The initial stages of preprotein import into chloroplasts are mediated by the receptor GTPase Toc159. In Arabidopsis thaliana, Toc159 is encoded by a small gene family: atTOC159, atTOC132, atTOC120, and atTOC90. Phylogenetic analysis suggested that at least two distinct Toc159 subtypes, characterized by atToc159 and atToc132/atToc120, exist in plants. atTOC159 was strongly expressed in young, photosynthetic tissues, whereas atTOC132 and atTOC120 were expressed at a uniformly low level and so were relatively prominent in nonphotosynthetic tissues. Based on the albino phenotype of its knockout mutant, atToc159 was previously proposed to be a receptor with specificity for photosynthetic preproteins. To elucidate the roles of the other isoforms, we characterized Arabidopsis knockout mutants for each one. None of the single mutants had strong visible phenotypes, but toc132 toc120 double homozygotes appeared similar to toc159, indicating redundancy between atToc132 and atToc120. Transgenic complementation studies confirmed this redundancy but revealed little functional overlap between atToc132/atToc120 and atToc159 or atToc90. Unlike toc159, toc132 toc120 caused structural abnormalities in root plastids. Furthermore, when proteomics and transcriptomics were used to compare toc132 with ppi1 (a receptor mutant that is specifically defective in the expression, import, and accumulation of photosynthetic proteins), major differences were observed, suggesting that atToc132 (and atToc120) has specificity for nonphotosynthetic proteins. When both atToc159 and the major isoform of the other subtype, atToc132, were absent, an embryo-lethal phenotype resulted, demonstrating the essential role of Toc159 in the import mechanism.

A multisubunit translocon of the inner envelope membrane, termed Tic, mediates the late stages of protein import into chloroplasts. Membrane proteins, Tic110 and Tic40, and a stromal chaperone, Hsp93, have been proposed to function together within the Tic complex. In Arabidopsis, single genes, atTIC110 and atTIC40, encode the Tic proteins, and two homologous genes, atHSP93-V and atHSP93-III, encode Hsp93. These four genes exhibited relatively uniform patterns of expression, suggesting important roles for plastid biogenesis throughout development and in all tissues. To investigate the roles played by these proteins in vivo, we conducted a comparative study of T-DNA knockout mutants for each Tic gene, and for the most abundantly expressed Hsp93 gene, atHSP93-V. In the homozygous state, the tic110 mutation caused embryo lethality, implying an essential role for atTic110 during plastid biogenesis. Homozygous tic110 embryos exhibited retarded growth, developmental arrest at the globular stage and a 'raspberry-like' embryo-proper phenotype. Heterozygous tic110 plants, and plants homozygous for the tic40 and hsp93-V mutations, exhibited chlorosis, aberrant chloroplast biogenesis, and inefficient chloroplast-import of both photosynthetic and non-photosynthetic preproteins. Non-additive interactions amongst the mutations occurred in double mutants, suggesting that the three components may cooperate during chloroplast protein import.

SummaryIn Arabidopsis, Hsp93 is encoded by two genes, atHSP93-V and atHSP93-III. We identified two T-DNA mutants for atHSP93-III: one being a partial 'knockdown' (hsp93-III-1) and the other a complete 'knockout' (hsp93-III-2). Homozygotes for both mutants were indistinguishable from wild type. We crossed each mutant to an atHSP93-V knockout, and identified double mutants with strongly chlorotic phenotypes. This implied redundancy, which was confirmed by the complementation of mildly chlorotic hsp93-V plants by atHSP93-III over-expression. While the hsp93-V hsp93-III-1 mutant was doubly homozygous, the second double mutant was heterozygous for hsp93-III-2 (genotype: hsp93-V/hsp93-V; +/hsp93-III-2). Attempts to identify an hsp93-V hsp93-III-2 double homozygote were unsuccessful, indicating that the Hsp93 pool is essential for viability. Consistently, siliques of the second double mutant contained aborted seeds (because of a block in the zygoteembryo transition) and failed ovules (because of a moderate defect in female gametophytes). Double-mutant plants were chlorophyll-deficient, contained under-developed chloroplasts, and exhibited stunted growth. In import assays using a chimeric pre-protein (plastocyanin transit peptide fused to dihydrofolate reductase; PC-DHFR), a clear defect was observed in hsp93-V hsp93-III-1 chloroplasts. Interestingly, while denaturation or stabilization of the DHFR moiety had a strong effect on import efficiency in the wild type, no such effects were observed with double-mutant (or tic40) chloroplasts. This indicated that pre-protein unfolding is not ratelimiting for import into mutant chloroplasts, and suggested that (unlike the situation in mitochondria) the inner membrane import machinery does not contribute to pre-protein unfolding at the organellar surface.

This study was carried out to examine the possibility of initiation of lipid peroxidation in the lung of Wistar albino male rats stressed by immobilization. The effects of vitamin E supplementation were also investigated. We found that immobilization of rats with normal pulmonary content of vitamin E caused lipid peroxidation in the lung. Decrease of the lung content of unsaturated fatty acids and vitamin E was also established. The immobilization-induced changes of all of these parameters were significantly inhibited by vitamin E injection (100 mg/kg body weight) for 7 days. A possible sequence of events leading to the initiation of lipid peroxidation and lung cell membrane damage in rats stressed by immobilization is discussed.