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Chloroplast ribulose-5-phosphate-3-epimerase (RPE) is a critical enzyme involved in the Calvin-Benson cycle and oxidative pentose phosphate pathways in higher plants. Three Arabidopsis rpe mutants with reduced level of RPE were identified through their high NPQ (nonphotochemical quenching) phenotype upon illumination, and no significant difference of plant size was found between these rpe mutants and WT (wild type) plants under growth chamber conditions. A decrease in RPE expression to a certain extent leads to a decrease in CO 2 fixation, V cmax and J max . Photosynthetic linear electron transport was partially inhibited and activity of ATP synthase was also decreased in the rpe mutants, but the levels of thylakoid protein complexes and other Calvin-Benson cycle enzymes in rpe mutants were not affected. These results demonstrate that some degree of reduction in RPE expression decreases carbon fixation in chloroplasts, which in turn feedback inhibits photosynthetic electron transport and ATP synthase activity due to the photosynthetic control. Taken together, this work provides evidence that RPE plays an important role in the Calvin-Benson cycle and influences the photosynthetic capacity of chloroplasts.

In chloroplasts, insertion of proteins with multiple transmembrane domains (TMDs) into thylakoid membranes usually occurs in a co-translational manner. Here, we have characterized a thylakoid protein designated FPB1 (Facilitator of PsbB biogenesis1) which together with a previously reported factor PAM68 (Photosynthesis Affected Mutant68) is involved in assisting the biogenesis of CP47, a subunit of the Photosystem II (PSII) core. Analysis by ribosome profiling reveals increased ribosome stalling when the last TMD segment of CP47 emerges from the ribosomal tunnel in fpb1 and pam68 . FPB1 interacts with PAM68 and both proteins coimmunoprecipitate with SecY/E and Alb3 as well as with some ribosomal components. Thus, our data indicate that, in coordination with the SecY/E translocon and the Alb3 integrase, FPB1 synergistically cooperates with PAM68 to facilitate the co-translational integration of the last two CP47 TMDs and the large loop between them into thylakoids and the PSII core complex. The assembly of the Photosystem II proximal antenna CP47 remains a challenging question. Here the authors show that FPB1 and PAM68 act coordinately with Alb3 and the SecY/E translocon to facilitate the co-translational integration of specific regions of CP47 into thylakoids.

Thylakoid membrane-localized chloroplast ATP synthases use the proton motive force generated by photosynthetic electron transport to produce ATP from ADP. Although it is well known that the chloroplast ATP synthase is composed of more than 20 proteins with α₃β₃γ₁ϵ₁δ₁I₁II₁III₁₄IV₁ stoichiometry, its biogenesis process is currently unclear. To unravel the molecular mechanisms underlying the biogenesis of chloroplast ATP synthase, we performed extensive screening for isolating ATP synthase mutants in Arabidopsis (Arabidopsis thaliana). In the recently identified bfa3 (biogenesis factors required for ATP synthase 3) mutant, the levels of chloroplast ATP synthase subunits were reduced to approximately 25% of wild-type levels. In vivo labeling analysis showed that assembly of the CF₁ component of chloroplast ATP synthase was less efficient in bfa3 than in the wild type, indicating that BFA3 is required for CF₁ assembly. BFA3 encodes a chloroplast stromal protein that is conserved in higher plants, green algae, and a few species of other eukaryotic algae, and specifically interacts with the CF₁β subunit. The BFA3 binding site was mapped to a region in the catalytic site of CF₁β. Several residues highly conserved in eukaryotic CF₁β are crucial for the BFA3–CF₁β interaction, suggesting a coevolutionary relationship between BFA3 and CF₁β. BFA3 appears to function as a molecular chaperone that transiently associates with unassembled CF₁β at its catalytic site and facilitates subsequent association with CF₁α during assembly of the CF₁ subcomplex of chloroplast ATP synthase

RNA editing is a posttranscriptional process that covalently alters the sequence of RNA molecules and plays important biological roles in both animals and land plants. In flowering plants, RNA editing converts specific cytidine residues to uridine in both plastid and mitochondrial transcripts. Previous studies identified pentatricopeptide repeat (PPR) motif-containing proteins as site-specific recognition factors for cytidine targets in RNA sequences. However, the regulatory mechanism underlying RNA editing was largely unknown. Here, we report that protoporphyrinogen IX oxidase 1 (PPO1), an enzyme that catalyzes protoporphyrinogen IX into protoporphyrin IX in the tetrapyrrole biosynthetic pathway, plays an unexpected role in editing multiple sites of plastid RNA transcripts, most of which encode subunits of the NADH dehydrogenase-like complex (NDH), in the reference plant Arabidopsis thaliana. We identified multiple organellar RNA editing factors (MORFs), including MORF2, MORF8, and MORF9, that interact with PPO1. We found that two conserved motifs within the 22-aa region at the N terminus of PPO1 are essential for its interaction with MORFs, its RNA editing function, and subsequently, its effect on NDH activity. However, transgenic plants lacking key domains for the tetrapyrrole biosynthetic activity of PPO1 exhibit normal RNA editing. Furthermore, MORF2 and MORF9 interact with three PPRs or related proteins required for editing of ndhB and ndhD sites. These results reveal that the tetrapyrrole biosynthetic enzyme PPO1 is required for plastid RNA editing, acting as a regulator that promotes the stability of MORF proteins through physical interaction.

Chloroplast thylakoid protein rubredoxin 1 (RBD1) in Chlamydomonas and its cyanobacterial homolog RubA contain a rubredoxin domain. These proteins have been proposed to participate in the assembly of photosystem II (PSII) at early stages. However, the effects of inactivation of RBD1 on PSII assembly in higher plants are largely unclear. Here, we characterized an Arabidopsis rbd1 mutant in detail. A drastic reduction of intact PSII complex but relatively higher levels of assembly intermediates including PSII RC, pre-CP47, and pre-CP43 were found in rbd1 . Polysome association and ribosome profiling revealed that ribosome recruitment of psbA mRNA is specifically reduced. Consistently, in vivo protein pulse-chase labeling showed that the rate of D1/pD1 synthesis is significantly reduced in rbd1 compared with WT. Moreover, newly synthesized mature D1 and pD1/D2 can assemble into the PSII reaction center (RC) complex but further formation of larger PSII complexes is nearly totally blocked in rbd1 . Our data imply that RBD1 is not only required for the formation of a functional PSII core complex during the early stages of PSII assembly but may also be involved in the translation of D1 in higher plants.

As a fascinating and complicated nanomotor, chloroplast ATP synthase comprises nine subunits encoded by both the nuclear and plastid genomes. Because of its uneven subunit stoichiometry, biogenesis of ATP synthase and expression of plastid-encoded ATP synthase genes requires assistance by nucleus-encoded factors involved in transcriptional, post-transcriptional, and translational steps. In this study, we report a P-class pentatricopeptide repeat (PPR) protein BFA2 (Biogenesis Factor required for ATP synthase 2) that is essential for accumulation of the dicistronic transcript in Arabidopsis chloroplasts. A loss-of-function mutation in results in a specific reduction of more than 3/4 of chloroplast ATP synthase, which is likely due to the absence of dicistronic transcript. BFA2 protein contains 22 putative PPR motifs and exclusively localizes in the chloroplast. Bioinformatics and Electrophoretic Mobility Shift Assays (EMSA) analysis showed that BFA2 binds to the consensus sequence of the intergenic region in a sequence-specific manner. However, translation initiation of the was not affected in the mutant. Thus, we propose that the chloroplast PPR protein BFA2 mainly acts as barrier to prevent the transcript degradation by exoribonucleases by binding to the consensus sequence of the intergenic region.

Objective This study aimed to assess the safety and feasibility of bile duct-guided right hepatic inflow occlusion and associated extended procedures in laparoscopic liver surgery (LLS). Methods A cohort of 23 patients who underwent laparoscopic right hemihepatectomy, which involved laparoscopic dissection and occlusion of the right hepatic artery and right portal vein using the bile duct-guided approach (primary procedure) were included in this study. Ten additional patients with insufficient future liver remnant (FLR) volume in the left hemiliver, as determined by preoperative assessment, underwent a staged approach. In the first stage, laparoscopic right portal vein ligation (LRPVL) was performed via the bile duct-guided approach (extended procedure). Following an observation interval, and upon confirmation of sufficient hypertrophy of the left hemiliver, either laparoscopic or open right hemihepatectomy, with or without combined procedures, was performed as the second stage. Results All procedures were completed without major postoperative complications. In the primary procedure cohort, the mean operative duration was 4.2 ± 0.4 h, the mean right hepatic portal occlusion time was 18 ± 8 min, intraoperative blood loss ranged from 200 to 400 ml, and the length of hospital stay was 7–12 days. For the first-stage extended procedure, the mean operative duration was 1.2 ± 0.3 h, the mean right portal vein occlusion was 14 ± 5 min, intraoperative blood loss ranged from 20 to 80 ml, and the length of hospital stay was 3–5 days. The second-stage procedure was performed 3 to 5 days after the most recent three-dimensional imaging assessment. The mean left liver volume increased from 456.14 ± 84.42 cm 3 before the first-stage procedure to 649.06 ± 102.26 cm 3 before the second-stage procedure, corresponding to a 44 ± 18% increase. Conclusion Bile duct-guided right hepatic inflow occlusion and its extended procedures demonstrated safety and feasibility in the context of LLS.

To gain insights into the molecular details of photosystem I (PSI) biogenesis, we characterized the PsbP-domain protein1 (ppd1) mutant of Arabidopsis thaliana that specifically lacks PSI activity. Deletion of PPD1 results in an inability of the mutant to grow photoautotrophically and a specific loss of the stable PSI complex. Unaltered transcription and translation of plastid-encoded PSI genes indicate that PPD1 acts at the posttranslational level. In vivo protein labeling experiments reveal that the rate of synthesis of PSI reaction center proteins PsaA/B in ppd1 is comparable to that of wild-type plants, whereas the rate of turnover of PsaA/B proteins is higher in ppd1 than in wild-type plants. With increasing leaf age, PPD1 content decreases considerably, while PSI content remains constant. PPD1 is a nuclear-encoded thylakoid lumenal protein and is associated with PSI but is not an integral subunit of PSI. Biochemical and molecular analyses reveal that PPD1 interacts directly and specifically with PsaB and PsaA. Yeast two-hybrid experiments show that PPD1 interacts with some lumenal loops of PsaB and PsaA. Our results suggest that PPD1 is a PSI assembly factor that assists the proper folding and integration of PsaB and PsaA into the thylakoid membrane.

Although numerous studies have been carried out on chloroplast development and biogenesis, the underlying regulatory mechanisms are still largely elusive. Here, we characterized a chloroplast stromal protein Chloroplast Development and Biogenesis1 (CDB1). The knockout cdb1 mutant exhibits a seedling-lethal and ivory leaf phenotype. Immunoblot and RNA blot analyses show that accumulation of chloroplast ribosomes is compromised in cdb1 , resulting in an almost complete loss of plastid-encoded proteins including the core subunits of the plastid-encoded RNA polymerase (PEP) RpoB and RpoC2, and therefore in impaired PEP activity. Orthologs of CDB1 are found in green algae and land plants. Moreover, a protein shows high similarity with CDB1, designated as CDB1-Like (CDB1L), is present in angiosperms. Absence of CDB1L results in impaired embryo development. While CDB1 is specifically located in the chloroplast stroma, CDB1L is localized in both chloroplasts and mitochondria in Arabidopsis . Thus, our results demonstrate that CDB1 is indispensable for chloroplast development and biogenesis through its involvement in chloroplast ribosome assembly whereas CDB1L may fulfill a similar function in both mitochondria and chloroplasts.