The Stromal Processing Peptidase of Chloroplasts is Essential in Arabidopsis, with Knockout Mutations Causing Embryo Arrest after the 16-Cell Stage

Stromal processing peptidase (SPP) is a metalloendopeptidase located in the stroma of chloroplasts, and it is responsible for the cleavage of transit peptides from preproteins upon their import into the organelle. Two independent mutant Arabidopsis lines with T-DNA insertions in the SPP gene were analysed (spp-1 and spp-2). For both lines, no homozygous mutant plants could be detected, and the segregating progeny of spp heterozygotes contained heterozygous and wild-type plants in a ratio of 2∶1. The siliques of heterozygous spp-1 and spp-2 plants contained many aborted seeds, at a frequency of ∼25%, suggesting embryo lethality. By contrast, transmission of the spp mutations through the male and female gametes was found to be normal, and so gametophytic effects could be ruled out. To further elucidate the timing of the developmental arrest, mutant and wild-type seeds were cleared and analysed by Nomarski microscopy. A significant proportion (∼25%) of the seeds in mutant siliques exhibited delayed embryogenesis compared to those in wild type. Moreover, the mutant embryos never progressed normally beyond the 16-cell stage, with cell divisions not completing properly thereafter. Heterozygous spp mutant plants were phenotypically indistinguishable from the wild type, indicating that the spp knockout mutations are completely recessive and suggesting that one copy of the SPP gene is able to produce sufficient SPP protein for normal development under standard growth conditions

Molecular and genetic analyses of Tic20 homologues in Arabidopsis thaliana chloroplasts.

The Tic20 protein was identified in pea (Pisum sativum) as a component of the chloroplast protein import apparatus. In Arabidopsis, there are four Tic20 homologues, termed atTic20-I, atTic20-IV, atTic20-II and atTic20-V, all with predicted topological similarity to the pea protein (psTic20). Analysis of Tic20 sequences from many species indicated that they are phylogenetically unrelated to mitochondrial Tim17-22-23 proteins, and that they form two evolutionarily conserved subgroups [characterized by psTic20/atTic20-I/IV (Group 1) and atTic20-II/V (Group 2)]. Like psTic20, all four Arabidopsis proteins have a predicted transit peptide consistent with targeting to the inner envelope. Envelope localization of each one was confirmed by analysis of YFP fusions. RT-PCR and microarray data revealed that the four genes are expressed throughout development. To assess the functional significance of the genes, T-DNA mutants were identified. Homozygous tic20-I plants had an albino phenotype that correlated with abnormal chloroplast development and reduced levels of chloroplast proteins. However, knockouts for the other three genes were indistinguishable from the wild type. To test for redundancy, double and triple mutants were studied; apart from those involving tic20-I, none was distinguishable from the wild type. The tic20-I tic20-II and tic20-I tic20-V double mutants were albino, like the corresponding tic20-I parent. In contrast, tic20-I tic20-IV double homozygotes could not be identified, due to gametophytic and embryonic lethality. Redundancy between atTic20-I and atTic20-IV was confirmed by complementation analysis. Thus, atTic20-I and atTic20-IV are the major functional Tic20 isoforms in Arabidopsis, with partially overlapping roles. While the Group 2 proteins have been conserved over approximately 1.2 billion (1.2 × 10(9) ) years, they are not essential for normal development