS5), which caused a dramatically reduced abundance of chloroplast 16S rRNA and seriously impaired 16S rRNA processing to affect ribosome function and plastid translation. Comparative proteomic analysis revealed that the rps5 mutation suppressed the expression of a large number of core components involved in photosystems I and II as well as many plastid ribosomal proteins. Unexpectedly, a number of proteins associated with cold stress responses were greatly decreased in rps5, and overexpression of the plastid RPS5 improved plant cold stress tolerance. Our results indicate that RPS5 is an important constituent of the plastid 30S subunit and affects proteins involved in photosynthesis and cold stress responses to mediate plant growth and development.

Keywords: Arabidopsis; RPS5.; cold stress tolerance; photosynthesis; plastid ribosomal protein; proteomics.

Fig. 1.

Molecular cloning of rps5. (A) Growth phenotype of wild-type and rps5 mutant plants at 20 and 40 days old, respectively. Scale bars=1cm. (B) Positional cloning of rps5. Homozygous rps5 F₂ seedlings from a total of 1258 plants were used for fine mapping. (C) RPS5 gene structure and the locations of the missense mutation for rps5 and T-DNA insertion for Salk_095863. Exons and introns are shown as solid boxes and lines, respectively. The T-DNA insertion is indicated with a triangle and the single nucleotide change in rps5 is indicated with a vertical arrow. (D) Complementation of the rps5 mutant. The rps5 phenotype was complemented with RPS5 cDNA (35S:RPS5) or genomic DNA (RPS5:RPS5). Plants were 4 weeks old. (E, F) Expression of RPS5 in wild-type and rps5 plants detected by semi-quantitative RT-PCR and qRT-PCR, respectively. Data represent means±SD from three biological replicates with three technical repeats. (This figure is available in colour at JXB online.)

Fig. 2.

Partial sequence alignment, secondary structure prediction, and subcellular localization of RPS5. (A) N-terminal domain sequence alignment of RPS5 from Arabidopsis (A. thaliana, AEC08888), rice (Oryza sativa, NP_001050474), maize (Zea mays, NP_001150762), potato (Solanum tuberosum, XP_006347020), tomato (Solanum lycopersicum, XP_004232896), Methanococcus vannielii (P14036), Chlamydia trachomatis (P0A4C8), Shigella flexneri (P0A7W6), and Cyanophora paradoxa (P23402). The vertical arrow indicates the mutation site. (B) Predicted secondary structure of the RPS5 N-terminal domain. The vertical arrow indicates the Glu substitution site in the mutant protein. (C) Wild-type RPS5 (top) and mutant rps5 (bottom) are localized in chloroplasts of 4-week-old tobacco leaves transiently expressing GFP fusion proteins. Bars=25 µm. (This figure is available in colour at JXB online.)

Fig. 3.

Chloroplast rRNA accumulation and processing in wild-type (WT), mutant lines (rps5 and Salk_095863), and a complementation RPS5-overexpressing line (35S:RPS5). (A) rRNA accumulation pattern in an ethidium bromide-stained gel. The mutant lines had reduced levels of 16S rRNA, identified based on Kleinknecht et al. (2014) and Yu et al. (2008). (B) Schematic diagram of the chloroplast rrn operon in Arabidopsis. The probes used for northern blot analysis are marked by black lines under the operon with asterisks. (C) Northern blot analysis of chloroplast 4.5S, 5S, 16S, 23S, and cytoplasmic18S rRNAs. Total RNA (3 µg) from 4-week-old leaves was used for northern blot experiments. Equal loading controls for northern blot analysis are shown in Supplementary Figure S5. (D) Quantification of the mature bands in C using ImageJ (http://www.di.uq.edu.au/sparqimagejblots). Asterisks in 16S indicate significant differences (p<0.05).

Fig. 4.

Comparative proteomic analysis of the differentially expressed proteins in the rps5 proteome. (A) Numbers of up- and down-regulated proteins showing significant differences between the wild-type and rps5 proteome. (B) Relative level of RPS5 protein in wild-type and rps5 plants. Values are means±SD from three biological replicates. (C, D) Subcellular localization of (C) down-regulated and (D) up-regulated proteins in the rps5 proteome. (E, F) Functional classification of (E) down-regulated and (F) up-regulated proteins in the rps5 proteome. (This figure is available in colour at JXB online.)

Fig. 5.

2D gel-based proteomic analysis of wild-type (top) and rps5 (bottom) plants. The differentially expressed proteins in wild-type and mutant plants are labelled by black circles and the enlarged 1507/1508 spots are indicated in red circles. Spots 1507/1508 indicate RPS5 protein. (This figure is available in colour at JXB online.)

Fig. 6.

Role of RPS5 in cold stress tolerance. (A) Relative levels of proteins associated with the cold stress response in rps5 plants. Values represent means±SD from three biological replicates. (B) Phenotypes of wild-type (WT), Salk_095863, rps5, and two RPS5 overexpression lines grown on Murashige and Skoog medium at 4 °C for 6 weeks. (C) Average weight per seedling grown at 4 °C for 6 weeks. (D) Chlorophyll content of seedlings grown at 4 °C for 6 weeks. Values are means±SD from four biological replicates. (This figure is available in colour at JXB online.)