Abstract
The plastid ClpPRT protease consists of two heptameric rings of ClpP1/ClpR1/ClpR2/ClpR3/ClpR4 (the R-ring) and ClpP3/ClpP4/ClpP5/ClpP6 (the P-ring) and peripherally associated ClpT1/ClpT2 subunits. Here, we address the contributions of ClpP3 and ClpP4 to ClpPRT core organization and function in Arabidopsis (Arabidopsis thaliana). ClpP4 is strictly required for embryogenesis, similar to ClpP5. In contrast, loss of ClpP3 (clpp3-1) leads to arrest at the hypocotyl stage; this developmental arrest can be removed by supplementation with sucrose or glucose. Heterotrophically grown clpp3-1 can be transferred to soil and generate viable seed, which is surprising, since we previously showed that CLPR2 and CLPR4 null alleles are always sterile and die on soil. Based on native gels and mass spectrometry-based quantification, we show that despite the loss of ClpP3, modified ClpPR core(s) could be formed, albeit at strongly reduced levels. A large portion of ClpPR subunits accumulated in heptameric rings, with overaccumulation of ClpP1/ClpP5/ClpP6 and ClpR3. Remarkably, the association of ClpT1 to the modified Clp core was unchanged. Large-scale quantitative proteomics assays of clpp3-1 showed a 50% loss of photosynthetic capacity and the up-regulation of plastoglobules and all chloroplast stromal chaperone systems. Specific chloroplast proteases were significantly up-regulated, whereas the major thylakoid protease (FtsH1/FtsH2/FtsH5/FtsH8) was clearly unchanged, indicating a controlled protease network response. clpp3-1 showed a systematic decrease of chloroplast-encoded proteins that are part of the photosynthetic apparatus but not of chloroplast-encoded proteins with other functions. Candidate substrates and an explanation for the differential phenotypes between the CLPP3, CLPP4, and CLPP5 null mutants are discussed.
Highlights
The plastid ClpPRT protease consists of two heptameric rings of ClpP1/ClpR1/ClpR2/ClpR3/ClpR4 and ClpP3/ ClpP4/ClpP5/ClpP6 and peripherally associated ClpT1/ClpT2 subunits
Heterozygous clpp4-1 had a wild-type phenotype, indicative of recessive alleles without a gene dosage effect. clpp4-1 null alleles were recovered in lines transformed with CLPP4 complementary DNA driven by a 13 35S promoter, and reverse transcription (RT)-PCR confirmed complementation, further showing that loss of CLPP4 is responsible for the embryo-lethal phenotype (Fig. 1D)
ClpP4 is essential for embryogenesis similar to ClpP5 but unlike the other nucleusencoded chloroplast Clp core proteins for which null mutants have been tested so far (CLPR1, CLPR2, and CLPR4; Kim et al, 2009)
Summary
The plastid ClpPRT protease consists of two heptameric rings of ClpP1/ClpR1/ClpR2/ClpR3/ClpR4 (the R-ring) and ClpP3/ ClpP4/ClpP5/ClpP6 (the P-ring) and peripherally associated ClpT1/ClpT2 subunits. The Clp protease system is the most abundant and complex stromal protease family in the plastid It consists of five Ser-type Clp proteases (P1 and P3–P6) and four nonproteolytic ClpR subunits (R1–R4), which together constitute the tetradecameric and asymmetric approximately 350-kD Clp protease core formed by two heptameric rings. Complete loss of CLPP5 gene expression is embryo lethal, whereas complete loss of CLPR2 or CLPR4 delayed embryogenesis and resulted in developmental arrest at the cotyledon stage (Kim et al, 2009) This arrest could be broken by growth on Suc, but seedlings remained sterile (Kim et al, 2009; Olinares et al, 2011b). A mutant in CLPR2 with approximately 20% residual CLPR2 mRNA and ClpR2 protein (clpr2-1; Rudella et al, 2006) and antisense lines against CLPP4 and CLPP6 (Sjögren et al, 2006; Zheng et al, 2006) in Arabidopsis exhibited delayed chloroplast and plant development and a virescent or variegated phenotype
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