Showed a moderately decreased synthesis rate for the chloroplast-encoded proteins, which may account for the accumulation of ABBV-075 site 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 MedChemExpress 370-86-5 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 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 23388095 insights into the conserved function of LEPA in chloroplast protein synthesis.maintained at 22uC throughout the 1480666 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 elect.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 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 23388095 insights into the conserved function of LEPA in chloroplast protein synthesis.maintained at 22uC throughout the 1480666 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 elect.