Subunits of the Plastid ClpPR Protease Complex Have Differential Contributions to Embryogenesis, Plastid Biogenesis, and Plant Development inArabidopsis   

Abstract

The plastid ClpPR protease complex in Arabidopsis thaliana consists of five catalytic ClpP and four noncatalytic ClpR subunits. An extensive analysis of the CLPR family and CLPP5 is presented to address this complexity. Null alleles for CLPR2 and CLPR4 showed delayed embryogenesis and albino embryos, with seedling development blocked in the cotyledon stage; this developmental block was overcome under heterotrophic conditions, and seedlings developed into small albino to virescent seedlings. By contrast, null alleles for CLPP5 were embryo lethal. Thus, the ClpPR proteins make different functional contributions. To further test for redundancies and functional differences between the ClpR proteins, we overexpressed full-length cDNAs for ClpR1, R2, R3, R4 in clpr1, clpr2 and clpr4 mutants. This showed that overexpression of ClpR3 can complement for the loss of ClpR1, but not for the loss of ClpR2 or ClpR4, indicating that ClpR3 can functionally substitute ClpR1. By contrast, ClpR1, R2 and R4 could not substitute each other. Double mutants of weak CLPR1 and 2 alleles were seedling lethal, showing that a minimum concentration of different ClpR proteins is essential for Clp function. Microscopy and large-scale comparative leaf proteome analyses of a CLPR4 null allele demonstrate a central role of Clp protease in chloroplast biogenesis and protein homeostasis; substrates are discussed. Lack of transcriptional and translational feedback regulation within the CLPPR gene family indicates that regulation of Clp activity occurs through Clp complex assembly and substrate delivery.

APO1 Promotes the Splicing of Chloroplast Group II Introns and Harbors a Plant-Specific Zinc-Dependent RNA Binding Domain

“…Analysis of psaA/B mRNA translation and NDH and LHC1 protein levels in ycf3 mutants (Ruf et al, 1997;Landau et al, 2009) can be used to address whether Ycf3 indeed plays these unanticipated roles. The reduced splicing of the clpP mRNA may also contribute to the protein defects in apo1 mutants, as the Clp protease complex plays wide-ranging roles in chloroplast biogenesis and homeostasis (Kim et al, 2009). …”

Modified Clp Protease Complex in the ClpP3 Null Mutant and Consequences for Chloroplast Development and Function in Arabidopsis

“…Interestingly, the severity of the phenotypes for the various CLPPR null mutants differs greatly (for review, see Olinares et al, 2011b). 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).…”

“…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). The Arabidopsis clpr1-1 null mutant could be maintained on soil and produced viable seeds, even if they showed a virescent phenotype (Koussevitzky et al, 2007).…”

“…The Arabidopsis clpr1-1 null mutant could be maintained on soil and produced viable seeds, even if they showed a virescent phenotype (Koussevitzky et al, 2007). Overexpression of CLPR3, but not CLPR2 or CLPR4, in clpr1-1 led to full complementation, indicating that ClpR1 is partially redundant to ClpR3 (Kim et al, 2009). In addition, ClpP1, the only plastid-encoded Clp subunit, was shown to be essential for leaf development in tobacco (Nicotiana tabacum; Shikanai et al, 2001;Kuroda and Maliga, 2003).…”

“…Furthermore, we quantified ClpT1/ClpT2 in the various Clp assemblies using immunodetection. Previously, we determined the leaf proteome phenotypes of clpr2-1 (Zybailov et al, 2009a) and clpr4-1 (Kim et al, 2009) using MS-based spectral counting methodology. These studies identified approximately 2,800 proteins, and quantification revealed that the strongest effects occurred within the chloroplast, which is consistent with their yellow/pale-green phenotype and delayed growth.…”