Chloroplast-encoded transcripts were also investigated by RNA gel blot hybridization using the samecpLEPA in Chloroplast TranslationFigure 2. Immunolocalization and Expression of cpLEPA. A: Immunolocalization CUDC-907 analysis of cpLEPA. The chloroplast, thylakoid, stroma and envelope fractions were subjected to immunoblot analysis with specific antisera against cpLEPA. Equal amounts of protein (20 mg) were loaded in each lane. The lanes marked cplepa-1, cplepa-2 and cplepa-1/35s::cpLEPA were loaded with equal amounts of total protein (20 mg). B: Salt washing of the membranes. The thylakoid membranes were incubated with 250 mM NaCl, 200 mM Na2CO3, 1 M CaCl2 and 6 M urea for 30 min at 4uC. Then, the thylakoid proteins were separated by SDS-PAGE and immunoblotted with anti-LEPA, anti-RbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit) and anti-CP47 antibodies. RbcL and CP47 were used as markers. Thylakoid membrane preparations that had not been subjected to treatment were used as controls. C: Expression patterns of cpLEPA. Upper panel: cpLEPA expression levels in different organs of Arabidopsis, ascpLEPA in Chloroplast Translationdetermined by RT-PCR analysis. RNA samples isolated from seedlings, rosettes, flowers, roots, petiole, cauline tissue and siliques of wild-type plants were reverse-transcribed and subjected to PCR analysis. Middle panel: Transcript levels of cpLEPA in Arabidopsis leaves at 5, 15, 25, 35 and 45 d. Bottom panel: Light-induced accumulation of cpLEPA transcripts. Three-week-old plants grown under medium light (120 mmol m22 s21), low light (40 mmol m22 s21) or high light (500 mmol m22 s21) were used. ACTIN is shown as a control. doi:10.1371/journal.pone.0049746.gmaterial as described in the polysome association experiments. Our results showed that the levels of mRNAs encoding the PsaA subunit of PSI (psaA-psaB-rps14) were reduced to 20 of wild-type levels in the mutant (Figure 6). Except for 23s rRNA, an approximately two fold decrease was also observed in the levels of transcripts encoding the following photosynthetic proteins: D1 (psbA), CP47 (psbB-psbT-psbH-petB-petD), D2 (psbD-psbC), atpB (CF1 b), and RBcL (rbcL) (Figure 6). The levels of chloroplast transcripts examined were not affected in the mutant plants when grown on MS (Figure S4).Increased Sensitivity of the cplepa Mutants to High LightWhen wild-type and cplepa-1 mutant plants that were initially grown at 120 mmol m22 s21 were transferred to low-light and order Crenolanib high-light growth conditions for another two weeks, the growth of the mutants was greatly inhibited under high light. The mutants did not differ from the wild-type plants under low light (Figure 7A). To further determine whether the cplepa-1 mutant is sensitive tohigh light, Fv/Fm was measured in the wild-type and cplepa-1 plants under high-light illumination of 1,000 mmol m22 s21. In the 15857111 absence of lincomycin, within 2 h of illumination at a light intensity of 1,000 mmol m22 s21, Fv/Fm declined in the wild-type and mutant leaves to approximately 73 and 55 of the darkadapted values, respectively. After 4 h of illumination, Fv/Fm declined in the wild-type and mutant leaves to approximately 60 and 40 of the dark-adapted values, respectively (Figure 7B). These results clearly demonstrated the increased photosensitivity of the mutants. In the presence of lincomycin, the decrease in Fv/ Fm was more rapid and continued until the Fv/Fm values approached approximately 10 of the dark-adapted values.Chloroplast-encoded transcripts were also investigated by RNA gel blot hybridization using the samecpLEPA in Chloroplast TranslationFigure 2. Immunolocalization and Expression of cpLEPA. A: Immunolocalization analysis of cpLEPA. The chloroplast, thylakoid, stroma and envelope fractions were subjected to immunoblot analysis with specific antisera against cpLEPA. Equal amounts of protein (20 mg) were loaded in each lane. The lanes marked cplepa-1, cplepa-2 and cplepa-1/35s::cpLEPA were loaded with equal amounts of total protein (20 mg). B: Salt washing of the membranes. The thylakoid membranes were incubated with 250 mM NaCl, 200 mM Na2CO3, 1 M CaCl2 and 6 M urea for 30 min at 4uC. Then, the thylakoid proteins were separated by SDS-PAGE and immunoblotted with anti-LEPA, anti-RbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit) and anti-CP47 antibodies. RbcL and CP47 were used as markers. Thylakoid membrane preparations that had not been subjected to treatment were used as controls. C: Expression patterns of cpLEPA. Upper panel: cpLEPA expression levels in different organs of Arabidopsis, ascpLEPA in Chloroplast Translationdetermined by RT-PCR analysis. RNA samples isolated from seedlings, rosettes, flowers, roots, petiole, cauline tissue and siliques of wild-type plants were reverse-transcribed and subjected to PCR analysis. Middle panel: Transcript levels of cpLEPA in Arabidopsis leaves at 5, 15, 25, 35 and 45 d. Bottom panel: Light-induced accumulation of cpLEPA transcripts. Three-week-old plants grown under medium light (120 mmol m22 s21), low light (40 mmol m22 s21) or high light (500 mmol m22 s21) were used. ACTIN is shown as a control. doi:10.1371/journal.pone.0049746.gmaterial as described in the polysome association experiments. Our results showed that the levels of mRNAs encoding the PsaA subunit of PSI (psaA-psaB-rps14) were reduced to 20 of wild-type levels in the mutant (Figure 6). Except for 23s rRNA, an approximately two fold decrease was also observed in the levels of transcripts encoding the following photosynthetic proteins: D1 (psbA), CP47 (psbB-psbT-psbH-petB-petD), D2 (psbD-psbC), atpB (CF1 b), and RBcL (rbcL) (Figure 6). The levels of chloroplast transcripts examined were not affected in the mutant plants when grown on MS (Figure S4).Increased Sensitivity of the cplepa Mutants to High LightWhen wild-type and cplepa-1 mutant plants that were initially grown at 120 mmol m22 s21 were transferred to low-light and high-light growth conditions for another two weeks, the growth of the mutants was greatly inhibited under high light. The mutants did not differ from the wild-type plants under low light (Figure 7A). To further determine whether the cplepa-1 mutant is sensitive tohigh light, Fv/Fm was measured in the wild-type and cplepa-1 plants under high-light illumination of 1,000 mmol m22 s21. In the 15857111 absence of lincomycin, within 2 h of illumination at a light intensity of 1,000 mmol m22 s21, Fv/Fm declined in the wild-type and mutant leaves to approximately 73 and 55 of the darkadapted values, respectively. After 4 h of illumination, Fv/Fm declined in the wild-type and mutant leaves to approximately 60 and 40 of the dark-adapted values, respectively (Figure 7B). These results clearly demonstrated the increased photosensitivity of the mutants. In the presence of lincomycin, the decrease in Fv/ Fm was more rapid and continued until the Fv/Fm values approached approximately 10 of the dark-adapted values.
