CpLEPA in efficient photosynthesis in higher plants. In addition, we have

CpLEPA in efficient photosynthesis in higher plants. In addition, we have presented evidence highlighting the importance of this protein for chloroplast translation, which provides further insights into the conserved function of LEPA in chloroplast protein synthesis.maintained at 22uC throughout the photoinhibitory treatments. The synthesis of HIV-RT inhibitor 1 manufacturer chloroplast-encoded proteins was blocked by incubating detached leaves with 1 mM lincomycin at low light (20 mmol m22 s21) for 3 h before photoinhibition treatment. To investigate the effects of high light on plant growth, we transferred 2-week-old Arabidopsis plants grown on soil under normal illumination of 120 mmol m22 s21 to 500 mmol m22 s21for 1485-00-3 chemical information another 2 weeks.ComplementationTo complement the cpLEPA mutation, a full-length cpLEPA cDNA was amplified using nested antisense primers (LEPAH-F, LEPAH-R1 and LEPAH-R2) with HIS tags, and the product was subcloned into the pSN1301 vector under the control of the CAMV 35S promoter. The constructed plasmids were then transformed into Agrobacterium tumefaciens strain C58 and introduced into the cplepa-1 mutant plants by a floral dip method, as described previously [25]. Transgenic plants were selected on MS medium containing 50 mg/mL hygromycin. Complemented plants were selected and transferred to soil to produce seeds. The success of the complementation was confirmed by PCR, immunoblot and chlorophyll fluorescence analysis.Chloroplast UltrastructureWild type and mutant leaves from 3-week-old plants grown on soil were used for transmission electron microscopy analysis. The leaves were chopped into 162 mm pieces and immersed in fixative solution (2.4 glutaraldehyde in phosphate buffer) for 4 h at 4uC. After fixation, the samples were rinsed and postfixed in 1 OsO4 overnight at 4uC and then dehydrated in an ethanol series, infiltrated with a graded series of epoxy resin in epoxy propane, and embedded in Epon 812 resin. Thin (80?00 nm) sections were obtained using a diamond knife on a Reichert OM2 ultramicrotome. The sections were stained with 2 uranyl acetate, pH 5.0, followed by 10 mM lead citrate, pH 12, and observed with a transmission electron microscope (Jem-1230; JEOL).Materials and Methods Plant Material and Growth 1081537 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 16574785 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. Afte.CpLEPA in efficient photosynthesis in higher plants. In addition, we have presented evidence highlighting the importance of this protein for chloroplast translation, which provides further insights into the conserved function of LEPA in chloroplast protein synthesis.maintained at 22uC throughout the photoinhibitory treatments. The synthesis of chloroplast-encoded proteins was blocked by incubating detached leaves with 1 mM lincomycin at low light (20 mmol m22 s21) for 3 h before photoinhibition treatment. To investigate the effects of high light on plant growth, we transferred 2-week-old Arabidopsis plants grown on soil under normal illumination of 120 mmol m22 s21 to 500 mmol m22 s21for another 2 weeks.ComplementationTo complement the cpLEPA mutation, a full-length cpLEPA cDNA was amplified using nested antisense primers (LEPAH-F, LEPAH-R1 and LEPAH-R2) with HIS tags, and the product was subcloned into the pSN1301 vector under the control of the CAMV 35S promoter. The constructed plasmids were then transformed into Agrobacterium tumefaciens strain C58 and introduced into the cplepa-1 mutant plants by a floral dip method, as described previously [25]. Transgenic plants were selected on MS medium containing 50 mg/mL hygromycin. Complemented plants were selected and transferred to soil to produce seeds. The success of the complementation was confirmed by PCR, immunoblot and chlorophyll fluorescence analysis.Chloroplast UltrastructureWild type and mutant leaves from 3-week-old plants grown on soil were used for transmission electron microscopy analysis. The leaves were chopped into 162 mm pieces and immersed in fixative solution (2.4 glutaraldehyde in phosphate buffer) for 4 h at 4uC. After fixation, the samples were rinsed and postfixed in 1 OsO4 overnight at 4uC and then dehydrated in an ethanol series, infiltrated with a graded series of epoxy resin in epoxy propane, and embedded in Epon 812 resin. Thin (80?00 nm) sections were obtained using a diamond knife on a Reichert OM2 ultramicrotome. The sections were stained with 2 uranyl acetate, pH 5.0, followed by 10 mM lead citrate, pH 12, and observed with a transmission electron microscope (Jem-1230; JEOL).Materials and Methods Plant Material and Growth 1081537 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 16574785 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. Afte.

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