Ron microscope (Jem-1230; JEOL).Materials and Methods Plant Material and Growth ConditionsThe cplepa-1 (T-DNA insertion line, Salk_140697) and cplepa-2 (T-DNA insertion line, CS464145) MedChemExpress Fruquintinib mutants were obtained from ABRC, and the homozygous mutants were verified by PCR using the primer pairs LEPA-LP and LEPA-RP as well as LEPAGKF+LEPA-GKR (for primer sequences, see Table S1). The TDNA insertion was confirmed by PCR and sequencing with the primers SALKLBb1 and LEPA-LP for the cplepa-1 MedChemExpress 1113-59-3 mutant and with the primers GABILB and LEPA-GKR for the cplepa-2 mutant. Wild type and mutant seeds were sterilized with 10 sodium hypochlorite for 15 min, washed five times with distilled water, and placed on solid MS medium [24] supplemented with sucrose as needed. Wild type and mutant seeds were sown and grown on soil according to a standard protocol. To ensure synchronized germination, the seeds were kept in the dark at 4uC for two days. The Arabidopsis plants were kept in a growth chamber at 22uC with a 12-h photoperiod at a photon flux density of 120 mmol m22 s21.In vivo Protein Labeling AssaysIn vivo protein labeling was performed essentially according to Meurer et al [26]. For pulse labeling, primary leaves from 12-d-old plants were labeled with 1 mCi/mL [35S]-Met in the presence of 20 mg/mL cycloheximide for 20 min at 25uC. After labeling, the leaves were washed twice with homogenization buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 2 mM EDTA) and ground with 300 mL of the same buffer. The thylakoid membranes were isolated and separated by SDS-PAGE, and the labeled proteins were visualized by autoradiography.Chloroplast and Thylakoid Membrane PreparationIntact chloroplasts were fractionated into envelope, stromal and thylakoid membrane fractions as described previously [27?9]. The thylakoid membranes were isolated according to Cai et al [30]. The chlorophyll contents were measured as described previously [31].Photoinhibitory TreatmentDetached wild type and cplepa-1 mutant leaves were floated face down on water and illuminated under a photon flux density of 1,000 mmol m22 s21, and the chlorophyll fluorescence was measured at 0.5 h, 1 h, 2 h, 3 h, and 4 h after exposure to high light using a PAM-2000 fluorometer (Walz). The temperature wascpLEPA in Chloroplast TranslationFigure 6. 24272870 Northern Blot Analysis for Chloroplast Transcripts in Wild-Type and cplepa-1 Plants. Northern blot analysis of the chloroplast transcripts psbA, psbB, psbD, atpB, psaA, petB, rbcL, rpoA, rpoB and rrn23 in wild-type and cplepa-1 mutant plants. Each lane was loaded with 10 mg of total RNA. The plants were grown on soil for 3 weeks under 120 mmol m22 s21 illumination. Additionally, 25S rRNA stained with EtBr was loaded as a control. The size of the transcript (in kb) is shown. doi:10.1371/journal.pone.0049746.gcpLEPA in Chloroplast TranslationFigure 7. Photosensitivity Analysis of cplepa-1 Plants. A: The phenotypes of wild-type (WT) and cplepa-1 mutant plants grown in a growth chamber at 120 mmol m22 s21 in the first two weeks, then transferred to low light (40 mmol m22 s21) or high light (500 mmol m22 s21) for another two weeks. B: The Fv/Fm ratio was measured for detached leaves from wild-type (WT) plants (red circles) and cplepa-1 mutant plants (black squares) following high-light illumination (1,000 mmol m22 s21) in the absence of lincomycin (Lin). C: The Fv/Fm ratio was measured for detached leaves from wild-type (WT) plants (red circles) and cplepa-1 mutant plants (black.Ron microscope (Jem-1230; JEOL).Materials and Methods Plant Material and Growth ConditionsThe cplepa-1 (T-DNA insertion line, Salk_140697) and cplepa-2 (T-DNA insertion line, CS464145) mutants were obtained from ABRC, and the homozygous mutants were verified by PCR using the primer pairs LEPA-LP and LEPA-RP as well as LEPAGKF+LEPA-GKR (for primer sequences, see Table S1). The TDNA insertion was confirmed by PCR and sequencing with the primers SALKLBb1 and LEPA-LP for the cplepa-1 mutant and with the primers GABILB and LEPA-GKR for the cplepa-2 mutant. Wild type and mutant seeds were sterilized with 10 sodium hypochlorite for 15 min, washed five times with distilled water, and placed on solid MS medium [24] supplemented with sucrose as needed. Wild type and mutant seeds were sown and grown on soil according to a standard protocol. To ensure synchronized germination, the seeds were kept in the dark at 4uC for two days. The Arabidopsis plants were kept in a growth chamber at 22uC with a 12-h photoperiod at a photon flux density of 120 mmol m22 s21.In vivo Protein Labeling AssaysIn vivo protein labeling was performed essentially according to Meurer et al [26]. For pulse labeling, primary leaves from 12-d-old plants were labeled with 1 mCi/mL [35S]-Met in the presence of 20 mg/mL cycloheximide for 20 min at 25uC. After labeling, the leaves were washed twice with homogenization buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 2 mM EDTA) and ground with 300 mL of the same buffer. The thylakoid membranes were isolated and separated by SDS-PAGE, and the labeled proteins were visualized by autoradiography.Chloroplast and Thylakoid Membrane PreparationIntact chloroplasts were fractionated into envelope, stromal and thylakoid membrane fractions as described previously [27?9]. The thylakoid membranes were isolated according to Cai et al [30]. The chlorophyll contents were measured as described previously [31].Photoinhibitory TreatmentDetached wild type and cplepa-1 mutant leaves were floated face down on water and illuminated under a photon flux density of 1,000 mmol m22 s21, and the chlorophyll fluorescence was measured at 0.5 h, 1 h, 2 h, 3 h, and 4 h after exposure to high light using a PAM-2000 fluorometer (Walz). The temperature wascpLEPA in Chloroplast TranslationFigure 6. 24272870 Northern Blot Analysis for Chloroplast Transcripts in Wild-Type and cplepa-1 Plants. Northern blot analysis of the chloroplast transcripts psbA, psbB, psbD, atpB, psaA, petB, rbcL, rpoA, rpoB and rrn23 in wild-type and cplepa-1 mutant plants. Each lane was loaded with 10 mg of total RNA. The plants were grown on soil for 3 weeks under 120 mmol m22 s21 illumination. Additionally, 25S rRNA stained with EtBr was loaded as a control. The size of the transcript (in kb) is shown. doi:10.1371/journal.pone.0049746.gcpLEPA in Chloroplast TranslationFigure 7. Photosensitivity Analysis of cplepa-1 Plants. A: The phenotypes of wild-type (WT) and cplepa-1 mutant plants grown in a growth chamber at 120 mmol m22 s21 in the first two weeks, then transferred to low light (40 mmol m22 s21) or high light (500 mmol m22 s21) for another two weeks. B: The Fv/Fm ratio was measured for detached leaves from wild-type (WT) plants (red circles) and cplepa-1 mutant plants (black squares) following high-light illumination (1,000 mmol m22 s21) in the absence of lincomycin (Lin). C: The Fv/Fm ratio was measured for detached leaves from wild-type (WT) plants (red circles) and cplepa-1 mutant plants (black.
