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.