The caseinolytic protease complex component CLPC1 in Arabidopsis maintains proteome and RNA homeostasis in chloroplasts

Abstract

Background: Homeostasis of the proteome is critical to the development of chloroplasts and also affects the expression of certain nuclear genes. CLPC1 facilitates the translocation of chloroplast pre-proteins and mediates protein degradation.

Results: We found that proteins involved in photosynthesis are dramatically decreased in their abundance in the clpc1 mutant, whereas many proteins involved in chloroplast transcription and translation were increased in the mutant. Expression of the full-length CLPC1 protein, but not of the N-terminus-deleted CLPC1 (ΔN), in the clpc1 mutant background restored the normal levels of most of these proteins. Interestingly, the ΔN complementation line could also restore some proteins affected by the mutation to normal levels. We also found that that the clpc1 mutation profoundly affects transcript levels of chloroplast genes. Sense transcripts of many chloroplast genes are up-regulated in the clpc1 mutant. The level of SVR7, a PPR protein, was affected by the clpc1 mutation. We showed that SVR7 might be a target of CLPC1 as CLPC1-SVR7 interaction was detected through co-immunoprecipitation.

Conclusion: Our study indicates that in addition to its role in maintaining proteome homeostasis, CLPC1 and likely the CLP proteasome complex also play a role in transcriptome homeostasis through its functions in maintaining proteome homeostasis.

Keywords: CLPC1; Chloroplast; Homeostasis; Proteome; SVR7; Transcriptome.

Fig. 1

Morphology of the wild type, clpc1, ΔN as well as CP seedlings in soil (left panels) and in the medium (right panel). WS, the wild type (WS ecotype); clpc1, the clpc1 mutant; ΔN, the clpc mutant expressing N-terminus-truncated CLPC1; CP, the clpc mutant expressing the full-length wild-type CLPC1

Fig. 2

Relative expression levels of sense transcripts in the clpc1 mutant and its complementation lines. Shown are means and SDs from three replicates. qRT-PCR was conducted using gene-specific primers (Additional file 1: Table S2) normalized against the expression of the ACTIN2 Gene. WS, the wild type; clpc1, the clpc1 mutant; ΔN, clpc1 expressing N-terminus-truncated CLPC1; CP, clpc1 expressing the full-length wild-type CLPC1

Fig. 3

Overexpressing CLPC2 in the hsp93V/clpc1 mutant causes chlorosis phenotypes under normal light conditions. Seedlings were transferred to soil from MS plates and the pictures were taken 10 days later. hsp93V, a clpc1 knockout allele in the Col-0 background; 1.4.3 and 1.4.4 are two independent transgenic lines overexpressing CLPC2 in the hsp93v/clpc1 knockout mutant background

Fig. 4

Four unique peptides were identified in a Co-IP experiment using anti-GFP antibody to pull down SVR7-GFP tag. Upper panels: alignment among CLPC1, CLPC2 and the identified peptides (P). Lower panels: Spectra of the four unique peptides

Fig. 5

The expression level of CLPC1 and CLPC2 in seedlings of the indicated genotypes relative to that in the wild type plants. Shown are means and SD from 3 replicates. qRT-PCR was conducted using gene-specific primers (Additional file 1: Table S2) normalized against the expression of the ACTIN2 Gene. WS, the wild type; clpc1, the clpc1 mutant; ΔN, clpc1 expressing N-terminus-truncated CLPC1; CP, clpc1 expressing the full-length wild-type CLPC1

Fig. 6

CLPC1’s possible roles in directly or indirectly mediating chloroplast protein and RNA homeostasis. Arrows indicate positive regulation of the abundance of the indicated proteins or RNAs; Bars indicate negative regulation of the abundance of the indicated proteins or RNAs, and double arrows indicate interaction. Solid lines represent regulation supported by experimental evidence; dashed lines denote hypothetical regulation