As a relative increase in nonpolysomal chloroplast mRNAs in the cps2 mutant, but a substantial fraction of mRNAs still remained associated with multiple ribosomes [11]. In this mutant, chloroplast protein translation was only very mildly affected. The effects of the cpLEPA mutation on the association ofcpLEPA in Chloroplast TranslationFigure 4. Accumulation and Synthesis of Chloroplast Proteins in cplepa-1 Plants. A:Immunoblot analysis of total protein extracts from wildtype and cplepa-1 plants. Wild-type and cplepa-1 plants grown on soil at a photon flux density of 120 mmol m22 s21 were used. For wild-type and cplepa-1 plants, 10 mg of total proteins were loaded. The antibodies used are indicated on the right. Actin served as a control to normalize the protein HIF-2��-IN-1 biological activity levels. Similar results were obtained in two additional independent experiments. B: Pulse labeling of thylakoid proteins. Primary leaves of 12-day-old plants were radiolabeled with [35] S-methionine in the presence of cycloheximide for 20 min. The thylakoid membranes were isolated, separated by SDS-urea-PAGE and visualized autoradiographically, lanes were loaded with equal protein contend. C: A coomassie blue-stained gel is presented to show that equal amounts of proteins were loaded. doi:10.1371/journal.pone.0049746.gcpLEPA in Chloroplast TranslationcpLEPA in Chloroplast TranslationFigure 5. Polysome Association Analysis for Chloroplast Transcripts in Wild-Type and cplepa-1 Plants. The association of psbA, psbB, atpB, psaA and rrn23 transcripts with polysomes. Total extracts from wild-type and cplepa-1 leaves grown on soil for 3 weeks at 120 mmol m22 s21 were fractionated on 15 ?5 sucrose gradients. Ten fractions of equal volume were collected from the top to the bottom of the sucrose gradients, and equal proportions of the RNA purified from each fraction were analyzed by northern-blot analysis. The rRNAs were detected by ethidium bromide (EtBr) staining. The size of the transcript (in kb) is shown. doi:10.1371/journal.pone.0049746.gthe psbA, psbB, atpB, and psaA/B mRNAs with ribosomes were similar to those of cps2 [11] (Figure 5). In vivo protein labeling experiments showed a moderately decreased synthesis rate for the chloroplast-encoded proteins, which may account for the accumulation of photosynthetic proteins (Figure 4B). Biochemical analysis of LEPA in E. coli has demonstrated its function as a translation factor in vitro. The elongation cycle of protein synthesis is characterized by tRNA movement between pre-translocation (PRE) and post-translocation (POST) complexes. Under stress conditions, such as high salt concentration or low temperature, translocation could be blocked, possibly by perturbation of the ribosome structure [9]. LEPA could effectively compete with EFG for binding to the PRE complex. This binding could lead to 
the formation of an intermediate complex, I3, which could allow for the JSI-124 price correction of an incorrect translocation event by replacing LEPA?GDP with EF-G?GTP (EF-G is present at considerably higher concentrations in bacterial cells compared with LEPA) [10]. A high Mg2+concentration could stabilize the I3 complex by inhibiting the conversion of I3 to a PRE complex, which explains why LEPA accelerates protein synthesis at increased Mg2+concentrations [6,10]. Our study is consistent with the proposed function of LEPA as a translation factor that contributes to the efficiency of protein synthesis. In summary, we have demonstrated the physiological role of.As a relative increase in nonpolysomal chloroplast mRNAs in the cps2 mutant, but a substantial fraction of mRNAs still remained associated with multiple ribosomes [11]. In this mutant, chloroplast protein translation was only very mildly affected. The effects of the cpLEPA mutation on the association ofcpLEPA in Chloroplast TranslationFigure 4. Accumulation and Synthesis of Chloroplast Proteins in cplepa-1 Plants. A:Immunoblot analysis of total protein extracts from wildtype and cplepa-1 plants. Wild-type and cplepa-1 plants grown on soil at a photon flux density of 120 mmol m22 s21 were used. For wild-type and cplepa-1 plants, 10 mg of total proteins were loaded. The antibodies used are indicated on the right. Actin served as a control to normalize the protein levels. Similar results were obtained in two additional independent experiments. B: Pulse labeling of thylakoid proteins. Primary leaves of 12-day-old plants were radiolabeled with [35] S-methionine in the presence of cycloheximide for 20 min. The thylakoid membranes were isolated, separated by SDS-urea-PAGE and visualized autoradiographically, lanes were loaded with equal protein contend. C: A coomassie blue-stained gel is presented to show that equal amounts of proteins were loaded. doi:10.1371/journal.pone.0049746.gcpLEPA in Chloroplast TranslationcpLEPA in Chloroplast TranslationFigure 5. Polysome Association Analysis for Chloroplast Transcripts in Wild-Type and cplepa-1 Plants. The association of psbA, psbB, atpB, psaA and rrn23 transcripts with polysomes. Total extracts from wild-type and cplepa-1 leaves grown on soil for 3 weeks at 120 mmol m22 s21 were fractionated on 15 ?5 sucrose gradients. Ten fractions of equal volume were collected from the top to the bottom of the sucrose gradients, and equal proportions of the RNA purified from each fraction were analyzed by northern-blot analysis. The rRNAs were detected by ethidium bromide (EtBr) staining. The size of the transcript (in kb) is shown. doi:10.1371/journal.pone.0049746.gthe psbA, psbB, atpB, and psaA/B mRNAs with ribosomes were similar to those of cps2 [11] (Figure 5). In vivo protein labeling 
experiments showed a moderately decreased synthesis rate for the chloroplast-encoded proteins, which may account for the accumulation of photosynthetic proteins (Figure 4B). Biochemical analysis of LEPA in E. coli has demonstrated its function as a translation factor in vitro. The elongation cycle of protein synthesis is characterized by tRNA movement between pre-translocation (PRE) and post-translocation (POST) complexes. Under stress conditions, such as high salt concentration or low temperature, translocation could be blocked, possibly by perturbation of the ribosome structure [9]. LEPA could effectively compete with EFG for binding to the PRE complex. This binding could lead to the formation of an intermediate complex, I3, which could allow for the correction of an incorrect translocation event by replacing LEPA?GDP with EF-G?GTP (EF-G is present at considerably higher concentrations in bacterial cells compared with LEPA) [10]. A high Mg2+concentration could stabilize the I3 complex by inhibiting the conversion of I3 to a PRE complex, which explains why LEPA accelerates protein synthesis at increased Mg2+concentrations [6,10]. Our study is consistent with the proposed function of LEPA as a translation factor that contributes to the efficiency of protein synthesis. In summary, we have demonstrated the physiological role of.